Reagent and method for detection of ADP-ribosyl-linked ubiquitylation

Abstract

The present invention relates to a protein being capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP- ribosyl-linked ubiquitylation.

Description

[0001] New PCT-Patent Application

[0002] Max-Planck-Gesellschaft zur Fbrderung der Wissenschaften e. V.

[0003] Vossius Ref.: AK1290 PCT

[0004] Reagent and method for detection of ADP-ribosyl-linked ubiquitylation

[0005] The present invention relates to a protein being capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation.

[0006] In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0007] By greatly augmenting the functional versatility of the proteome, far beyond what is encoded by the genome, post translational modifications (PTMs) are pivotal regulators of all signaling pathways1. Within complex and dynamic cellular networks, distinct PTMs often engage in crosstalk, occasionally interacting directly at the chemical level to generate composite signals. An emerging example of the combined chemistries of two distinct PTMs is the interplay between ADP-ribosylation (ADPr) and ubiquitylation2.

[0008] ADPr is a modification of protein, RNA and DNA that plays crucial roles in regulating key biological functions across all forms of life3. Catalyzed by various ADP-ribosyltransferases, including Poly(ADP-ribose) Polymerases (PARPs), ADPr involves the covalent addition of ADP-ribose from NAD+to diverse amino acid side chains and nucleotides through different conjugation chemistries, ranging from O-glycosidic to ester-linked ADPr4-5. This chemical diversity and versatility results in high complexity, leading to decades of technical challenges that have only recently been addressed through the development of effective tools and approaches6-7. Research on ADPr has primarily focused on PARPl's role in the DNA damage response (DDR). PARP1 detects DNA breaks and modifies various targets, mainly itself and histones, to recruit DNA repair factors and chromatin remodelers to the DNA lesions8. Historically, poly-ADPr on aspartate and glutamate was considered as the only signal generated by PARP1810. Our initial identification of histone serine ADPr (Ser-ADPr)11led us and others to establish Ser-ADPr by PARP1 / HPF1 as a widespread and functionally significant PTM12 17. This discovery hasprompted a profound reinterpretation of PARP1 signaling18, illustrating how uncovering the chemical nature of ADPr in cells can transform a research field. In a transient complex with HPF113, PARP1 synthesizes mono-ADPr on serine residues12. In contrast, without HPF1, PARP1 extends the initial ADP-ribose to poly-ADPr14-19and, as we have recently shown, catalyzes mono-ADPr on aspartate and glutamate, which are the main target residues in cells lacking HPF120. Several hydrolases remove different forms of ADPr. While ARH3 cleaves O-glycosidic serine ADPr (Ser-ADPr)21-22, PARG breaks down poly-ADPr23and for some targets, especially PARP1 itself, acts as a poly-to-mono converting enzyme24-25. The inability of PARG to remove the initial ADP-ribose from serine residues14, combined with the PARP / HPFl-mediated generation of mono-ADPr on these residues, gives rise to serine mono-ADPr24. This second wave of PARP1 signaling recruits the ubiquitin E3 ligase RNF114 to DNA lesions, where it regulates the DNA damage response and telomere maintenance24. The recruitment of RNF114 to DNA lesions depends on its C-terminal zfDil9 domain and Ubiquitin Interaction Motif (UIM), which recognize mono-ADPr and ubiquitin, respectively24-26. It has also been reported that RNF114 can recognize poly-ADPr27. Beyond its role in PARP1 signaling, RNF114 has recently been shown to recognize and stabilize mono-ADP-ribosylated tankyrase28.

[0009] In addition to functioning as a PTM on its own, ADPr can also directly contribute to the conjugation chemistry of ubiquitylation2. A notable example of this interplay is a chemically unique form of ubiquitylation catalyzed by an effector of Legionella pneumophila, which ADP-ribosylates Arg42 of ubiquitin. This modification is then processed to enable conjugation of ubiquitin to serine residues via a phosphodiester bond, resulting in phosphoribose-linked serine ubiquitylation29. Another example is the unconventional ester-linked ubiquitylation of ADP-ribose on the 3'-hydroxy group of its adenine-proximal ribose, recently reported in studies using biochemical reactions with recombinant DELTEX ubiquitin E3 ligases30. Given that DELTEX enzymes catalyze the ubiquitylation of free ADP-ribose and ADP-ribose attached to nucleic acid and proteins, as well as direct ubiquitylation of nucleic acids and canonical lysine-linked ubiquitylation30-34, it remains unclear which of these potential modifications occur in cells and if ADP-ribosyl-linked ubiquitylation truly constitutes a protein PTM. Crucially, a series of technical challenges have hindered mass spectrometry - the gold standard for PTM characterization - from detecting ester-linked ubiquitylation of ADP-ribose not only in cellular contexts but even in supposedly less challenging biochemical reactions.

[0010] The aim of the present invention is to provide the first means and method for the identification of ADP-ribosyl-linked ubiquitylation in cells and in particular the ADP-ribosyl-linked ubiquitylation sites in an amino acid sequence.Accordingly, the present invention relates in first aspect to protein being capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation, wherein the protein comprises or consists of

[0011] (a) the amino acids of positions 140 to 228, preferably 138 to 228 of any one of SEQ ID NO: 1, 4, 5 and 6 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or amino acids of positions 141 to 229, preferably 139 to 229 of SEQ ID NO: 2 or 3 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; or amino acids of positions 142 to 230, preferably 140 to 229 of SEQ ID NO: 7 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or amino acids of positions 135 to 223, preferably 133 to 223 of SEQ ID NO: 8 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto,

[0012] (b) the amino acids of positions 149 to 237, preferably 147 to 237 of any one of SEQ ID NOs 9 to 11, 14 and 15 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 156 to 244, preferably 154 to 244 of SEQ ID NO: 12 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 153 to 241, preferably 151 to 241 of SEQ ID NO: 13 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto;

[0013] (c) the amino acids of positions 138 to 225, preferably 136 to 225of SEQ ID NO: 16 or 20 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 139 to 226, preferably 137 to 226 of SEQ ID NO: 17 or 19 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 137 to 224, preferably 135 to 224 of SEQ ID NO: 18 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 127 to 217, preferably 125 to 217of SEQ ID NO: 21 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; or

[0014] (d) the amino acids of positions 156 to 244, preferably 154 to 244 of any one of SEQ ID NOs 22 to 24 or 27 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 133 to 217, preferably 131 to 217 of SEQ ID NO: 25 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 156 to 209, preferably 154 to 209 of SEQ ID NO: 26 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 157 to 245, preferably 155 to 245 of SEQ ID NO: 28 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical.ADP-ribosylation (ADPr) is the addition of one or more ADP-ribose moieties to a biomolecule (protein, peptide, or nucleic acid molecule (RNA or DNA)) and preferably to one or more amino acids of a protein or peptide. ADPr may be mono-ADP-ribosylation or poly-ADP-ribosylation. Serine-mono-ADP ribosylation is the addition of only one ADP-ribose to a serine side chain. Serine-poly-ADP ribosylation is the addition of two or more ADP-riboses to a serine side chain.

[0015] ADP-ribosyl-linked ubiquitylation comprises the ester-linked ubiquitylation of ADP-ribose (Fig. 2a). While ADP-ribosyl-linked ubiquitylation may exist as a free molecule or may be found as a PTM on a biomolecule (protein, peptide, or nucleic acid molecule (RNA or DNA)), the ester-linked ubiquitylation of ADP-ribose is preferably on mono-ADP-ribosylation and more preferably on serine mono-ADP-ribosylation. Hence, ADP-ribosyl-linked ubiquitylation is most preferably a serine post-translational modification. Accordingly, the protein being of the fist aspect is capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids (preferably comprising or being one or more serines) being modified by ADP-ribosyl-linked ubiquitylation.

[0016] The terms "protein" (wherein "protein" is interchangeably used with "polypeptide") and "peptide" as used herein describe a group of molecules consisting of amino acids. Whereas peptides consist of up to 30 amino acids, "proteins" consist of more than 30 amino acids. Peptides and proteins may further form dimers, trimers and higher oligomers, i.e. consisting of more than one molecule which may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo-or heterodimers, homo- or heterotrimers etc. Peptides and proteins are preferably composed of the 20 naturally-occurring amino acids being encoded by the genetic code optionally plus selenocysteine. However, the peptides and proteins may also comprise one or more non-natural amino acids, noting that about 500 amino acids are known in the art. Any non-natural amino acid is preferably an a-amino acids (generic formula H2NCHRCOOH, where R is an organic substituent known as a "side chain" of the amino acid).

[0017] In accordance with the present invention, the term "percent (%) sequence identity" describes the number of matches ("hits") of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the template amino acid sequences. In other terms, using an alignment, for two or more sequences or subsequences the percentage of amino acid residues that are the same (e.g. 80%, 85%, 90% or 95% identity) may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences whichare compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids.

[0018] The skilled person is also aware of suitable programs to align amino acid sequences. The percentage sequence identity of amino acid sequences can, for example, be determined with programmes such as CLUSTLAW, FASTA and BLAST. Preferably the BLAST programme is used, namely the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), " Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402).

[0019] SEQ ID NOs 1 to 8 are the amino acid sequences of the protein RNF114 from human, mouse, rat, chimpanzee, pig, macaque, cow and frog, respectively.

[0020] RNF114 is a E3 ubiquitin-protein ligase (EC 2.3.2.27) that is known to comprise a Ubiquitin-lnteracting Motif (UIM). As shown in Fig. 1A the UIM can be found at the N-terminus of RNF114. Is furthermore known from Longarini et al (2023), Molecular Cell, 83(10):1743-1760.ell that RNF114 is a mono-ADPr reader in telomere maintenance and DNA repair signaling. It is shown therein that the Dil9 zinc-binding domain interacts with serine mono-ADPr. As shown in Fig. 1A the Dil9 zinc-binding domain can be found N-terminally of the UIM in the C-terminal part of RNF114.

[0021] SEQ ID NOs 9 to 15 are the amino acid sequences of the protein RNF166 from human, mouse, rat, chicken, frog, macaque and cow, respectively. SEQ ID NOs 16 to 21 are the amino acid sequences of the protein RNF125 for human, mouse, macaque, rat, cow and frog, respectively. SEQ ID NOs 22 to 28 are the amino acid sequences of the protein RNF138 for human, mouse, cow, frog, rat, macaque and chicken, respectively.

[0022] Just as RNF114 also RNF166, RNF125 and RNF138 are E3 ubiquitin-protein ligases (EC 2.3.2.27). Just as RNF114 also RNF166, RNF125 and RNF138 comprise in the C-terminal part Dil9 zinc -binding domain followed by a UIM. A sequence alignment of these RNFs from human is:

[0023] CLUSTAL 0 ( 1. 2. 4 ) multiple sequence alignment

[0024] RNF

[0025] 125 - MGSVLSTDSGKSAPASATARALERRRDPELPVTSFDCAVCLEVLHQPVRT-RC

[0026] 52

[0027] 138 - MAED - LSAATSYTEDDFYCPVCQEVLKTPVRTTAC114 - MAAQQRDCGGAAQLAG - PA-AEADPLGRFTCPVCLEVYEKPVQV-PC 44

[0028] 166 MAMERS LVASAQQRQ - PPAG - PAGGDSGLEAQYTCPICLEVYHRPVAIGSC

[0029] 49

[0030] RNF

[0031] 125 GHVFCRSCIATSLKNNKWTCPYCRAYLPSEG - VPATDVAKRMKSEYKNCAECDTLVC 108

[0032] 138 QHVFCRKCFLTAMRESGAHCPLCRGNVTRRERACPERALDLENIMRKFSGSCRCCAKQIK

[0033] 94

[0034] 114 GHVFCSACLQECLKPKKPVCGVCRSALAPG V — RAVELERQIESTETSCHGCRKNFF

[0035] 99

[0036] 166 GHTFCGECLQPCLQVPSPLCPLCRLPFDPK KVDKATHVEKQLSSYKAPCRGCNKKVT 106

[0037] RNF

[0038] 125 LSEMRAHIRTCQKYIDKYGPLQELE - ET 135

[0039] 138 FYRMRHHYKSCKKYQDEYGVSSI-IPNFQISQDSVGNSNRSETSTSDNTETYQENTSSSG 153

[0040] 114 LSKIRSHVATCSKYQNYIMEGVK — ATIKDAS - LQPRNVPN 137

[0041] 166 LAKMRVHISSCLKVQEQMANCPKFVPVVPTSQ - PIPSNIPN 146

[0042] RNF

[0043] 125 AARCVCPFCQREL - YEDSLLDHC ITHHRSERRPVFCPLCR IPDENPSSFSGSL IRHLQV 194

[0044] 138 HPTFKCPLCQESNFTRQRLLDHCNSNHLFQIVPVTCPICVSLPWGDPSQITRNFVSHLNQ 213

[0045] 114 RYTFPCPYCPEKNFDQEGLVEHCKLFHSTDTKSWCPICASMPWGDPNYRSANFREHIQR 197

[0046] 166 RSTFACPYCGARNLDQQELVKHCVESHRSDPNRWCPICSAMPWGDPSYKSANFLQHLLH 206

[0047] ★ ★ ★ • ★ • ' ★ ★ ★ • • ★ • • RNF

[0048] 125 SHTLFYDDFIDFNIIEEALIRRVLDRSLLEYVNHSNTT 232

[0049] 138 RHQFDYGEFVNLQLDEETQYQTAVEESFQVNI - 245

[0050] 114 RHRFSYDTFVDYDVDEEDMMNQVLQRSIIDQ - 228

[0051] 166 RHKFSYDTFVDYSIDEEAAFQAALALSLSEN - 237

[0052] * • *, * • • • ** ' • ★ •

[0053] In the above alignment the C-terminal parts of the RNFs harboring the Dil9 zinc-binding domain followed by a UIM is shown in bold. The alignment shows that the amino acid sequences of the C-terminal parts of the RNFs are highly conserved.

[0054] The amino acid sequences of the C-terminal parts of the RNFs are not only highly conserved among the RNFs, but also for each RNF across different vertebrate specie, including human, mouse, rat, chimpanzee, pig, macaque, cow, chicken and frog. In this connection the RNFs sequences of mammals are preferred (human, mouse, rat, chimpanzee, pig, macaque and cow), the RNFs sequence of primates (human and macaque) are more preferred and the RNFs sequences of human are most preferred.Among RNF114, RNF166, RNF125 and RNF138, RNF114 is most preferred because RNF114 is illustrated by the appended examples.

[0055] The protein of the first aspect of the invention comprises and preferably consists of the amino acids of positions of the full-length RNFs as indicated in the above first aspect of the invention. The amino acids of positions of the full-length RNFs as indicated in the above aspect of the invention harbor the Dil9 zinc-binding domain (zfDil9) and the UIM. For this reason, the protein of the invention is capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation.

[0056] With regard to the sequence identity language of the first aspect of the invention it is to be understood that all proteins falling under the first aspect of the invention are not only structurally defined by reference to the RBF sequences but also functionally as being capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation. All proteins falling under the first aspect of the invention harbor binding domains for ADP-ribose and ubiquitin.

[0057] With regard to the protein of the first aspect of the invention that comprise the amino acids of positions of the full-length RNFs as indicated in the above first aspect of the invention is preferred that the protein does not comprise the RING finger and Zinc finger 1 domain (Znl) required for catalytic activity of RNFs (Fig la), and more preferably no other binding domains than the binding domains for ADP-ribose and ubiquitin. In molecular biology, binding domain is a protein domain which binds to a specific atom or molecule, such as a PTM, calcium or DNA. Is it likewise preferred that the protein of the first aspect of the invention does not have ubiquitin E3 ligase activity, and more preferably no enzymatic activity.

[0058] The above is preferred and more preferred, because the protein of the first aspect of the invention shall only bind ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation but shall not bind to other specific atoms or molecules within cells and shall not display other biologic activities than this binding activity.

[0059] It is shown in the appended examples based on human RNF114 that the N-terminal part of RNFs RNF114, RNF166, RNF125 and RNF138 harboring and the Dil9 zinc-binding domain and the UIM is a means for the specific detection of ADP-ribosyl-linked ubiquitylation and can be used in methods for the specific detection of ADP-ribosyl-linked ubiquitylation. In the examples a short, acidic ArgCdigestion method that was recently developed by the inventors of the present application (Longarini and Matic, Nature Communications volume 15, Article number: 4239 (2024)) is combined with an enrichment strategy based on the zfDil9-UIM domains of RNF114 for the specific detection of ADP-ribosyl-linked ubiquitylation and the enrichment of proteins being modified by ADP-ribosyl-linked ubiquitylation. As a key step in the enrichment procedure, specific chemical elution is achieved through zinc ion chelation by EDTA, disrupting the binding of the zfDil9 domain to mono-ADPr substrates, thereby ensuring specificity for ADP-ribosyl-linked ubiquitylated proteins. This biochemical enrichment strategy is complemented by proteomics approaches tailored to the unique chemical features of the composite PTM ADP-ribosyl-linked ubiquitylation. Beyond proteomics, the spytag protein ligation technology is applied in the appended examples to convert the zfDil9-U IM domains into a reagent that enables the detection of cellular ADP-ribosyl-linked ubiquitylation by western blotting following enrichment and the specific EDTA elution. Through these multilevel methodological advances, ADP-ribosyl-linked serine ubiquitylation has been unveiled in cells within the context of PARP1 signaling, identifying sites on histones, PARP1 and additional targets. The finding of first protein being capable of specifically binding to ADP-ribosyl-linked ubiquitylation, along with the repertoire of approaches that use this protein as shown in the appended examples, paves the way for the identification of chemical variants of the complex composite PTM ADP-ribosyl-linked ubiquitylation in diverse signaling pathways.

[0060] The present invention relates in a second aspect to a protein conjugate or fusion protein comprising the protein of the first aspect.

[0061] The definitions and preferred embodiments of the above first aspect apply mutatis mutandis to the second aspect as far as being amendable for combination therewith.

[0062] A "fusion protein" according to the present invention contains at least one additional heterologous amino acid sequence as fusion partner. Often, but not necessarily, these additional sequences will be located at the N- or C-terminal end of the protein of the invention. It may e.g. be convenient to initially express the protein of the invention as a fusion protein from which the additional amino acid residues can be removed, e.g. by a proteinase capable of specifically trimming the fusion protein and releasing the protein of the invention. Those at least one additional heterologous amino acid sequence of said fusion proteins includes amino acid sequences which confer desired properties such as modified / enhanced stability, modified / enhanced solubility and / or the ability of targeting one or more specific cell types. The amino acid sequence compound can either be directly or indirectly fused to thenucleic acid molecule of the invention. In case of an indirect fusion generally a peptide linker may be used for the fusion, such that a GS-linker.

[0063] A "protein conjugate" according to the present invention contains at least one additional heterologous as conjugation partner other than an amino acid sequence. While the fusion protein can be encoded by a nucleic acid sequence the protein conjugate cannot be encoded by a nucleic acid sequence. The heterologous non-proteinaceous compound can either be directly or indirectly fused to the nucleic acid molecule of the invention. For example, chemical linker may be used. Chemical linkers may contain diverse functional groups, such as primary amines, sulfhydryls, acids, alcohols and bromides. Many of our crosslinkers are functionalized with maleimide (sulfhydral reactive) and succinimidyl ester (NHS) or isothiocyanate (ITC) groups that react with amines. The heterologous non-proteinaceous compound can be, for example, a pharmaceutically active compound or diagnostically active compound.

[0064] As discussed above and as shown in the appended examples conjugating or fusing the protein of the first aspect to one or more conjugation or fusion partner might be advantageous for specific applications of the protein of the first aspect for the detection of ADP-ribosyl-linked ubiquitylation, preferably of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acid sequence that are modified by ADP-ribosyl-linked ubiquitylation.

[0065] While the conjugation or fusion partners are not particularly limited preferred examples and their technical advantages will be provided herein below.

[0066] In accordance with a preferred embodiment of the second aspect the protein of the first aspect is fused to a spytag, preferably the spytagB or a sequence being at least 80%, preferably at least 90% identical thereto.

[0067] In accordance with a more preferred embodiment of the second aspect the protein conjugate or fusion protein is coupled via an isopeptide bond to a spycatcher protein, preferably spycatcherB, wherein the spycatcher protein is preferably a dimer to which two copies of the protein of claim 1 are linked.

[0068] The spytag / spycatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide spytag (generally 13 amino acids) spontaneously reacts with the protein spycatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair. DNA sequence encoding either spytag or spycatcher can be recombinantly introduced into the DNA sequence encoding a protein ofinterest, forming a fusion protein. These fusion proteins can be covalently linked when mixed in a reaction through the spytag / spycatcher system. Spytag / spycatcher react with high specificity even when in the presence of bacterial and mammalian cell environments. The spytag / spycatcher system is, for example, described in Hentrich et al. (2021); Cell Chemical Biology, 28(6):813-824.e6 and WO 2020 / 188350. The spycatcher protein can be produced by bacterial cytoplasmic expression and purified via Ni NTA as described by Zakeri et al. (2012), PNAS, 109: E690-697. The nucleotide and amino acid sequence of a spycatcher protein has been deposited by Zakeri et al. (2012), loc. lit. under Gen Bank: JQ478411.1. Spytags and spycatcher are commercially available, for example, from kerafast und BioRad.

[0069] By the spytag / spycatcher technology bioconjugation can be achieved between two recombinant proteins that would otherwise be restrictive or impossible with traditional direct genetic fusion between the two proteins. For example, issues regarding protein folding, suboptimal expression host, and specialized post-translational modifications can be alleviated by separating the production of the proteins with the modularity of the tag / catcher system.

[0070] The amino acid sequence of the spytag3 is RGVPHIVMVDAYKRYK (SEQ ID NO: 29). spytag3 / spycatcher3 reacts up to 400-fold faster than the original pair with a rate constant of 5.5 ± 0.6 x 105M-1s-1. Spycatcher3 (S49C) contains an additional unique cysteine residue for precise labeling with dye or precise attachment to surfaces or beads. Via S49C (S-S bond) also a dimeric Spycatcher3 (S49C) can be prepared.

[0071] The amino acid sequence of spycatcher3 is SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSS GKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS (SEQ ID NO: 30)

[0072] The amino acid sequence of spycatcher3 (S49C) is: SYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDCS GKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS (SEQ ID NO: 31)

[0073] The spycatcher as used herein preferably shares a sequence identity - with increasing preference - of at least 80%, at least 85% at least 90, at least 95%, at least 97,5% and at least 99% with SEQ ID NO: 30or 31. The spycatcher as used herein most preferably comprises or consists of SEQ. ID NO: 30 or 31, or a dimer thereof.

[0074] The fusion protein with the spytag of the second aspect is preferably coupled via an isopeptide bond to a spycatcher protein. In this connection it is preferred that the spycatcher protein is a dimer. For the spycatcher protein dimer preferably two spycatcher protein are fused via a linker, preferably a flexible linker (e.g. peptide linker). By this way two copies of the fusion protein with the spytag of the second aspect can be coupled via the dimeric spycatcher protein (Fig. 5a). The spycatcher protein in turn can be conjugated or fused to further conjugation or fusion partners. This is illustrated in Fig. 5a, wherein one copy of the spycatcher protein of the dimer is bound to one and the other copy of the spycatcher protein of the dimer is bound to two copies of biotinylated peroxidase horse radish peroxidae (HRP / biotin).

[0075] In accordance with a preferred embodiment of the second aspect the protein of the first aspect and / or the spycatcher protein is fused or conjugated to (a) a fluorescent protein, preferably GFP, RFP, or YFP or a fluorophore, preferably, an Alexa- or Cy-fluorophore, (b) a tag, preferably a GST-tag, His-tag, Flagtag, Strep-tag or ALFA-tag, (c) biotin, (d) an antibody or an antibody fragment, preferably an Fc part, and / or (e) an enzyme or truncated version thereof being enzymatically active, preferably horse radish peroxidase (HRP) or alkaline phosphatase (ALP).

[0076] A fusion / conjugation to a fluorescent protein or fluorophore is advantageous for the detection of the protein, protein conjugate or fusion protein of the invention, for example by FACS or fluorescent microscopy.

[0077] A fusion / conjugation to a tag is advantageous for the isolation of the protein, protein conjugate or fusion protein of the invention, for example by chromatographic methods.

[0078] A fusion / conjugation to biotin is advantageous, because thereby the protein, protein conjugate or fusion protein of the invention can be coupled to avidin or streptavidin. The strept(avidin)-biotin complex is the strongest known non-covalent interaction (Kd = 10-15M) between a protein and ligand. Applications for which the strept(avidin)-biotin interaction is used include enzyme linked immunosorbent assay (ELISA), immunohistochemistry (IHC), Western, Northern and Southern blotting, immunoprecipitation, cell surface labeling, affinity purification, fluorescence-activated cell sorting (FACS) and electrophoretic mobility shift assays (EMSA).A fusion / conjugation to an antibody or an antibody fragment, preferably an Fc part is advantageous because it allows for stabilizing the protein, protein conjugate or fusion protein (e.g. via an Fc part) or also allows for binding the protein, protein conjugate or fusion protein of the invention to a desired antigen (e.g. via an antibody or antigen binding fragment therefore).

[0079] A fusion / conjugation to an enzyme or truncated version thereof being enzymatically active is advantageous because the enzymes can catalyze chemical reactions in order to visualize the protein, protein conjugate or fusion protein. For instance, HRP and ALP enzymes can catalyze a colorimetric reaction, which is, for example, the typical readout of an ELISA.

[0080] It is preferred that the above fusion or conjugation constructs are prepared by conjugating the protein of the invention to the one or three cysteines of the spycatcher protein (which is how HRP and biotin are added in Fig5a).

[0081] Among the above options it is preferred to fuse or conjugate to the spycatcher protein to HRP, ALP, biotin, fluorophores, while protein / peptide tags are preferably fused or conjugated directly to the protein of the first aspect.

[0082] It is also particularly preferred to fuse the protein of the first aspect to an Fc. This is because such a fusion protein can then be used for various applications by combining it with secondary antibodies with HRP, fluorophores, protein A / G for immunoprecipitation.

[0083] It is also particularly preferred to use the spytag system with an extra simple spycatcher conjugation step but without secondary antibodies for most applications. The case of the fusion of the protein to the invention to a spytag the Fc is preferably introduced via fusion to the spycatcher. The same secondary antibodies as described before could then be used.

[0084] In the appended examples the following fusion protein of zf Di 19-UIM of RNF114 with the fluorescent protein GFP is used.

[0085] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYP DHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYKSGSGPDQTS / . Y AGFNRYTFPCPYCPEKNFDQEGLVEHCKLFHSTDTKSVVCPICASMPW GDPNYRSANFREHIQRRHRFSYDTFVDYDVDEEDMMNQVLQRSIIDQ* (SEQ ID NO: 33)1-239: GFP

[0086] 240-242: Linker

[0087] 243-255: attBl site (used for cloning; no functional relevance)

[0088] 256-347: zfDil9-UIM of RNF114

[0089] The above fusion and conjugation partners can also be combined. For instance, In the appended examples also the following fusion protein is illustrated:

[0090] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPD FMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEV7. FQ GPLGSHHHHHHGSGRGVPHIVMVDAYKRYKGSGDYKDDDDKGSGRYTFPCPYCPEKNFDQEGLVEHCKLFHSTD TKSVVCPICASMPWGDPNYRSANFREHIQRRHRFSYDTFVDYDVDEEDMMNQVLQRSIIDQ (SEQ ID NO 32)

[0091] 1-218: GST tag

[0092] 221-229: HRV3C site

[0093] 232-237: Hexa-Histidin tag

[0094] 241-256 SpyTag3

[0095] 260-267 Flag tag

[0096] 271-361: zfDil9-UIM domain of RNF114

[0097] In SEQ ID NO: 32 the spy-tagged zfDil9-UIM domain was designed to contain GST-tag followed by an HRV3C site a Hexa-Histidine tag, spytagS and a Flag-tag N-terminal of the zfDil9-UIM domain (AA:138-228). The fusion protein of SEQ ID NO: 32 is a preferred example of the fusion protein of the invention. It is particularly suitable for the pulldown of proteins carrying ADP-ribosyl-linked ubiquitylation in the methods of the invention that will be described herein below.Also the conjugation to beads (polymeric (e.g. agarose) beads and / or magnetic beads) of the protein or the conjugation construct or fusion protein (e.g. via the spycatcher protein) of the invention is described herein. For example, the spycatcher protein could also be directly conjugated to beads or the protein of the invention could also be directly conjugated to beads.

[0098] The present invention relates in a third aspect to a nucleic acid molecule encoding the protein of claim or the fusion protein of the invention.

[0099] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the third aspect as far as being amendable for combination therewith.

[0100] The term "nucleic acid molecule" in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, " DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. " RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001, 8:1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- noncoding regions, and the like.

[0101] The present invention relates in a fourth aspect to a vector comprising the nucleic acid molecule of the invention.

[0102] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the fourth aspect as far as being amendable for combination therewith.

[0103] The term "vector" in accordance with the invention means preferably a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering which carries the nucleic acid molecule of the invention. The nucleic acid molecule of the invention may, for example, be inserted into several commercially available vectors. Non-limiting examples include prokaryotic plasmid vectors, such as of the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXlN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pAO815, pPIC9K and pPIC3.5K (all Invitrogen).

[0104] The nucleic acid molecules inserted into the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and / or to other amino acid encoding sequences can also be carried out using established methods. Transcriptional regulatory elements (parts of an expression cassette) ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of transcription (e. g., translation initiation codon, promoters, such as naturally-associated or heterologous promoters and / or insulators; see above), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.Additional regulatory elements may include transcriptional as well as translational enhancers. Preferably, the polynucleotide encoding the polypeptide / protein or fusion protein of the invention is operatively linked to such expression control sequences allowing expression in prokaryotes or eukaryotic cells. The vector may further comprise nucleic acid sequences encoding secretion signals as further regulatory elements. Such sequences are well known to the person skilled in the art. Furthermore, depending on the expression system used, leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention. Such leader sequences are well known in the art.

[0105] Furthermore, it is preferred that the vector comprises a selectable marker. Examples of selectable markers include genes encoding resistance to neomycin, ampicillin, hygromycine, and kanamycin. Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria-fungal cells or bacteria-animal cells (e. g. the Gateway system available at Invitrogen). An expression vector according to this invention is capable of directing the replication, and the expression, of the protein or fusion protein of this invention. Apart from introduction via vectors such as phage vectors or viral vectors (e.g. adenoviral, retroviral), the nucleic acid molecules as described herein above may be designed for direct introduction or for introduction via liposomes into a cell. Additionally, baculoviral systems or systems based on vaccinia virus or Semliki Forest virus can be used as eukaryotic expression systems for the nucleic acid molecules of the invention.

[0106] In the appended examples the use of plasmids to overexpress GFP-Di19UIM U2OS cells to directly enrich ADP-ribosyl-linked ubiquitylation from these cells, and to produce recombinant SpyTag-Dil9U IM in E. coli is illustrated.

[0107] The present invention relates in a fifth aspect to a host cell transformed with the nucleic acid molecule or the vector of the invention.

[0108] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the fifth aspect as far as being amendable for combination therewith.

[0109] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of the protein or peptide or fusion protein of the invention by the cell.The host cell of the invention is typically produced by introducing the nucleic acid molecule or vector(s) of the invention into the host cell which upon its / their presence mediates the expression of the nucleic acid molecule of the invention encoding the protein or peptide or fusion protein of the invention. The host from which the host cell is derived or isolated may be any prokaryote or eukaryotic cell or organism, preferably with the exception of human embryonic stem cells that have been derived directly by destruction of a human embryo.

[0110] Suitable prokaryotes (bacteria) useful as hosts for the invention are, for example, those generally used for cloning and / or expression like E. coli (e.g., E coli strains BL21, HB101, DH5a, XL1 Blue, Y1090 and J MIDI), Salmonella typhimurium, Serratia marcescens, Burkholderia glumae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas stutzeri, Streptomyces lividans, Lactococcus lactis, Mycobacterium smegmatis, Streptomyces coelicolor or Bacillus subtilis. Appropriate culture mediums and conditions for the above-described host cells are well known in the art.

[0111] A suitable eukaryotic host cell may be a vertebrate cell, an insect cell, a fungal / yeast cell, a nematode cell or a plant cell. The fungal / yeast cell may a Saccharomyces cerevisiae cell, Pichia pastoris cell or an Aspergillus cell. Preferred examples for host cell to be genetically engineered with the nucleic acid molecule or the vector(s) of the invention is a cell of yeast, E. coli and / or a species of the genus Bacillus (e.g., B. subtilis). In one preferred embodiment the host cell is a yeast cell (e.g. S. cerevisiae).

[0112] In a different preferred embodiment, the host cell is a mammalian host cell, such as a Chinese Hamster Ovary (CHO) cell, mouse myeloma lymphoblastoid, human embryonic kidney cell (HEK-293), human embryonic retinal cell (Crucell's Per. C6), or human amniocyte cell (Glycotope and CEVEC). The cells are frequently used in the art to produce recombinant proteins. CHO cells are the most commonly used mammalian host cells for industrial production of recombinant protein therapeutics for humans.

[0113] The present invention relates in a sixth aspect to a method for the production of the protein or the fusion protein of the invention, the method comprising culturing the host cell of the invention under suitable conditions (wherein the protein or the fusion protein of the invention is expressed) and isolating the produced protein or the fusion protein of the invention.

[0114] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the sixth aspect as far as being amendable for combination therewith.Means and methods for the recombinant production and isolation (or purification) of proteins and fusion proteins are known in the art. The first step, protein expression aims at a large amount of proteins. This gene-to-protein process contains two main steps: Transcription and Translation.

[0115] Protein isolation may involve a number of processes, including pumping and ultrafiltration, which involve significant shear environments. More importantly, protein tags are a useful and convenient tool for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification. For example, the main chromatography methods used in protein purification are affinity chromatography, ion exchange chromatography and HPLC. A wide variety of protein purification methods is available that can also be combined to generate a suitable purification scheme.

[0116] The present invention relates in a seventh aspect to a composition, preferably a pharmaceutical composition or a diagnostic composition comprising at least one of (i) the protein; (ii) the protein conjugate or fusion protein; (iii) the nucleic acid molecule; (iv) the vector; and / or (v) the host cell of according to the foregoing aspects.

[0117] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the seventh aspect as far as being amendable for combination therewith.

[0118] The term "composition" as used herein refers to a composition comprising at least one of (i) the protein; (ii) the protein conjugate or fusion protein; (iii) the nucleic acid molecule; (iv) the vector; and / or (v) the host cell of according to the foregoing aspects, or combinations thereof which are also collectively referred in the following as compounds.

[0119] In accordance with the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. In accordance with the present invention, the term "diagnostic composition" relates to a composition for the detection of a disease or disease state in a patient, preferably a human patient.

[0120] The pharmaceutical or diagnostic composition of the invention comprises the compounds recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and / or activating their function. The composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). The pharmaceutical or diagnosticcomposition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical or diagnostic compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically or diagnostically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. Generally, the regimen as a regular administration of the pharmaceutical or diagnostic composition should be in the range of 1 pg to 5 g units per day. However, a more preferred dosage might be in the range of 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg per day. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

[0121] The present invention relates in an eighth aspect to the use of the protein or the protein conjugate or fusion protein of the invention for the detection of ADP-ribosyl-linked ubiquitylation, preferably of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acid sequence that are modified by ADP-ribosyl-linked ubiquitylation.

[0122] The present invention relates in a ninth aspect to a method for the detection of ADP-ribosyl-linked ubiquitylation, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation comprising:

[0123] (a) contacting a composition comprising one or more ADP-ribosyl-linked ubiquitylated compounds, preferably one or more amino acid sequences with the protein or the protein conjugate or fusion protein of the invention such that the protein can bind to ADP-ribosyl-linked ubiquitylation; and (b) optionally isolating or further enriching ADP-ribosyl-linked ubiquitylated compounds, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation.

[0124] The definitions and preferred embodiments of the above aspects apply mutatis mutandis to the ninth and tenth aspect as far as being amendable for combination therewith.As discussed herein above, the protein and the protein conjugate or fusion protein of the invention are both capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation.

[0125] For this reason the protein and the protein conjugate or fusion protein of the invention can be applied in uses and methods for the detection of ADP-ribosyl-linked ubiquitylation, preferably of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acid sequence that are modified by ADP-ribosyl-linked ubiquitylation.

[0126] The contacting step (a) is cried out under conditions, wherein the protein, protein conjugate or fusion protein of the invention can bind within a composition to the one or more ADP-ribosyl-linked ubiquitylated compounds.

[0127] The (optional) means and methods for isolating or further enriching ADP-ribosyl-linked ubiquitylated compounds, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation are not particularly limited and prefer examples will be described herein below.

[0128] In accordance with a preferred embodiment of the ninth aspect after step (a) and before step (b) bivalent cations, preferably Zn2+are removed by chelator, preferably EDTA, EGTA, iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or methylglycinediacetic acid (MGDA).

[0129] In accordance with a related preferred embodiment of the eight aspect the use in addition uses a chelator, preferably EDTA (ethylenediaminetetraacetic acid), EGTA (thylene glycol-bis(P-aminoethyl ether)-N, N, N', N'-tetraacetic acid), iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or Methylglycinediacetic acid (MGDA).

[0130] It is of note that the binding of the protein or the protein conjugate or fusion protein to ADPr via the zinc finger domain (zf-Di 19) is dependent on divalent ion, in particular Zn2+. For this reason employing a chelator, preferably EDTA, EGTA, iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or methylglycinediacetic acid (MGDA) in the uses and methods of the invention is particularly advantageous, because thereby the binding to ubiquitylated ADP-ribose via the zinc finger domain (zf-Di 19) and UIM can be disrupted.In accordance with another preferred embodiment of the ninth aspect said compounds, preferably amino acid sequences are detected and / or enriched by immunoblotting (preferably western blotting), ELISA, RIA, immunoprecipitation, immunofluorescent cell staining, FACS, chromatin immunoprecipitation or streptavidin enrichment.

[0131] All of immunoblotting (preferably western blotting), ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunassay), immunoprecipitation, immunofluorescent cell staining, FACS, chromatin immunoprecipitation or streptavidin enrichment are well-established methods for the isolation of a protein of interest.

[0132] In this connection in particular the protein conjugate or fusion protein might be of interest because thereby additional functionalities can be introduced that are helpful for the isolation of a protein of interest. Some non-limiting but preferred examples are discussed herein below.

[0133] In accordance with a more preferred embodiment of the second aspect the protein and / or the spycatcher protein of the invention is fused to HRP for immunoblotting, Fc for immunofluorescence, Fc for protein A / G enrichment and biotin for (strept)avidin enrichment.

[0134] HRP for immunoblotting allows for chemiluminescence detection of ADP-ribosyl-linked ubiquitylated compounds, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation via the enzyme HRP and its substrate. Also here the immunoblot is preferably a Western blot. HRP fusion to the spycatcher protein is preferred, because HRP conjugation via the spytag / spycatcher technology gives higher sensitivity than other ways of conjugating to HRP (e.g. to the protein of the invention), as is illustrated by the appended examples. A comparison between HRP via spytag / spycatcher technology and the use of secondary antibodies with HRP revealed that HRP via the spytag / spycatcher is more sensitive (Longarini and Matic (2024), Nature Communications, 15: Article number: 4239 (2024)).

[0135] An antibody Fc part facilitates immunofluorescence detection via secondary antibodies. These secondary antibodies bind Fc and can be labelled by a fluorescent compound, for example fluorescent protein, preferably GFP, RFP, or YFP or a fluorophore, preferably, an Alexa- or Cy-fluorophore.

[0136] An antibody Fc part also facilitates A / G enrichment (i.e. protein A or protein G enrichment, usually via (column or resin) chromatography). Protein G and protein A are bacterial proteins from Group G Streptococci and Staphylococcus aureus, respectively. When coupled to a column or resin, protein Gand protein A create chromatography media. Protein G and protein A can specifically bind to Fc parts, in particular IgG Fc parts.

[0137] The biotin / (strept)avidin enrichment technology has been described herein above. The interaction of biotin and Avidin or Streptavidin has been exploited for use in many protein and nucleic acid detection and purification methods. For protein purification of biotinylated protein, for example, streptavidin coated beads are available.

[0138] In accordance with a preferred embodiment of the ninth aspect in step (c) the compounds, preferably the amino acid sequences that are bound by the protein are detected, isolated or enriched by an anti-ADPr antibody, an anti-ubiquitin antibody, an antibody to a target compound, preferably target amino acid sequence.

[0139] Anti-ADPr antibodies are commercially available, for example, the poly / mono-ADP Ribose (D9P7Z) Rabbit mAb #89190 of cell signalling technology.

[0140] Likewise anti-ubiquitin antibodies are commercially available, for example, the Ubiquitin (P4D1) Mouse mAb (HRP Conjugate) #14049.

[0141] The antibody to a target compound may be directed against conjugation partner or fusion partner, such as the spycatcher protein. An example of a spycatcher antibody is AbD41909kg from BioRad.

[0142] The present invention relates in a tenth aspect to a method for identifying within one or more amino acid sequences the amino acids that are modified by ADP-ribosyl-linked ubiquitylation

[0143] (a) contacting one or more amino acid sequences with the protein or the protein conjugate or fusion protein of the invention such that the protein can bind to those amino acids of the one or more amino acid sequences being modified by ADP-ribosyl-linked ubiquitylation; and

[0144] (b) identifying the amino acids that are modified by ADP-ribosyl-linked ubiquitylation and their positions within the one or more amino acid sequences by mass spectrometry.

[0145] The method for the tenth aspect of the invention is a mass spectrometry-based method that allows for the detection of exactly those amino acids with an amino acid sequence that are modified by ADP-ribosyl-linked ubiquitylation. For the enablement of this method provision of the protein or the protein conjugate or fusion protein of the invention is a key issue, because it allows the specific enrichment of those amino acid sequences wherein one or more amino acids are modified by ADP-ribosyl-linkedubiquitylation, optionally followed by the specific elusion based on chelation of zinc ions, and preferably subjecting these amino acid sequences to the mass spectrometry analysis.

[0146] In accordance with a preferred embodiment of the tenth aspect in step a) the protein of the invention is (a) fused to a fluorescent protein, preferably GFP, RFP, or YFP and in addition nanobeads being coupled to an antibody or antibody fragment binding to the fluorescent protein are used, (b) conjugated to a spycatcher protein coupled to a Fc fragment for protein A / G enrichment and / or biotin for streptavidin enrichment, (c) fused to a tag, preferably a GST-tag, His-tag, Flag-tag, Strep-tag or ALFA-tag, and / or (d) fused an antibody or an antibody fragment, preferably an Fc part.

[0147] All the above fusion partners allow of the enrichment or isolation of amino acid sequences wherein one or more amino acids are modified by ADP-ribosyl-linked ubiquitylation in the state where they are bound by the protein of the invention. All the fusion partners have already been described in more detail herein above.

[0148] In accordance with another preferred embodiment of the tenth aspect after step (a) and before step (b) bivalent cations, preferably Zn2+are removed by a chelator, preferably EDTA, EGTA, iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or Methylglycinediacetic acid (MGDA) to specifically elute the ADP-ribosyl-linked ubiquitylation compounds.

[0149] As already described in connection with the ninth aspect and as it also applies to the tenth aspect, the chelator can be used to achieve specific elution of the binding of the protein or the protein conjugate or fusion protein to ADPr-ubiquitinylated substrates.

[0150] In accordance with a more preferred embodiment of the tenth aspect the method further comprises the isolation of the nanobeads and optionally eluting the more amino acid sequences being modified by ADP-ribosyl-linked ubiquitylation from the nanobeads.

[0151] It is of note that in the above preferred embodiment the fluorescent protein is used together with nanobeads being coupled to an antibody of antibody fragment binding to the fluorescent protein. Via this combination and not the fluorescent protein alone the enrichment or isolation of amino acid sequences wherein one or more amino acids are modified by ADP-ribosyl-linked ubiquitylation is achieved.The nanobeads can be isolated. For example, magnetic nanobeads may be isolated by magnetic filed and polymeric nanobeads may be isolated by centrifugation or filtration.

[0152] For example, GFP-Trap® Nanobodies are commercially available from proteintech and Anti-GFP Nanobody Immunomagnetic Beads from MSE Supplies LLC.

[0153] In accordance with an even more preferred embodiment of the tenth aspect amino acid sequences being modified by ADP-ribosyl-linked ubiquitylation are eluted from the nanobeads with about 8M urea.

[0154] Urea has a denaturing effect on individual IgG domains and can therefore be used for eluting the more amino acid sequences being modified by ADP-ribosyl-linked ubiquitylation from the nanobeads. The use of about 8M urea for this purpose is illustrated by the appended example. The term "about as used herein preferably means ±20%, more preferably ±10% and most preferably ±5%.

[0155] In accordance with a preferred embodiment of the tenth aspect the mass spectrometry comprises a sample preparation protocol that comprises about 3h incubation at acidic pH with the protease Arg-C, Lys-C, trypsin or any combination thereof, thereby preserving ester-linked ADP-ribosyl amino acid modifications.

[0156] The above preferred embodiment preserves the ester bond linking ubiquitin to ADP-ribose. The combination with ArgC and LysC is preferred because it is illustrated in the appended examples. It is particularly preferred that the sample preparation protocol is applied after the specific elution via the removal of bivalent cations as described herein above.

[0157] In accordance with another preferred embodiment of the tenth aspect the mass-spectrometry comprises electron transfer dissociation (ETD) and high-energy collisional dissociation (HCD) fragmentation methods and the generation of ETD and HCD spectra.

[0158] The generation of both, ETD and HCD spectra is advantageous because the appended examples show that this leads to the confident identification of ADP-ribosyl-linked ubiquitylation sites in various proteins; histones H3 and H2B, PARP1, HMGAl and HNRNPU.

[0159] The present invention relates in an eleventh aspect to a kit for the detection of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acidsequence that are modified by ADP-ribosyl-linked ubiquitylation comprising the protein or the protein conjugate or fusion protein of the invention.

[0160] The kit of the invention comprises the components required to carry out the methods and uses of the invention packed into one or more container(s). For this reason the definitions and the preferred examples of the components as described herein above in connection with the other aspects of the invention apply mutatis mutandis to the eleventh aspect of the invention.

[0161] The one or more containers may be, for example, one or more vials. The vials may, in addition to the components, comprise preservatives or buffers for storage. In addition, the kit may contain instructions for use.

[0162] It is particularly preferred that the kit comprises the protein of the invention fused to spytag and in this case the kit optionally comprises spycatcher protein as described herein above.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. In case of conflict, the patent specification including definitions, will prevail.

[0164] Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0165] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter ofclaims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0166] This also holds true for alternatives in different claims that depend from each other. Thus, if claim 1 recites three alternatives of the same category and claim 2 recites three alternatives of a different category as recited in claim 1, and refers back to claim 1, all combinations of the alternatives as recited in claims 1 and 2 are explicitly disclosed herein.

[0167] The above considerations apply mutatis mutandis to all appended claims.

[0168] The figures show.

[0169] Figure 1: GFP-Pulldown of GFP-tagged RNF114 WT and GFP-tagged RNF114 C176A. (a) RNF114 domain structure. RNF114 contains a RING finger and Zinc finger 1 domain (Znl) required for catalytic activity. Zn2 and Zn3 build the zfDil9 domain required for mono-ADPr binding. The Ubiquitin interaction motif (UIM) at the C-terminus binds ubiquitin. zfDil9 and UIM domains are required for recruitment towards DNA damage sites. The C176A mutation shown below abolishes mono-ADPr binding. Numbers indicate the motifs amino-acid positions, (b) Experimental set-up of the GFP-Pulldown. RNF114 KO U2OS cells complemented with inducible GFP-RNF114 WT or GFP-RNF114 C716A were left untreated or treated with ImM H2O2 for 1 h. Each condition consisted of 4 biological replicates. The pulldown was performed in non-denaturing conditions to identify interactors dependent on an intact zfDil9 domain and DNA damage, (c) Volcano plot showing the Iog2-fold change of the interactors identified in RNF114 WT overexpressing cells comparing the DNA damage treated and untreated conditions.

[0170] (d) Volcano plot showing the Iog2-fold change of the interactors identified in untreated conditions comparing the cell lines overexpressing either GFP-RNF114 WT or GFP-RNF114 C176A. (e) Heatmap showing the fold change of identified interactors in the comparisons in (c) and (d).

[0171] Figure 2: Analysis of published data sets for combinations of ADPr and ubiquitylation. (a) Expected fragmentation pattern of ADP-ribosyl-linked ubiquitylation and ADPr. ADP-ribosyl-linked ubiquitylation results in specific diagnostic ions, consisting of ADP-GlyGly, AMP-GlyGly, Adenosine-GlyGly and Adenine. Conventional ADPr on substrates with or without other modifications should result in a well-known set of diagnostic ions emerging from ADPr, consisting of the whole ADPr modification, ADP, AMP, Adenosine as well as Adenine. The masses between the two sets of diagnostic ions differ only slightly in case of ADP-GlyGly and ADPr. Adenine is a shared diagnostic ion. Adenosine-GlyGly and AMP-GlyGly can be clearly distinguished from AMP and ADP of conventional ADPr. (b) Results of an open search of the dataset PXD023835, searching for masses combining ADPr (541.0611 Da) and Ubiquitylation after digestion (GlyGly = 114.0429 Da; LRGG = 383.2281 Da). The open search results were filtered on less stringent masses to adjust for potential mass errors, (c) Identified spectra of neighbouring ubiquitylation (GlyGly) on lysine 5 and ADP-ribosylation on serine 6 on H2B. (d) Identified spectra of neighbouring ubiquitylation (LRGG) on lysine 9 and ADP-ribosylation on serine 10 on H3. Both the spectra in (c) and (d) show the conventional ADPr diagnostic ions, indicating that ADPr is present in an unmodified version.

[0172] Figure 3: Reducing the contamination of the bait and unspecific binders in the GFP-zfDil9-UIM pulldown to allow detection of ADP-ribosyl-linked-ubiquitylation. (a) Scheme of the full-length GFP-RNF114 WT pulldown, illustrating that this approach also enriches proteins binding the beads, the GFP-tag and other regions of RNF114. (b) Performing the GFP-pulldown with the zfDil9-UIM domain focuses the pulldown on binders of these two domains of RNF114, but still allows enrichment of unspecific binders of the beads and the GFP-tag. This can be partially circumvented by using a specific EDTA elution step to remove Zn2+ions from the zfDil9 domain, thereby releasing ADP-ribosylated proteins and proteins carrying ADP-ribosyl-linked-ubiquitylation, simplifying the MS / MS analysis, (c) EDTA specific elution of the GFP-zfDil9-UIM pulldown leads to the elution of mono-ADP-ribosylated proteins (as shown in the AbD43647 HRP-coupled blot) as well as PARP1 and H3. To elute GFP-zfDil9-UIM, the EDTA eluted beads were heated to 95°C for 15 min. (d) Comparison of the intensity during MS / MS analysis of the eluted proteins after EDTA elution and subsequent on beads digest of the EDTA eluted beads. The results clearly show that the bait remains on the beads, while PARP1, XRCC1, and Lig3 are specifically released by EDTA.

[0173] Figure 4: Optimized mass spectrometry methods enable confident localization of ADP-ribosyl-linked ubiquitylation using ETD and HCD fragmentation, (a) The detection of ADP-ribosyl-linked ubiquitylation relies on triggering a medium-quality HCD scan upon detecting a m / z corresponding to Adenine. If the subsequent triggered MS2 scan reveals a mass fitting to AMP GlyGly or Adenosine GlyGly, it triggers ETD or HCD, dependending on the chosen method, (b) & (c) HCD and ETD spectra revealing ADPr-GlyGly diagnostic ions and localizing ADP-ribosyl-linked-Ubiquitylation to serine 499 of PARP1. (d) ADP-ribosyl-linked ubiquitylation was also identified on serine 519 of PARP1. (e) & (f) HCD and ETD spectra revealing ADPr-GlyGly diagnostic ions and localizing ADP-ribosyl-linked ubiquitylation to serine 10 of H3. (g) Additional sites of ADP-ribosyl-linked-ubiquitylation were found on serine 28 of H3 and serine 6 of H2B.Figure 5: Converting the zfDil9-UI M domain into a detection reagent using the SpyTag / SpyCatcher technology, (a) The Spytag / SpyCatcher technology enables spontaneous isopeptide bond formation between the amino group of lysine side chains and the carboxyl groups of aspartate side chains. This allows spy-tagged proteins like the zf Dil9-U IM domain to be coupled to HRP for immunoblotting, Fc for immunofluorescence and biotin for streptavidin-based enrichment, (b) Immunoblotting images of ARH3 KO cells after GFP pulldown of GFP-tagged zfDil9-UIM. zfDil9-UIM HRP coupled reagent does not give any signal after heating the elution to 95°C for 15 min. (c) Streptavidin pulldown of biotin-coupled zfDil9-UIM from untreated, DNA damage treated (2mM H2O2, 30 min) and DNA damage + Olaparib-treated (lpM) U2OS WT and ARH3 KO cells. U2OS WT cells were treated with Olaparib for 1 hour, while AHR3KO cells for 24 hours.

[0174] Figure 6: (a) GFP immunoblot before and after 24-hour doxycycline induction in inducible RNF114 KO U2OS cells complemented with GFP-RNF114 WT and GFP-RNF114 C176A expression, (b) Correlation analysis output of DIANN, showing relationships within and between different conditions of the interactor screen, with a general separation of the different conditions, (c) PCA analysis confirming clear separation conditions in the GFP-Pulldown. (d) Volcano plot showing the logj-fold change of the interactors identified in GFP-RNF114 C176A overexpressing cells comparing the DNA damage treated and untreated conditions. The interactors from Figure 1 are marked if significant, (e) Comparison of DNA damaged conditions between cell lines overexpressing GFP-RNF114 WT and GFP- RNF114 C176A. The interactors from Figure 1 are marked if significant.

[0175] Figure 7: Representative Freestyle layout showing the abundance of conventional ADPr diagnostic ions (Adenine, AMP, ADP) and ADP-ribosyl-linked-ubiquitylation (Adenine, AMP-GlyGly, Adenosine-GlyGly) diagnostic ions throughout one run of an MS / MS analysis of the published dataset PXD023835. The examples illustrate the invention.

[0176] Figure 8: First spectra of ADP-ribosyl-linked ubiquitylation identified and optimization of GFP-pulldown. (a) HCD spectrum of ADP-ribosyl-linked-ubiquitylation identified on HMGA1 after 8M Urea elution, (b) EDTA elution optimization. Elution was tested under various conditions, including different temperatures (4°C, room temperature, and 37°C) and elution times (30 seconds and 15 minutes), (c) HCD spectrum of ADP-ribosyl-linked ubiquitylation on H2B identified after optimizing the elution.

[0177] Figure 9: Incubating ADP-ribosylated H3 peptide with CAA did not induce the presence of artifact ADP-ribosyl-linked- ubiquitylation diagnostic ions caused by alkylation.Figure 10: Additional spectra of ADP-ribosyl-linked ubiquitylation carrying peptides, (a) & (b) HCD and ETD spectra of ADP-ribosyl-linked ubiquitylation on serine 519 of PARP1, showing the diagnostic ions of ADP-ribosyl-linked ubiquitylation in HCD spectra and the localization in ETD spectra, (c) & (d) HCD and ETD spectra of ADP-ribosyl-linked ubiquitylation on serine 6 of H2B, showing the diagnostic ions of ADP-ribosyl-linked ubiquitylation in HCD spectra and the localization in ETD spectra, (e) and (f) HCD spectra of ADP-ribosyl-linked-ubiquitylation probably on HMGAl serine 8 or serine 9 and of HNRNPU serine 187, 188 or 192. These spectra illustrate that ETD data are critical to detect the fully labile modification on peptides, however their presence can still be validated by diagnostic ions with HCD. Further illustrating how HCD and ETD data complement each other.

[0178] Figure 11: Additional spectra of ADP-ribosyl-linked ubiquitylation carrying peptides, (a) & (b) HCD and ETD spectra of ADP- ribosyl-linked ubiquitylation on serine 10 of H3, showing the diagnostic ions of ADP-ribosyl-linked ubiquitylation in HCD spectra and the localization in ETD spectra, (c) & (d) HCD and ETD spectra of ADP-ribosyl-linked-ubiquitylation on serine 10 of H3 occurring in close proximity to acetylation of lysine 14, both modifications are confidently localized by ETD. (e) & (f) HCD spectra of ADP-ribosyl-linked-ubiquitylation probably on H3 serine 8 (e) or serine 28 (f) occurring with dimethylation of lysine 9 and lysine 36, respectively.

[0179] Figure 12: The presence of ADPr-GlyGly and conventional ions can be quickly identified by inspecting the raw data with Freestyle, using a layout that highlights the respective masses observed in MS2 spectra. This method allows for the rapid assessment of ADP-ribosyl-linked-ubiquitylation in raw files. This is particularly useful when peptides carry additional modifications, such as di-methylation, as seen in the case of H3.

[0180] Figure 13: Coupling of SpyCatcher in different formats to the SpyTag of zf Dil9-U I M. (a) Scheme of the reaction and the bivalent antibody-like reagent, (b) The coupling was analysed on 8% Bis-Tris gels by comassie staining. On the left, the Biotin SpyCatcher coupling to the SpyTag of zfDil9-U IM is shown, and on the right, the Fc SpyCatcher coupling to the SpyTag of zf Di 19-U I M is depicted. Both gels display the uncoupled zfDil9-UIM as the input, the uncoupled respective SpyCatcher, and the result of spontaneous coupling, from left to right. 2 pg of each sample were loaded, (c) HRP coupled zfDil9-UIM domain in whole cell lysate of untreated, H2O2 treated and Olaparib + H2O2 treated ARH3 KO cells.

[0181] Figure 14: (a) Streptavidin pulldown of biotin-coupled ZUD from untreated, DNA-damage-treated (2 mM H2O2, 30 min) or DNA-damage-treated and olaparib-treated (1 pM) untransfected U2OS WT anduntransfected ARH3-KO cells (two 15-cm dishes per condition were used). Immunoblots of the elutions for ZUD-HRP, AbD43647-HRP-coupled and PARP1 antibodies reveal a DNA-damage-dependent increase in ZUD-HRP signal and in mono-ADPr and PARP1. The bands likely to correspond to PARP1, PARP1 ADPr and PARP1 ADPrUb are labeled on each elution blot. An 8% Bis— Tris gel was used for the anti-PARPl and AbD43647-HRP-coupled blots. A 4-12% Bis— Tris gradient gel was used for the ZUD-HRP-coupled blot. Shown is a representative result from three independent experiments, (b) GFP-ZUD pulldown of ARH3-KO cells (four 500-cm2dishes) transfected with GFP-ZUD and treated with 2 mM H2O2 (30 min). One sample was processed and the elution was split in two as described in the scheme (top). One half was treated with 1 M hydroxylamine (NH2OH) and the other half was left untreated (-). The ZUD-HRP signal is abolished after treating the elution with NH2OH for 2 h. This treatment preserves the mono-ADPr pattern (AbD43647-HRP-coupled) and the overall ubiquitin signal. Shown is a representative result from three independent experiments, (c) GFP-ZUD pulldown of ARH3 KO cells (eight 500 cm2dishes) transfected with GFP-ZUD and treated with 2 mM H2O2(30 min). Immunoblotting the elution with an H2B antibody reveals an NHzOH-sensitive ADPrUb band. 20% BisTris gel was used to better resolve H2B. Shown is a representative result from three independent experiments. Following the EDTA elution, GFP-ZUD was eluted by heating the beads at 95 °C for 15 min (d) GFP-ZUD pulldown of ARH3-KO cells (eight 500-cm2dishes) transfected with GFP-ZUD and treated with 2 mM H2O2(30 min). The elution was split in two and half of it was treated with hydroxylamine (NH2OH) as described in b. The H3 blot reveals NH2OH-sensitive ADPr-monoUb and ADPr-diUb bands. A 20% Bis— Tris gel was used to better resolve H3. A 4-12% Bis— Tris gradient gel was used for the ZUD-HRP-coupled blot. Following the EDTA elution, GFP-ZUD was eluted by heating the beads at 95 °C for 15 min.

[0182] Figure 15: GFP-ZUD pulldown of ARH3 KO cells (four 500 cm2dishes) transfected with GFP-ZUD and treated with 2 mM H2O2 (30 min). Half of the EDTA elution (5 min RT) was heated to 95 °C for 5 min. The ZUD-HRP signal is abolished after boiling. This treatment preserves the mono-ADPr pattern (AbD43647 HRP-coupled) and the overall ubiquitin signal. Shown is a representative result from three independent experiments.

[0183] Figure 16: GFP pulldown of ARH3 KO cells (two 500 cm2 dishes per condition) transfected with GFP-ZUD or GFP-zfDil9 and treated with 2 mM H2O2 (30 min). The elutions (5 min RT) of both pulldowns were immunoblotted for ZUD HRP-coupled, AbD43647 HRP-coupled and ubiquitin antibody. zfDil9 (lacking UIM) shows negligible signal for AbD43647 HRP-coupled, ubiquitin and HRP-ZUD compared to ZUD (zfDil9-UIM), showing the specificity of ZUD for ADP-ribosyl-ubiquitylation. Shown is a representative result from three independent experiments. The GFP input and 95 °C after EDTA elutionblots were cropped for clarity. The asterisk (*) indicates the position where the image was cut. Following the EDTA elution, GFP-ZUD was eluted by heating the beads at 95 °C for 15 min.

[0184] The examples illustrate the invention.

[0185] Example 1 - Results

[0186] Mono-ADPr-dependent interactome of RNF114

[0187] Recently, we showed that RNF114 binding to mono-ADPr depends on its zfDil9 zinc-binding domain, which is necessary for its recruitment to DNA lesions24. Considering that hundreds of proteins are mono-ADP-ribosylated upon DNA damage12-15, we sought to identify RNF114 interactors specifically dependent on its mono-ADPr-binding ability. To achieve this, we employed the Sleeping Beauty transposon system to generate cell lines with inducible expression of GFP-tagged RNF114 WT and GFP-tagged RNF114 C176A (Fig. la; Fig. 6a). As we previously reported24, the mutation of C176 completely abolishes the ability of RNF114 to bind mono-ADPr. Thus, the comparison between RNF114 WT and C176A allows for the specific identification of the interactors dependent on its ADPr-binding ability. To identify ADPr-dependent binders of RNF114 in the context of DNA damage, we treated cells to H2O2 for 60 minutes, condition that maximizes the induction of serine mono-ADPr24, and performed the GFP pulldown followed by data-independent acquisition (DIA) quantitative proteomics analysis (Fig. lb, Fig. 6b-6f). Among the identified interactors, we detected several DNA repair proteins, including PARP1, XRCC1 and LIG3, as well as the E3 ubiquitin ligase and DTX3L (Fig. lc). Interestingly, in the absence of DNA damage we also identified the ADP-ribosyl transferases PARP12 and tankyrase, suggesting that RNF114 binds ADPr targets beyond the DNA damage response (Fig. Id).

[0188] A computational proteomics approach for identifying peptides co-modified by ubiquitin and ADPr Having identified mono-ADPr targets recognized by RNF114, we aimed to investigate the molecular basis of this interaction by pinpointing the specific modification sites read by this ubiquitin ligase. While we showed that the zfDil9 domain of RNF114 is necessary for binding in vitro to a mono-ADP-ribosylated peptide24, a UIM domain is also required for the recruitment of RNF114 to DNA lesions (Djerir et al. (2024), JBC, 300(8):107545). This suggests that RNF114 functions as a reader of both mono-ADPr and ubiquitylation, motivating us to develop a computational proteomics approach capable of distinguishing between the various possible ubiquitylation / ADPr chemistries. Our identification of DTX3L in RNF114 interactome (Fig. 1) and recruitment of DTX3L to serine mono-ADPr upon DNA damage24suggest that RNF114 may act as a reader of ester-linked ubiquitylation of ADP-ribose, catalyzed in vitro by DTX ligases. However, we sought an approach that also considers other potential dual ADP-ribose / ubiquitin signals, including ADPr of ubiquitin and separate modificationsoccurring in close proximity (Fig. 2a). This differs from conventional proteomics data analysis, which makes it difficult to identify and distinguish between different possible composite ubiquitin / ADPr chemistries. The first step of our computational proteomics approach involves open search to determine clusters of delta masses, followed by the inspection of diagnostic ions in HCD fragmentation spectra. The presence of standard ADP-ribose-derived diagnostic ions indicates that any additional peptide modification is on the peptide backbone, rather than on ADP-ribose itself. In contrast, if these diagnostic ions show mass shift - potentially due to the GlyGly remnant from digested ubiquitin - it indicates that ADP-ribose is the direct target of the additional modification (Fig. 2a). Given the availability of several large-scale ADPr proteomics studies and the successful application of our reanalysis approaches to uncover hidden forms of ADPr12-35-36, we reasoned that we could obtain cellular evidence of ubiquitylation / ADPr through our tailored reanalysis strategy. Intriguingly, through open search analysis (Fig 2b), and inspection of EThcD MSMS spectra37we discovered that histone peptides are co-modified by ubiquitin on the lysine immediately preceding the ADP-ribosylated serine (Fig 2c, d), suggesting an interplay that could be as significant as the crosstalk observed between histone phosphorylation, acetylation and ADPr35. Despite the two modifications occurring on adjacent amino acids, the exclusive detection of ADP-ribose specific diagnostic ions alongside the peptide backbone fragment ions clearly indicates that ubiquitylation is a separate modification occurring on the preceding lysine. We did not find evidence of ubiquitylated ADP-ribose in our reanalysis of published datasets (Fig. 7). This is expected, given the high chemical lability of the ester bond linking ubiquitin to ADP-ribose. The reanalyzed published datasets rely on experimental conditions, such as high pH for protein digestion and peptide fractionation, which cleave ester bonds20-38. In fact, these studies have identified a negligible number of ester-linked aspartate and glutamate ADPr sites15-37-39, further underscoring the general inadequacy of standard proteomics approaches for ester-linked modifications.

[0189] Identification of ADP-ribosyl-linked ubiquitylation sites using zfDil9-UIM-based enrichment and specific EDTA elution

[0190] While our computational proteomics approach can identify and distinguish between different combinations of ubiquitin and ADPr (Fig. 2), ester-linked modifications, including ubiquitylation of ADP-ribose, are largely lost in conventional proteomics preparation procedures20-38. Therefore, specialized sample preparation and pull-down protocols are essential to preserve, enrich and identify the exact ADPr / ubiquitin sites recognized by RNF114. To this end, we combined our recently introduced methods for preserving ester-linked modifications20with the development of a specific elution strategy.In general, identifying PTM sites in complex peptide mixtures, e.g. from whole cell extracts or insufficiently enriched samples, is significantly more challenging than detecting substrate proteins. In fact, the on-bead digestion protocol that enabled the identification of RNF114 interactors (Fig. 1) did not yield any ADPr sites. For this reason, we first switched from full-length, GFP-tagged RNF114 to GFP-tagged zfDil9-UI M domains to focus our proteomics analyses on the interactors of the C-terminal region of RNF114 (Fig. 3a, b). Second, we reasoned that an elution approach preventing the elution of the bait would increase the likelihood of successfully identifying the modification sites (Fig.3b). Hoping to exploit the stability of GFP and its strong binding to nanobody-coupled beads, we tested elution with 8M urea20, which leaves the abundant GFP-zfDil9-UIM bait on the beads while eluting both specific and nonspecific binders. Analysis of the resulting peptide mixture, largely freed from abundant bait peptides, enabled the initial identification of an ADP-ribosyl-linked ubiquitylated peptide on HMGA1 in WT U2OS cells (Fig.8a). This result encouraged us to pursue a more specific elution method to prevent the elution of both the bait and nonspecific binders. Since RNF114 recognizes mono-ADPr via the two zinc fingers of its zfDil9 domain, we reasoned that sequestration of zinc ions by EDTA -previously shown to disrupt the interaction between recombinant RNF114 and a mono-ADPr peptide24- might specifically elute mono-ADPr targets from zfDil9, and consequently, from zfDil9-UI M (Fig.

[0191] 3b). This disruption of the structural integrity of the zfDil9 zinc fingers reduces the elution of the abundant bait and nonspecific binders, thereby focusing the proteomic analyses on proteins carrying the modification. By western blotting, we confirmed that EDTA specifically elutes mono-ADPr targets from GFP-zfDil9-UIM beads (Fig. 3c) and optimized the conditions for this pull-down. We opted for EDTA elution at room temperature to preserve ester bonds and to avoid nonspecific elution observed at 37 °C in the absence of EDTA (Fig. 8b). Moreover, to improve the detectability of ADP-ribosyl-linked serine ubiquitylation by increasing its cellular levels, we also employed ARH3 KO cells21, as the absence of this hydrolase elevates serine mono-ADPr levels and prolongs RNF114 recruitment at DNA lesions24-25. To preserve the ester bond linking ubiquitin to ADP-ribose, we used our short acidic digestion protocol with ArgC and LysC proteases20to the EDTA-eluted proteins. As anticipated, mass spectrometric analysis showed that, compared to on-bead digestion, this specific elution method focused the mass spectrometric analysis on RNF114 interactors (Fig. 3d). Notably, alongside conventional serine ADPr sites, we observed HCD MSMS spectra containing adenine but with further diagnostic fragment ions shifted by a di-glycine mass, the typical ubiquitin signature that remains after tryptic digestion. These ions appear consistent with a form of ADP-ribose ubiquitylated on its adenine-proximal ribose, and thus, these spectra provide evidence of ADP-ribosyl-linked ubiquitylation sites (Fig. 2). This fragment ion signature appears specific for ubiquitylated ADP-ribose: ADP-GlyGly, AMP-GlyGly and adenosine-GlyGly (Fig. 2a). By including these masses in a standard MaxQuant search, we achieved the initial identification of an H2B peptide as target of ADP-ribosyl-linked ubiquitylation incellular contexts (Fig. 8c). Alkylation, a common step in protein digestion protocols, is a potential artifact that can mimic ubiquitin attachment to lysine residues in proteomics analyses40. While hydroxyl groups, including the hydroxyl of the adenine-proximal ribose targeted by ester-linked ubiquitylation, have not been reported to form alkylation artifacts, we used chloroacetamide, which effectively prevents such alkylation artifacts compared to the traditionally used iodoacetamide40. To completely exclude the possibility that our identification of GlyGly-modified ADP-ribose is due to an alkylation artifact, we incubated a serine mono-ADP-ribosylated peptide with the chloroacetamide, following the same protocol used for protein digestion. We detected the mono-ADPr peptide and a very minor amount of peptide with a mass shift potentially corresponding to single alkylation, a modification that does not mimic the GlyGly remnant of ubiquitin. Importantly, while we detected many spectra with the standard diagnostic ions of ADPr, we found no evidence of spectra containing the diagnostic ion pattern specific to ADP-ribosyl-linked ubiquitylation - the key criterion introduced here for confidently identifying this composite modification on peptides (Fig. 9). This confirms that alkylation was not responsible for the modification corresponding to the mass of GlyGly on ADP-ribose observed on target peptides.

[0192] A double-triggering mass spectrometric strategy tailored to the chemical signatures of ADP-ribosyl-linked ubiquitylation

[0193] Our biochemical protocol based on specific EDTA elution allowed us to uncover the first sites of this composite modification. However, the abundance of these peptides remained low compared to the predominance of unmodified contaminating peptides, hindering mass spectrometric analysis of this composite PTMs. Proteomics of simpler PTMs typically involves enriching modified peptides and depleting those peptides that do not carry the modification of interest, including those from target proteins. But since UIM motifs interact with the hydrophobic patch of intact, undigested ubiquitin41, peptide-level enrichment cannot be used with zfDil9-UIM. Thus, while EDTA elution significantly simplifies the sample at the protein-level, the peptide mixture remains complex. Furthermore, the potential partial cleavage of the ester bond may further decrease the relative abundance of modified peptides, despite our efforts to preserve it. To improve our means of detecting ADP-ribosyl-linked ubiquitylation within this remaining complexity, we have developed strategies to focus mass spectrometry more precisely on this composite PTM.

[0194] Building on our successful adenine-triggering to obtain high-quality HCD and ETD spectra24-25, we designed a more sensitive and specific mass spectrometric approach. This involves first detecting adenine in fast, low-quality HCD spectra to trigger acquisition of medium-quality HCD spectra. Detection of either ADPr-GlyGly or AMP-GlyGly in these spectra triggers high-quality MS2 (Fig.4a). Thisapproach enabled the acquisition of high-quality ETD and HCD spectra, leading to confident identification of ADP-ribosyl-linked ubiquitylation on histones H3 and H2B, PARP1, HMGAl and HNRNPU (Fig. 4b-g; and Figs. 10 and 11). Importantly, the presence of several prominent specific diagnostic ions clearly rules out the possibility that ADPr and ubiquitylation co-occur on different residues of the target peptide. In such a case the HCD spectra would contain exclusively standard ADP-ribose diagnostic ions, as shown above (Fig. 2). Specifically, for ADP-ribosyl-linked ubiquitylation we observed ions corresponding to singly charged adenine (136.061 m / z), Adenosine with GlyGly (364.136 m / z), AMP with GlyGly (462.113 m / z) and ADP with GlyGly (542.079 m / z). In contrast, conventional serine-mono-ADPr yields diagnostic ions of Adenine (136.061 m / z), AMP (348.068 m / z), ADP (428.034) and full ADP-ribose (542.068 m / z) (Fig. 2b). While ADP-GlyGly and unmodified ADP-ribose are nearly identical (542.079 m / z vs. 542.068 m / z), the presence of AMP with GlyGly and Adenosine with GlyGly clearly distinguishes ADP-ribosyl-linked ubiquitylation from conventional ADPr (Fig. 12). Furthermore, high resolution ETD, which does not fragment the modification11, confirmed site identification by confidently pinpointing the exact modified residue. Following MaxQuant42analysis, we manually inspected and annotated all the representative MSMS spectra (Fig. 4b, c, e, f; Figs. 10 and 11). All the reported peptides are confidently identified as modified by GlyGly-ADP-ribose, based on the presence of multiple diagnostic ions in the HCD spectra. ETD spectra precisely localized the composite PTM to S499 and S519 of PARP1 (Fig. 4b, c; Fig. 10a, b), H3S10 (Fig. 4e, f; Fig. 11 a-e) and H2BS6 (Fig. 10 c, d).

[0195] However, pinpointing the modification site for H3S28, HMGAl and HNRNPU - also confident targets of ADP-ribosyl-linked ubiquitylation - was less definitive, as only HCD spectra were obtained (Fig. 10 e, f) and due to the lability of Ser-ADPr to HCD43. Nevertheless, serines on all identified peptides are established sites of HPFl / PARPl-dependent ADPr11-12-15-25-44-45. Similarly to our analysis of the interplay between conventional Ser-ADPr and other histone marks35, we identified the ADP-ribosyl-ubiquitylated H3S10 and H3S28 in combination of additional histone marks. This includes mono- and di-methylation on K9 and acetylation of K14 in the vicinity of ADP-ribosyl-ubiquitylated S10 (Fig. 4e, f; Fig. 11 c-e). Interestingly, the ADP-ribosyl-ubiquitylated peptide with the H3S28 site was identified only when co-occurring with K36 di-methylation (Fig. Ilf).

[0196] Conversion of zfDil9-UIM into a modular reagent for detecting cellular ADP-ribosyl-linked ubiquitylation

[0197] Recently, we advanced mono-ADPr detection by integrating SpyTag / SpyCatcher protein ligation technology46into our recombinant antibodies24-25. This approach allows for rapid and straightforward coupling of a Fab antibody to enzymes, probes and multiple Fc regions24-47. Among these formats are a sensitive HRP-coupled tool for western blotting and rabbit and mouse IgG-like antibodies. Inspiredby our development of SpyTag modular antibodies24and the successful conversion of ADPr-binding domains to detection reagents by the Kraus lab48-49, we reasoned that combining the ability of zfDil9-UIM to recognize ADP-ribosyl-linked ubiquitylation with SpyTag technology could yield a versatile and sensitive detection reagent for this composite PTM. The concept of zf Dil9-U IM as a detection reagent for ADP-ribosyl-linked ubiquitylation stems from our successful pull-down and site-specific identification of targets modified with this dual PTM (Figs. 3 and 4) and is further supported by the detailed characterization of its binding properties presented in the accompanying manuscript.

[0198] Applying SpyTag protein ligation technology to zfDil9-UIM offers three unique advantages. First, it enables the generation of a dimeric zfDil9-UIM reagent, mimicking the bivalent nature of standard immunoglobulins (Fig. 5a). The resulting avidity effect, arising from the synergistic binding of two zfDil9-UIM copies, increases sensitivity, as demonstrated for recombinant Fab antibodies24-47. Second, a further increase in immunoblotting sensitivity is achieved with a specialized format, site-specifically coupled to three horseradish peroxidase (HRP) moieties, compared to standard IgG or IgG-like recombinant antibodies20-24-47. Third, the modularity of the SpyTag system ensures that any reagent with a SpyTag can be quickly adapted to additional formats, including human, rabbit and mouse IgG-like reagents as well as biotin-coupled tools47(Fig. 5a, Fig. 13a). Thus, by recombinantly expressing SpyTag-zfDil9-UIM, we introduce the first application of SpyTag technology to a domain that binds ADPr or ubiquitin.

[0199] To detect cellular ADP-ribosyl-linked ubiquitylation by western blotting, we engineered a reagent comprising two zfDil9-UIM moieties coupled to a Catcher domain. This reagent can be site-specifically labelled with three copies of biotin or HRP, and an FcCatcher to convert it into a synthetic IgG-like rabbit or mouse antibody (Fig. 5a, Fig. 13a, b). Building on our discovery that serine ADPr is a target of unconventional ester-linked ubiquitylation (Fig. 4), and the DNA damage-dependent recruitment of RNF114 to chromatin24, we sought to explore the patterns of ADP-ribosyl-linked ubiquitylation in response to DNA damage. No clear increase in the signal was observed upon DNA damage, and the background was high (Fig. 13c), suggesting that ADP-ribosyl-linked ubiquitylation may be challenging to detect in whole-cell lysate by immunoblotting. Building on the significant increase in the detection of low mono-ADPr levels previously achieved by combining AbD43647 IgG for immunoprecipitation and its SpyTag HRP-coupled format for immunoblotting24, we reasoned that a similar approach -employing zfDil9-UIM pulldown followed by western blotting with the HRP-coupled SpyTag-zfDil9-UIM reagent - could enable the detection of ADP-ribosyl-linked ubiquitylation. To assess the performance of the HRP-coupled SpyTag-zfDil9-UIM reagent, we exploited the high sensitivity of ester bonds to boiling20as a means to differentiate specific from background signals. To this end, we lysed DNA-damaged cells under ester-preserving conditions and then subjected half of the lysate to boiling, which cleaves the ester bond between ubiquitin and ADP-ribose while leaving the stable O-glycosidicbond between serine and ADP-ribose intact20. In the eluate of the pull-down, we observed complete removal of the signal upon boiling (Fig. 5b), indicating that the signal detected in the non-boiled sample, where ADP-ribosyl-linked ubiquitylation is preserved, is specific. To investigate the extent to which the levels of ADP-ribosyl-linked ubiquitylation depend on DNA damage, we created a bivalent biotin-coupled SpyTag-zfDil9-UIM reagent (Fig. 5a, Fig. 13b), exploiting the very strong interaction between biotin and streptavidin for pull-down. Subsequent western blotting with HRP-coupled SpyTag- zfDil9-UIM allowed us to observe a dramatic increase in ADP-ribosyl-linked ubiquitylation upon DNA damage (Fig. 5c). This was expected based on our proteomics results (Fig. 4) and previous evidence for the recruitment of RNF114 and DTX3L to DNA lesions24. Treatment with the PARP1 inhibitor olaparib largely abolished the DNA damage-induced increase in ADP-ribosyl-linked ubiquitylation (Fig. 5c), indicating that this composite PTM depends on PARP1 signaling. This finding is consistent with our mass spectrometric evidence showing that Ser mono-ADPr, produced by PARP1, is a target of this unconventional ubiquitylation (Fig. 4). We observed a similar pattern in ARH3 KO cells with a higher signal for the DNA damage treated sample compared to WT cells. Our zfDil9-U IM reagent does not detect the main PARP1 band recognized by both the anti-PARPl and mono-ADPr antibodies, but rather an upper band, possibly corresponding to ADP-ribosyl-ubiquitylated PARP1 (Fig. 5c).

[0200] Overall, we have developed zfDil9-UIM into a versatile, modular reagent by applying application SpyTag protein ligation technology.

[0201] Example 2 - Discussion

[0202] Recent technological advances have resolved many challenges that have plagued the field of ADPr for over half a century6-7, opening up new research directions within established signaling pathways. This progress is exemplified by the reinterpretation of PARP1 signaling triggered by the discovery of Ser-ADPr mediated by the PARP1 / HPF1 complex11 14and, more recently, by the concept of mono-ADPr as the second wave of PARP1 signaling24. However, investigating the emerging interplay between ADPr and ubiquitylation in cellular contexts requires next-level development of specialized tools and approaches. Only by combining the analysis of the mass spectrometric behaviors of ADPr using multiple fragmentation techniques with approaches for detecting branched peptides were we able to provide evidence of phosphoribose-linked serine ubiquitylation by Legionella pneumophila11. Other forms of unconventional ubiquitylation, occurring through labile ester bonds, including the ubiquitylation of ADP-ribose, pose even greater challenges, spanning sample preparation, mass spectrometry and data analysis. Therefore, it is perhaps not surprising that reports on the exact modification generated by DELTEX enzymes have varied, ranging from conventional lysine ubiquitylation to ADPr of the ubiquitin C-terminus, ubiquitylation of the adenine-proximal ribose onvarious substrates, including NAD+, free ADP-ribose, ADP-ribosylated peptides and nucleic acids, as well as direct ubiquitylation of nucleotides without ADP-ribose30-34. Here, we began with our recently developed methods for preserving ester-linked modifications20. We introduced the zfDil9-UI M pulldown, designed a specific EDTA-based elution strategy and developed proteomic approaches precisely tailored to the unique chemistry of this composite modification. This methodology has enabled us to identify the first endogenous sites of ADP-ribosyl-ubiquitylation, previously unattainable by mass spectrometry, even in biochemical reactions using recombinant DELTEX ligases. Specifically, we have discovered that Ser mono-ADPr on histones, PARP1 and other proteins serves as a target for this unconventional PTM, establishing ADP-ribosyl-linked serine ubiquitylation as a cellular signal for the first time, including its role as a dual-modification of histones H3 and H2B. Given our findings that RNF114 is a reader of ADP-ribosyl transferases (Fig. 1), including PARP1, PARP12 and tankyrase, future applications of our methodology are likely to reveal this composite modification in other important biological processes, such as interferon signaling. Beyond PARP1 / 2 signaling, we expect diverse conjugation chemistries, including ADP-ribosyl-ubiquitylation on aspartate and glutamate, as presented in a recent preprint using chemical and enzymatic removal of western blotting signals, though without identifying the modification sites50. While the original mass spectrometric evidence of a PTM in cellular contexts represents a key step in fostering new lines of research, as illustrated by the discovery of Ser-ADPr11-18, the broad pursuit of such directions relies on the availability of tools that can be readily implemented in any biological laboratory. Complementing sophisticated tools developed by others6-49, we have previously integrated Ser-ADPr technology with the SpyTag protein ligation system to generate modular, recombinant antibodies24-25. In this work, we applied SpyTag technology46-47to engineer zfDil9-UIM domains as the first specific reagent for detecting this composite PTM by immunoblotting. Future development of complementary, high-affinity recombinant antibodies through the extension of Ser-ADPr technology, could further expand investigations of ADP-ribosyl-ubiquitylation in cellular contexts.

[0203] In conclusion, the intricate chemical interplay between ADPr and ubiquitylation is emerging as a promising avenue for both research fields2. While the much-studied phosphoribosyl-linked serine ubiquitylation has thus far been exclusively associated with Legionella host interactions, our discovery of ADP-ribosyl-linked serine ubiquitylation reveals a signaling mechanism for endogenous processes in mammalian cells. Beyond PARP1 signaling and the DNA damage response, we envision that our multifaceted methodology will uncover this composite PTM in additional biological processes and through diverse attachment chemistries. We propose ADP-ribosyl-ubiquitylation as a general mechanism for cell signaling, with RNF114 acting as its reader in different biological processes.Example 3 - Specificity of the ubiquitin interaction motif domain (ZUD) towards ADPr-Ub

[0204] In contrast to total protein antibodies, the zfDil9- U IM domain (ZUD) of RNF114 does not detect the unmodified protein bands, but instead only the corresponding ADPr-Ub-modified proteins. Many antibodies raised against their antigens mainly recognize the unmodified protein, e.g. the antibodies as used herein to detect PARP1, H3 & H2B. Compare the PARP1 immunoblot in Fig. 14a, the H2B immunoblot in Fig. 14c and the H3 immunoblot in Fig. 14d.

[0205] The ZUD signal is completely abolished upon hydroxylamine treatment and heating (95°C). This should not be the case if ZUD detects also serine mono-ADPr, since the O-glycosidic ADPr linkage to serine is resistant to both of these treatments. Indeed, the mono-ADPr antibodies show a signal after both treatments, including new bands post treatment. Additionally, a ubiquitin specific antibody demonstrates new low molecular weight ubiquitin bands upon these treatments, which are also not recognized by ZUD (Fig. 5b, Fig. 14b and Fig. 15).

[0206] ZUD is strictly required to enrich ADPr-Ub as demonstrated by pulldowns comparing ZUD to zfDil9 pulldowns (Fig. 16). Here, ZUD, immunoblotting against mono-ADPr or ubiquitin results in negligible signals when the pulldown is performed with the zfDil9 domain (Fig. 16). Demonstrating ZUD clearly prefers ADPr-Ub over mono-ADPr or Ubiquitin. This is in line with Kloet et al. (2025), Nature Communications volume 16, Article number: 6319, where dissociation constant measurements show that ZUD is the minimal domain to efficiently bind ADPr-Ub.

[0207] Example 4- Material and Methods

[0208] Cell culture and drug treatments

[0209] U2OS cell lines were obtained, authenticated by STR profiling and confirmed mycoplasma free by ATCC cell line authentication services. Cells were routinely tested for mycoplasma contamination. Each cell line was cultured in Glutamax-DMEM supplemented with 10% bovine serum and 100 U / ml penicillin / streptomycin at 37 °C and 5% CO2. To induce PARP1 inhibition, U2OS wt and ARH3ko cells were treated with 1 pM Olaparib for 1 hour and 24h respectively. To induce DNA damage, the cell medium was aspirated and replaced with 37 °C complete DMEM containing 2 mM H2O2 for the indicated times.Generation of doxycycline inducible GFP-RNF114 WT and GFP-RNF114 C176A expressing cells

[0210] Previously published RNF114 KO U2OS cells were complemented with an inducible system based on the sleeping beauty transposon system51. Full length N-terminal GFP-tagged RNF114 WT and GFP-tagged RNF114 C176A were subcloned from previously established plasmids24into piTR-TTP vector52. The plasmids were transfected with a pCMV-Trp plasmid expressing a Transposase. 24 h after transfection the cells were selected with 1.5 pg / ml for 5 days followed by 2 pg / ml puromycin for 7 days. The cells were then FACS sorted and GFP-negative cells were collected to exclude leaky expression. After sorting, the cells were expanded and tested for inducible expression by adding 1 pg / ml doxycycline for 24h. Clones showing inducible expression upon doxycycline addition were kept and stored.

[0211] GFP-Pulldown to identify mono-ADPr dependent interactors of RNF114

[0212] RNF114 KO cell lines24complemented with an inducible expression of either N-terminal GFP-tagged WT RNF114 or N-terminal GFP-tagged RNF114 C176A. The cells were induced using 1 pg / ml of doxycycline for 24 hours. Then, both cell lines were either treated with 1 mM H2O2 for 60 min or left untreated and of each condition 4 biological replicates each corresponding to a confluent 10 cm dish were collected and processed in parallel.

[0213] Collection of cells was performed by washing the cells two times with ice cold PBS, and scraping the cells off the dishes in 1 ml PBS. The cells were collected and pelleted at 500 g at 4 °C for 5 min. The samples were then processed in a GFP Pulldown. The pellets were lysed by resuspension in 200 pl of 20 mM HEPES pH 7.9, 300 mM NaCI, 2.5 mM MgCI2, 0.5 % NP-40, 20 % glycerol, 20 pM Olaparib, 20 pM ADP-HPD, 2x EDTA free protease inhibitor cocktail and 750 U / ml Benzonase and incubated for 1 h at 4°C on an end-to-end rotator. The Benzonase was quenched using 200 pl of 20 mM HEPES pH 7.9, 0.5% NP-40, 20 pM Olaparib, 20 pM ADP-HPD, 2x EDTA free protease inhibitor and 30 mM EDTA. The lysate was clarified by centrifuging for 10 min at 20 k x g and 4 °C. The supernatant was diluted in 20 mM HEPES pH 7.9, 150 mM NaCI, 0.5 mM EDTA, 20 pM Olaparib, 20 pM ADP-HPD and 2x EDTA-free protease inhibitor cocktail. Magnetic GFP-Beads (Chromotek) were pre-washed three times in 1 ml 10 mM HEPES pH 7.9 and 150 mM NaCI and 0.5 % NP-40 and then added to the diluted lysates. 10 pl of magnetic beads were used per sample and incubated at 4 °C on end-to end rotation for 1 h. After incubation the Flow-Through was collected and stored at -20 °C. The beads were washed four times in 500 pl of 10 mM HEPES pH 7.9 and 150 mM NaCI and 0.5 % NP-40 and four times in 500 pl of 10 mM HEPES pH 7.9 and 150 mM NaCI to remove residual detergent.For MS, the samples were digested overnight on-bead by resuspending beads of each sample in 100 pl of 5 ng / pl Trypsin, 50 mM Tris-HCI pH 7.5, 1 mM TCEP and 5 mM CAA.

[0214] The supernatant of the digested samples containing the peptides was then used for stage-tipping on 30 pg C18 stagetips activated with 200 pl methanol and pre-washed and equilibrated with two times 200 pl 30 % ACN / 0.1 % FA and 0.1 % FA, respectively. The samples were washed twice in 200 pl 0.1 % FA and eluted in 100 pl 30 % ACN / 0.1 % FA. After drying the samples in a speedvac, one fifth of each sample was used for MS / MS on Q-Exactive HF. DIA analysis was performed with a gradient starting at minute 1 from 4 % ACN to 30 % ACN after 20 min and increasing to 90 % ACN after 25 min, followed by a minute of 90 % ACN, resulting in a total duration of 26 min. The machine was run in positive mode with 15k resolution, AGC target of 3e6 and maximum injection time of 22 ms. The data was collected in centroid mode.

[0215] The resulting raw data were analyzed with DIA-NN and the following settings: UP000005640_9606.fasta was used for the generation of a library. " FASTA digest for library free search / library generation" and " Deep learning-based spectra, RTs and IM prediction" was switched on as well as reannotation of the fasta. 2 missed cleavages of a tryptic digest were allowed as well as 1 variable modification which could either be oxidation of methionine or acetylation of the n-terminus. Further " N-term M excision" and " C carbamidomethylation" were set as fixed modifications. Peptides between 5 and 30 amino acids, precursors of a charge between 1 - 4 in a m / z range between 300 and 1800 and fragment ions in a m / z range between 200 and 1800 were considered for the analysis. Mass accuracy and MSI accuracy were set to default. The use of isotopologues, match-between-runs, heuristic protein interference and no shared spectra options were switched on. Protein interference was set to "genes", neural network classifier to "single-pass mode", quantification strategy to " Robust LC (high precision)", cross-run normalization to " RT-dependent", library generation to "smart profiling" and speed and ram usage to "optimal results".

[0216] The protein groups output of DIA-NN was used for analysis of the interactors using a custom R-script and RStudio.

[0217] Open Search of published data sets.

[0218] An open search for potential combinations of ADPr and ubiquitin in published data sets was performed using MSfragger v22.053. For the presented open search, we used the previously published dataset PXD_02383537. All the corresponding raw files were analyzed using the following settings only using the MSFragger and database module of fragpipe54. As a database served an automatically downloaded database by MSfragger including contaminants and decoys. For "peak matching" a precursor mass tolerance form -1 to 1000 Da, a fragment mass tolerance of 20 ppm, mass calibration & parameteroptimization was switched on and isotope errors were set to 0 / 1 / 2. " Protein digestion" was allowed with trypsin as the enzyme allowing for 2 missed cleavages, a peptide length of 7-50 with a mass range of 500 - 5000 Da. The maximum number of variable modifications was set to 3 with oxidation of methionine as variable modifications and the "max combinations" parameter was kept on default settings. The only allowed fixed modification was cabamidomethylation of cysteine residues. " Mass offsets" were set to 0 and "delta masses" were allowed on all residues. " Glyco / Labile Mods" was switched off. For "spectral processing" activation type filter and analyze filter were set to "all", precursor mass mode was set to "selected", check spectral files and require precursors was enabled. The "min number of peaks" was set to 15 and the 150 top n peaks were used, the "min ratio" was set to 0.01. " Clear m / z range" was set to 0 and no intensity transform was enabled. Remove precursor peaks was set to "only peak with precursor charge" and the "removal m / z range" was set from -1.5 to 1.5. In the open search options "mass shift report as a variable mod" was turned off, "track zero top n" and "zero bin accept expect" were set to 0, "zero bin multiply expect" was set to 1 and " Delta mass exclude range" was -1.5 to 1.5. " Localize mass shift" was turned off. For advanced output options, "report top N for DDA" was set to 1, "report alternative proteins" was switched on and the output format was set to " TSV_PEPXML_PIN", " Report top N for DDA+" and " Report top N for DIA" were unchanged and at 5. " Write calibrated mzML" was checked and "write uncalibrated MGF" was unchecked, no group variable was selected and "output max expect" was at 50.

[0219] For advanced peak matching options "min frags modeling" was set to 2. " Deisotope" was switched on, "deneutralloss" remained on default, the "minimum matched frags" were set to 7 and the "maximum fragment charge" allowed was 4. " Fragment ion series" allowed were c,z,b and y. No custom ion series was added. The "precursor tolerance" was set to 20 ppm. " Override charge with precursor charge" was unchecked.

[0220] All the output files were merged into on excel table and filtered to remove "rev_sp" annotated proteins. Then the mass shift column was filtered for 541.0 as a proxy for ADPr, 655.1 as a proxy for combinations of ADPr and Gly Gly and 924.2 as a proxy for combinations of ADPr and LRGG. The resulting peptides were filtered to remove duplicates to determine the number of unique peptides identified with one of the three mass shifts.

[0221] To validate findings by localizing the modifications and for annotated spectra, MaxQuant42was used. The same dataset was analyzed with MaxQuant. Every setting was kept on default unless stated different. As variable modifications ADPr was allowed on serine residues, for ubiquitylation glycine glycine and LRGG on lysine residues was allowed additionally to oxidation of methionine and acetylation of the protein N-terminus. Carbamdiomethylation of cysteine residues was set as a fixed modification. The maximum number of modifications per peptide was set to 3. For digestion, trypsin was set and 5 missed cleavages were allowed. As a FASTA database served the human proteome fromuniport (UP000005640_9606). The maximum peptide length was set to 5 and the maximum peptide mass to 8000 Da. Second peptides option was enabled for identification.

[0222] GFP pulldown for site identification with Urea elution

[0223] WT U2OS cells equivalent to 2 confluent 15 cm dishes were transfected with 25 pg of plasmid encoding GFP-tagged zfDil9-UIM each (1:3 DNA: PEI ratio in Optimem, Gibco) and collected as described for the RNF114 interactome. To preserve ester-linked PTMs, we used the conditions previously described by us20. Until the elution every step was performed as described above (GFP-Pulldown to identify mono- ADPr dependent interactors of RNF114). Elution was then performed by resuspending the beads in 25 pl 8 M Urea, 20 mM HEPES pH 7.0 and 1 mM DTT for 10 min at 37 °C. The supernatant was then taken off the beads and diluted lOx using digestion buffer optimized for ArgC digestion (NH4Ac pH 5.0, 5mM DTT). Digestion was then carried out by adding 0.25 pg ArgC and 1 pg LysC for 3 h at 37 °C, and stopped by adding FA to a final concentration of 2 %. Additional alkylation with CAA was not performed in this experiment. Stage-tipping, drying and resuspending of the peptide was then performed as previously described. Seven tenth of the resuspended elution were injected into a Q Exactive HF Orbitrap mass spectrometer for DDA data collection. The machine was operated in positive mode. The gradient for reverse phase chromatography was increasing ACN to 50 % in 120 min followed by an increase to 90 % ACN after additional 30 min and followed by 3 min of 90 % ACN wash. Data was collected from 0 to 128 min. The scan range for full MS spectra was 400 to 1600 m / z with an AGC target of 100000 and a maximum injection time of 100 ms. For MS2 a Top5 method was chosen using a scan range from 200 to 2000 m / z with a fixed first mass of 120 m / z to ensure that adenine peaks are collected. The dynamic exclusion time was set to 15s.

[0224] Optimized GFP-Pulldown and EDTA elution

[0225] ARH3 KO U2OS cells were transfected as described above and then treated with 2 mM HzOzfor 30 min. After two washes in ice-cold PBS, the cells were harvested by scraping and recovered by centrifugation at 500 x g for 5 min at 4 °C. For each pulldown, a number of cells equivalent to a confluent 50 cm x 50 cm square dish was used. The cell pellet was resuspended in 450 pl of lysis buffer (20 mM HEPES pH 7.0, 300 mM NaCI, 2.5 mM MgCI2, 0.5% NP-40, 20 % glycerol, 750 U / ml benzonase, 1 pM Olaparib, 1 pM ADP-HPD, 50 mM PR619, IX EDTA-free protease inhibitor cocktail) and incubated with shaking for 1 hour at 4 °C. 450 pl of 20 mM HEPES pH 7.0, 0.5% NP-40, 1 pM Olaparib, 1 pM ADP-HPD, 50mM PR619, IX EDTA-free protease inhibitor cocktail was added and the lysate was clarified by centrifugation at 16,000 x g for 5 min at 4 °C, followed by collection of the supernatant. Thesupernatant was incubated with washed GFP-trap M-270 magnetic beads (Chromotek) for 1 h at 4 °C, 1400 rpm. 200 pl of beads were used for each pulldown. After incubation, the beads were washed first with 4 washes in 20 mM HEPES pH 7.0, 300 mM NaCI, 0.05 % NP-40, then 3 washes in 20 mM HEPES pH 7.0, 300 M NaCI on ice. One last wash was performed in 20 mM HEPES pH 7.0, 300 mM NaCI at RT for 15 min. Proteins were then eluted by incubating the beads with 100 pl 30 mM EDTA for 5 min at RT with shaking. The elutions were then alkylated in the same volume by adding 4 M Urea, 20 mM NH4AC pH 5.0, 5mm DTT and 15 mM CAA and incubating in the dark at RT for lh. Excess CAA was then quenched by adding DTT to a final concentration of 15 mM and incubation in the dark at RT for 10 min. The alkylated elutions were directly digested for 3 h with 0.3 pg ArgC and 1.2 pg LysC per dish at 37 °C, in case of digests containing only ArgC also 0.3 pg of ArgC were used. The digest was stopped by adding FA to a final concentration of 2 % and stored at -20 °C until stage tipping. Stage tipping was performed as described above and drying was performed in a speed-vac at room temperature.

[0226] Optimized GFP-Pulldown for Immunoblotting

[0227] After eluting with EDTA, 4x LDS with 100 mM DTT was added to the elution. The sample was incubated at RT for 5 to 15 min and then loaded on a gel. Unless otherwise stated input and elution samples were not heated prior to loading to ensure ester-linked modifications are preserved. SDS-PAGE and immunoblotting was then performed in pre-cooled buffers on ice. After SDS-PAGE, the gels were transferred onto nitrocellulose membranes (Amersham) using wet transfer at 110 V for 90 min. The transfer buffer is 1 x NuPAGE transfer buffer (Invitrogen), 20% ethanol in water. The membranes were blocked in 5 % milk in PBS buffer with 0.1 % Tween-20 for 1 h at room temperature and incubated overnight with primary antibody at 4 °C. For antibodies requiring secondary antibody, this was followed by a 1 h incubation at room temperature with peroxidase-conjugated secondary anti-mouse or anti-rabbit.

[0228] Triggered Mass Spectrometry Methods to detect ADPr-linked ubiquitylation

[0229] Methods aimed to trigger specifically in case of peptides carrying ADPr-linked ubiquitylation were set up for HCD and ETD and DDA proteomics. These methods were run on an Orbitrap Fusion Lumos Mass Spectrometer equipped with a FAIMS, which was run at -50 V and - 70 V.

[0230] The gradient used for the following methods were 1 % to 31 % ACN in 90 min followed by 10 min to 50 % ACN and another 10 min to 95 % ACN which was then followed by another 20 min wash of ACN at 95 %. To account for potential longer and more hydrophobic peptides in ArgC only digests thisgradient was adjusted to 1 % to 40 % ACN in 90 min followed by 10 min to 50 % ACN and another 10 min to 95 % ACN which was then followed by another 20 min wash of ACN at 95 %.

[0231] Triggered HCD spectra

[0232] The machine was operated in positive mode. For MSI an AGC target of 100000 and a maximum injection time of 50 ms was set. The scan range was from 350-1550 m / z and charge states of 2 to 7 were collected. The MS2 were collected in a Top20 method, and the first collected m / z was 120. The first MS2 scan was performed using HCD fragmentation a maximum injection time of 22 ms and an AGC target of 50000. This triggered a second HCD fragmentation on the same peak if a mass of 136.06 with a mas tolerance of 15 ppm was detected. For the second MS2 the maximum injection time was 300 ms and an AGC target of 150000.

[0233] High Quality triggered HCD and triggered ETD spectra

[0234] The machine was operated in positive mode. For MSI an AGC target of 100000 and a maximum injection time of 50 ms was set. Charge states of 3 to 10 were collected. The MS2 were collected in a Top20 method. The first MS2 scan was performed using HCD fragmentation a maximum injection time of 22 ms and an AGC target of 50000. This triggered a second HCD fragmentation on the same peak if a mass of 136.06 with a mass tolerance of 15 ppm was detected. For the second MS2 the maximum injection time was 200 ms and an AGC target of 150000. If this second MS2 yielded a m / z of either 364.134 (AMP-GlyGly) or 462.11 (ADP-GlyGly) an additional MS2 using ETD was performed on this precursor with a maximum injection time of 300 ms and an AGC target of 150000.

[0235] High Quality triggered HCD spectra

[0236] The machine was operated in positive mode. For MSI an AGC target of 100000 and a maximum injection time of 50 ms was set. Charge states of 2 to 7 were collected. The MS2 were collected in a Top20 method. The first MS2 scan was performed using HCD fragmentation a maximum injection time of 22 ms and an AGC target of 50000. This triggered a second HCD fragmentation on the same peak if a mass of 136.06 with a mass tolerance of 15 ppm was detected. For the second MS2 the maximum injection time was 200 ms and an AGC target of 150000. If this second MS2 yielded a m / z of either 364.134 (AMP-GlyGly) or 462.11 (ADP-GlyGly) an additional MS2 using HCD was performed on this precursor with a maximum injection time of 300 ms and an AGC Target of 150000.Alkylation assay with H3(22-44) S28-ADPr peptide

[0237] 1 pg of an H3 peptide 22-44 with ADPr on Serine 28, N-terminal amidation and C-terminal biotinylation was incubated in 4 M Urea, 20 mM NH4Ac, 5mm DTT and 15 mM CAA and incubated in the dark at RT for 1 h. Excess CAA was then quenched by adding DTT to a final concentration of 15 mM and incubation in the dark at RT for 10 min. In parallel the same experiment was performed without CAA. The peptides were then stage-tipped and dried as described and used for mass spectrometry.

[0238] MaxQuant analysis to detect ADPr-linked ubiquitylation

[0239] To detected ADP-ribosyl-linked ubiquitylation, MaxQuant42version 2.4.12 was used. A modification was set up integrating our observations on the fragmentation behavior of ADP-ribosyl-linked ubiquitylation. The composition was set to H(27)O(15)P(2)C(19)N(7) resulting in a neutral mass of 655.104 Da. Neutrallosses and diagnostic ions were setfor the whole modification (M+H+:656.111 Da), for ADP-GlyGly (H(21)N(7)O(12)P(2)C(14); 541.072 Da; M+H+: 542.079 Da), for AMP-GG (H(20)N(7)O(9)PC(14); 461.106 Da; M+H+: 462.113 Da) and for Adenosine-GlyGly (H(17)N(7)O(5)C(14); 363.129 Da; M+H+ 364.136 Da). Diagnostic ions were also set up for Adenine (H(5)N(5)C(5); 135.054 Da; M+H+ 136.061) the loss of GlyGly+H2O which was observed (H(8)O(3)N(2)C(4); 132.053 Da; M+H+:133.060 Da) and the corresponding Adenosine-H2O (H(10)C(10)Q(2)N(5); 232.083 Da; M+H+:233.090) and conventional Adenosine (H(12)C(10)O(3)N(5); 250.094 Da, M+H+:251.101) and a potential form of Adenosine that lost the GlyGly remnant (H(ll)C(10)O(3)N(5); 249.086 Da, M+H+:250.093). This modification was allowed on Serine residues. To perform the MaxQuant search, the set-up modification was used as a variable modification alone or in combination with e.g. mono- and di-methylation or acetylation which were predefined in the software. We allowed up to 5 variable modifications per search. The library digestion was adjusted accordingly to the digestion method with either ArgC or ArgC and LysC allowing up to 8 missed cleavages and a peptide range from 5 to 30 amino acids with a maximum mass of 8000 Da. As a sequence database the human proteome from uniprot was used. To boost identification rates match-between runs and second peptide search can be switched on.

[0240] Purification of Spy-tagged zfDi9-UIM domain

[0241] The spy-tagged zfDil9-UIM domain was designed to contain GST-tag followed by an HRV3C site a Hexa-Histidine tag, Spytag3 and a Flag-tag N-terminal of the zfDil9-UIM domain (AA:138-228). The design was sent to GeneScript for cloning and production. For purification we used the previously described purification protocol24. The elution buffer for the purified protein was exchanged to 50 mM HEPES, pH7.9, 200 mM NaCI and 1 mM DTI in a 10 kDa cutoff filter at 4 °C. The protein was then aliquoted and frozen on dry ice and stored at -80 °C.

[0242] Coupling of spy-tagged zfDil9-UIM domain with HRP and Biotin catcher

[0243] To couple the spy-tagged zfDil9-UIM domain with either HRP- or Biotin-SpyCatcher we followed the manufacturers protocol. The purified spy-tagged zfDil9-UIM was diluted to either 0.5 pg / pl in PBS to couple it to HRP-SpyCatcher or to 1 pg / pl to couple it to Biotin-SpyCatcher. Then the desired SpyCatcher was added in a ration of 1:10 (vol / vol) to the diluted recombinant protein, e.g. 10 pl SpyCatcher were added to 100 pl Spy-tagged zfDil9-UIM. The solution was left at RT for 1 h and then aliquoted as desired and stored at -20 °C until further use.

[0244] Streptavidin-Biotin pulldown of zfDil9-UIM domain

[0245] To pulldown endogenous proteins carrying ADP-ribosyl-linked ubiquitylation we used 25 pg Biotin coupled zfDil9-UIM per 15 cm dish. For the shown experiment we used 2 confluent 15 cm dishes per condition of either ARH3 KO U2OS or WT U2OS cells. ARH3 KO U2OS were treated with 1 pM Olaparib 24 h prior to DNA damage treatment and WT U2OS were treated with 1 pM Olaparib 30 min prior to DNA damage treatment. DNA damage was induced using 2 mM H2O2 for 30 min. Afterwards the cells were collected as described before. The pulldown followed the optimized pulldown protocol described above. However, the biotin coupled zfDil9-UIM domain was incubated during lysis with streptavidin magnetic beads (1:1, m(Biotin coupled reagent):vol(Streptavidin beads)). After preparing the beads, they were washed 3 times in NP-40% containing buffer to remove unbound biotin-zfDil9-UIM from the beads which could compete with the bead coupled biotin-zfDil9-UIM to bind endogenous proteins carrying ADP-ribosyl-linked ubiquitylation. The beads were then equally distributed to the samples and the protocol of the optimized pulldown was performed for immunoblotting.

[0246] Data Analysis

[0247] Data analysis was performed using Rstudio, Prism 9, Excel and Image Lab.

[0248] Data availability

[0249] The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE55partner repository with the dataset identifier PXD058858; username: reviewer_pxd058858@ebi.ac.uk, password: Vexi8NUp3bih.REFERENCES

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Claims

CLAIMS1. A protein being capable of binding to ADP-ribosyl-linked ubiquitylation, preferably within an amino acid sequence to amino acids being modified by ADP-ribosyl-linked ubiquitylation, wherein the protein comprises or consists of(a) the amino acids of positions 140 to 228, preferably 138 to 228 of any one of SEQ ID NO: 1, 4, 5 and 6 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or amino acids of positions 141 to 229, preferably 139 to 229 of SEQ ID NO: 2 or 3 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; or amino acids of positions 142 to 230, preferably 140 to 229 of SEQ ID NO: 7 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or amino acids of positions 135 to 223, preferably 133 to 223 of SEQ ID NO: 8 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto;(b) the amino acids of positions 149 to 237, preferably 147 to 237 of any one of SEQ ID NOs 9 to 11, 14 and 15 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 156 to 244, preferably 154 to 244 of SEQ ID NO: 12 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 153 to 241, preferably 151 to 241 of SEQ ID NO: 13 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical therefore) the amino acids of positions 138 to 225, preferably 136 to 225of SEQ ID NO: 16 or 20 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 139 to 226, preferably 137 to 226 of SEQ ID NO: 17 or 19 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 137 to 224, preferably 135 to 224 of SEQ ID NO: 18 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 127 to 217, preferably 125 to 217of SEQ ID NO: 21 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto; or(d) the amino acids of positions 156 to 244, preferably 154 to 244 of any one of SEQ ID NOs 22 to 24 or 27 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 133 to 217, preferably 131 to 217 of SEQ ID NO: 25 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the aminoacids of positions 156 to 209, preferably 154 to 209 of SEQ ID NO: 26 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto, or the amino acids of positions 157 to 245, preferably 155 to 245 of SEQ ID NO: 28 or a sequence being at least 80%, preferably at least 90% and most preferably at least 95% identical thereto.

2. A protein conjugate or fusion protein comprising the protein of claim 1.

3. The protein conjugate or fusion protein of claim 2, wherein the protein of claim 1 is fused to a spytag, preferably the spytag3 or a sequence being at least 80%, preferably at least 90% identical thereto;preferably wherein the protein conjugate or fusion protein is coupled via an isopeptide bond to a spycatcher protein, more preferably spycatcher3,preferably wherein the spycatcher protein is preferably a dimer to which two copies of the protein of claim 1 are linked.

4. The protein conjugate or fusion protein of claim 2 or 3, wherein the protein of claim 1 and / or the spycatcher protein is fused or conjugated to(a) a fluorescent protein, preferably GFP, RFP, or YFP or a fluorophore, preferably, an Alexa- or Cy-fluorophore,(b) a tag, preferably a GST-tag, His-tag, Flag-tag, Strep-tag or ALFA-tag,(c) biotin,(d) an antibody or an antibody fragment, preferably an Fc part, and / or(e) an enzyme or truncated version thereof being enzymatically active, preferably horse radish peroxidase (HRP) or alkaline phosphatase.

5. A nucleic acid molecule encoding the protein of claim 1 or the fusion protein of any one of claims 2 to 4.

6. Use of the protein of claim 1 or the protein conjugate or fusion protein of any one of claims 2 to 4 for the detection of ADP-ribosyl-linked ubiquitylation, preferably of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acid sequence that are modified by ADP-ribosyl-linked ubiquitylation.

7. A method for the detection of ADP-ribosyl-linked ubiquitylation, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation comprising:(a) contacting a composition comprising one or more ADP-ribosyl-linked ubiquitylated compounds, preferably one or more amino acid sequences with the protein of claim 1 or the protein conjugate or fusion protein of any one of claims 2 to 4 such that the protein can bind to ADP-ribosyl-linked ubiquitylation; and(b) optionally isolating or further enriching ADP-ribosyl-linked ubiquitylated compounds, preferably amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation.

8. The method of claim 7, wherein after step (a) and before step (b) bivalent cations, preferably Zn2+are removed by chelator, preferably EDTA, EGTA, iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or Methylglycinediacetic acid (MGDA).

9. The method of claim 7 or 8, wherein said compounds, preferably amino acid sequences are detected and / or enriched by immunoblotting (preferably western blotting), ELISA, RIA, immunoprecipitation, immunofluorescent cell staining, FACS, chromatin immunoprecipitation or streptavidin enrichment.

10. The method of claim 9, wherein the protein of claim 1 and / or the spycatcher protein is fused to HRP for immunoblotting, Fc for immunofluorescence, Fc for protein A / G enrichment and biotin for streptavidin enrichment.

11. The method of any one of claims 7 to 10, wherein in step (c) the compounds, preferably the amino acid sequences that are bound by the protein are detected, isolated or enriched by an anti-ADPr antibody, an antibody anti-ubiquitin, an antibody to a target compound, preferably target amino acid sequence.

12. A method for identifying within one or more amino acid sequences the amino acids that are modified by ADP-ribosyl-linked ubiquitylation(a) contacting one or more amino acid sequences with the protein of claim 1 or the protein conjugate or fusion protein of any one of claims 2 to 4 such that the protein can bind to those amino acids of the one or more amino acid sequences being modified by ADP- ribosyl-linked ubiquitylation; and(b) identifying the amino acids that are modified by ADP-ribosyl-linked ubiquitylation and their positions within the one or more amino acid sequences by mass spectrometry.

13. The method of claim 12, wherein in step a) the protein of claim 1 is(a) fused to a fluorescent protein, preferably GFP, RFP, or YFP and in addition nanobeads being coupled to an antibody or antibody fragment binding to the fluorescent protein are used,(b) conjugated to a spycatcher protein coupled to a Fc fragment for protein A / G enrichment and / or biotin for streptavidin enrichment,(c) fused to a tag, preferably a GST-tag, His-tag, Flag-tag, Strep-tag or ALFA-tag, and / or (d) fused an antibody or an antibody fragment, preferably an Fc part.

14. The method of claim 12 or 13, wherein after step (a) and before step (b) bivalent cations, preferably Zn2+are removed by a chelator, preferably EDTA, EGTA, iminodisuccinic acid (IDS), polyaspartic acid, S, S-Ethylenediamine-N, N'-disuccinic acid (EDDS) or Methylglycinediacetic acid (MGDA) to specifically elute the ADP-ribosyl-linked ubiquitylation compounds.

15. A kit for the detection of amino acid sequences that are modified by ADP-ribosyl-linked ubiquitylation and / or amino acids within an amino acid sequence that are modified by ADP- ribosyl-linked ubiquitylation comprising the protein of claim 1 or the protein conjugate or fusion protein of any one of claims 2 to 4.

Images