Active base probe

Abstract

The present invention is directed to the development and use of novel photocrosslinking activity-based probes (ABPs). Specifically, ubiquitin-charged E2 conjugating enzymes are engineered and shown to be effective ABPs for RING ubiquitin E1 and E3 ligases and deubiquitinating enzymes.

Description

[Technical Field]

[0001] (Field of the present invention) This invention is directed toward the development and use of novel photocrosslinking activity-based probes (ABPs). Specifically, ubiquitin-charged E2 conjugating enzymes are engineered and shown to be effective ABPs for RING ubiquitin E1 and E3 ligases and deubiquitinating enzymes. [Background technology]

[0002] (Background of the present invention) Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutic substances. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate numerous (pathological)physiological processes. The largest subtype is RING E3s, which has >600 members. RING E3s possess adapter-like activity, which can be conditional on diverse regulatory mechanisms and have made them attractive drug targets. To our knowledge, there are no activity-based probes (ABPs) for measuring RING E3 activity.

[0003] Ubiquitination is a fundamental post-translational modification that regulates normal cell physiology, and its dysfunction can lead to the development of disease (Rape, M. (2018). Nat Rev Molecular Cell Biology 19, 59-70). Ubiquitination is carried out by an enzyme cascade involving sequential activity of ubiquitin E1 activation (E1), ubiquitin E2 conjugation (E2), and ubiquitin E3 ligase (E3s) (Hershko, A. and Ciechanover, A. (1998). Annu Rev Biochem 67, 425-479). Ubiquitin (Ub) is covalently transferred from the cysteine ​​related to the catalyst in E1 to the cysteine ​​related to the catalyst in E2, forming a thioester-linked E2 intermediate (E2~Ub). Hundreds of E3s are known to exist that replenish E2~Ub and ubiquitinate specific substrates. The branching of the E3 mechanism can be broadly divided into two general classes. There are approximately 50 "Cys E3s" molecules, and they utilize cysteine ​​related to the catalyst to form a covalent thioester intermediate with Ub prior to substrate modification (KC Pao et al., Nature, 2018, 556, 381-385; Scheffner, M., Nuber, U., and Huibregtse, JM (1995). Nature 373, 81-83; Wenzel, DM, et al. (2011), Nature 474, 105-108).

[0004] However, the largest class of E3s is adapter-like E3s, of which >600 distinct forms exist (Deshaies, RJ, and Joazeiro, CA (2009). Annu Rev Biochem 78, 399-434). Adapter-like E3s lack a nucleophile for catalytic activity and catalyze the direct transfer of Ub from E2~Ub to the substrate. This adapter-like activity is utilized by the multi-subunit Cullin-RING E3s and the ~350 single polypeptide RING E3s (hereinafter simply referred to as RING E3s). The latter can exist as monomers, homodimers, or heterodimers (Metzger, MB et al. Biochim Biophys Acta 1843, 47-60). Activity regulation is a particularly important aspect of E3 biology, ensuring cellular homeostasis and adaptive signaling. Dysregulation can lead to disease development, and therefore RING E3s have become attractive therapeutic targets (Burgess, A. et al. (2016). Frontiers in Oncology 6, 7). However, the cellular roles and regulatory mechanisms of the vast number of RING E3s remain poorly understood. Furthermore, RING E3s have recently been shown to be compatible with targeted proteolytic strategies (e.g., the PROTAC methodology) (Naito, M., Ohoka, N., and Shibata, N. (2019). Drug Discovery Today Technology, 31, 35-42; Spradlin, JN et al. (2019). Nature Chemical Biology, 15, 747-755; Ward, CC et al. (2019). ACS Chem Biol.). Tools for determining which RING E3s are active in clinical settings are needed to further enhance this potential.

[0005] A characteristic of adapter-like E3s is that, when active, they shift their dynamic E2-Ub stereostructure ensemble toward a distinct group where the E2-Ub conjugate adopts a folded or "closed" stereostructure (see Dou, H. et al. (2012b). Nat Struct Mol Biol (Nature Structural & Molecular Biology), 19, 876-883; Plechanovova, A. et al. (2012). Nature 489, 115-120.; Pruneda, JN et al. (2012). Mol Cell (Molecular Cell), 47, 933-942; Pruneda, JN et al. (2011). Biochemistry 50, 1624-1633; see Figure 1a). This three-dimensional structure is necessary for activating the thioester bond within E2-Ub for nucleophilic attack and for efficient aminolysis activity. RING E3 activity can be regulated, and switching to the activated state is achieved by E3 acquiring structural features that engage with the Ub component, thereby promoting the induction of a closed three-dimensional structure. For example, RING E3s, such as RNF4 and BIRC7, are activated by dimerization of the RING domain, where the tail region of the second RING protomer engages with the Ub component (Dou, H. et al., 2012b; Plechanovova, A. et al., 2012, supra (op. cit.)). Dimerization can be regulated by cellular signaling, and in the case of RNF4, this is brought about by binding to the polySUMO chain (Rojas-Fernandez, A. et al., Mol Cell, 53, 880-892).

[0006] Regarding the activation of monomeric RING E3s, it has been shown that so-called non-RING elements play a role in binding the Ub component, and in the case of Cbl-b and c-Cbl, this is a phosphorylated tyrosine residue (Dou, H. et al., Nat Struct Mol Biol, 20, 982-986). Phosphorylation is carried out by the kinase c-Src in response to growth factor stimulation, and Cbl activation induces ubiquitination and degradation of receptor and non-receptor tyrosine kinases. The existence of RING E3s and non-RING elements that require dimerization has also been reported (Koliopoulos, MG et al., EMBO J, 35, 1204-1218). Additional RING E3 activation mechanisms exist, including allosteric binding of accessory proteins or ligands (DaRosa, PA et al., Nature, 517, 223-226; Dickson, C. et al. (2018). Elife, 7; Duda, DM et al. (2012). Mol Cell, 47, 371-382; Wright, JD, Mace, PD, and Day, CL (2016). Nat Struct Mol Biol, 23, 45-52). A great many crystal structures of E2-Ub bound to activated RING E3s have been solved, revealing a highly conserved binding mode (Dou, H. et al. 2012b and 2013; Koliopoulos, MG et al. 2016; Plechanovova, A. et al. 2012; Wright, JD, Mace, PD, and Day, CL, 2016, op. cit.). Importantly, the consensus region of the Ub component in a closed E2-Ub conjugate is located near the activated RING.Furthermore, biophysical analysis demonstrates that previously studied activated RING E3s can have even higher free energy binding for E2~Ub than their inactive forms (Berndsen, CE et al. (2013). Nat Chem Biol. 9, 154-156.; Buetow, L. et al. (2016). BMC Biol. (BMC Biology) 14, 76).

[0007] Activity-based probes (ABPs) are powerful chemical tools that allow for activity-dependent covalent labeling of enzyme family members (Hewings, DS et al., FEBS J., 284, 1555-1576; Niphakis, MJ and Cravatt, BF (2014). Annu Rev Biochem., 83, 341-377). This enables: (i) studies of enzyme regulation, (ii) discovery of novel enzyme classes, (iii) inhibitor screening, (iv) inhibitor-selective profiling, and (v) stabilization of enzyme intermediates for structural studies (Hu, M. et al., Cell, 111, 1041-1054). We and others have developed ABPs for Cys E3s, which have been deployed to elucidate the E3 activation mechanism and discover entirely novel E3 classes (Love, KR et al. ACS Chem. Biol., 4, 275-287; Mulder, MP et al. (2016). Nat. Chem. Biol., 12, 523-530; Pao, KC et al. (2016). Nat. Chem Biol., 12, 324-331; Xu, L. et al. (2019). Chem. Commun. (Cambridge), 55, 7109-7112). [Overview of the project] [Problems that the invention aims to solve]

[0008] To the best of our knowledge, there are currently no ABPs (Analytic Behavioral Processes) for evaluating RING E3 ligase activity. The present invention aims to address this issue by providing ABPs for evaluating RING E3 ligase activity. [Means for solving the problem]

[0009] (Outline of the present invention) Without being constrained by theory, the inventors believe that the conserved (and activity-dependent) consensus interaction of the Ub component within a closed E2-Ub, coupled with the improvement of the free energy of binding for activated RING E3s, could be utilized for the development of ABPs.

[0010] We hereby disclose ABPs based on engineered ubiquitin molecules. Activated ubiquitin molecules containing a photocrosslinking agent (MOYETY) in place of the glutamine residue at position 31 and / or the aspartic acid residue at position 32 of ubiquitin were found to be remarkably effective as ABPs when conjugated to E2 conjugating enzymes. Such ABPs are effective for activity profiling of RING E3 ligases in diverse sample types and may be useful, for example, for RING E3 regulation studies, target discovery, biomarker applications, modulator discovery, and structural studies.

[0011] Accordingly, in a first embodiment, the present invention provides an activated ubiquitin molecule comprising the photocrosslinking agent Moiety instead of a glutamine residue at position 31 and / or an aspartic acid residue at position 32 of ubiquitin.

[0012] In a second embodiment, the present invention provides activation of the first embodiment conjugated to an E2 conjugating enzyme. Ubiquitin We provide conjugate molecules containing molecules.

[0013] In a third aspect, the present invention provides the use of the conjugated molecule of the second aspect in a method for activity profiling of a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme.

[0014] In a fourth aspect, the present invention provides a method for detecting an interaction between the conjugate molecule of the second aspect and a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme, the method comprising contacting the conjugate molecule of the second aspect with the RING E3 enzyme, the RING E1 enzyme and / or the deubiquitinating enzyme, and detecting the formation of any new conjugate. For example, by adding a reporter group that can be enriched in the conjugate, such as biotin, activated RING E3s can be enriched from natural cells and identified by mass spectrometry.

[0015] Those skilled in the art will recognize that any reference to an aspect of the present invention includes every embodiment of that aspect. For example, any reference to the first aspect of the present invention includes the first aspect and all embodiments of the first aspect. (List of figures) BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Figure 1-1]Figure 1: Strategies and synthetic schemes for the production of photocrosslinked ABPs for RING E3 ligase. a) Binding of activated RING E3 induces a closed conformation of the otherwise sterically dynamic E2-Ub conjugate. Activation can be achieved by dimerization of RING, but monomeric RING E3s can be activated by the presence of a non-RING element (NRE). The Ub component of E2-Ub interacts with the RING / NRE region. b) Judicious incorporation of the p-benzoyl-L-phenylalanine (Bpa) crosslinking amino acid within the stabilized E2-Ub conjugate functions as an ABP for RING E3 activity. c) Crystal structure of E2-Ub in complex with activated, dimerized RING E3 (RNF4). Ten amino acid sites within Ub adjacent to activated E3 were tested for Bpa incorporation. Q31 was found to be optimal. d) Synthetic scheme for photocrosslinking ABP. [Figure 1-2] Refer to the explanation in Figure 1. [Figure 1-3] Refer to the explanation in Figure 1. [Figure 2-1] Figure 2: Assembly and characterization of photo-ABPs and biotin-tagged photo-ABP-UbBpa31 probes. a) SDS-PAGE analysis of representative enzyme conjugation of UbBpa31 E2 (UBE2D3 C85K S22K double mutant) by ubiquitin E1 activating enzyme. Asterisks correspond to the putative diubiquitin species. b) Representative purified fraction of probe product after size exclusion chromatography (SEC). c) RP-HPLC chromatogram of purified photo-ABP-UbBpa31. d) Deconvoluted mass spectrum of photo-ABP-UbBpa31. Observed mass = 27901 Da, Observed mass (-Met) = 27771 Da. Expected mass = 27908.87 Da, Expected mass (-Met) = 2777.67 Da. [Figure 2-2] See the explanation in Figure 2. [Figure 3-1]Figure 3: Activity-dependent profiling of RNF4 E3 ligase activity. a) Constitutively active RNF-RING fusion proteins can productively interlock two E2-Ub conjugates. b) Probe photoABP-UbBpa31 (40 μM) undergoes two crosslinking reactions with RNF4-RING (10 μM). c) Interlocking of one or both E2-Ub conjugates can be disrupted by M140A R181A double mutations introduced in one or both RING domains in RNF-RING. d) Crosslinking with probe photoABP-UbBpa31 (40 μM) is attenuated or lost depending on whether one or both RING domains are mutated. e) Introduction of the F62A mutation into the E2 component should result in loss of E3 binding. f) Crosslinking is lost with photoABP-UbBpa31 F62A (probe concentration 5 μM). g) Regarding dimerization, RNF4 is inactive at concentrations below Kd. Binding of polySUMO chains induces dimerization and E3 ligase activity. h) Photo-ABP-UbBpa31 (5 μM) undergoes innate polySUMO chain (10 μM)-dependent crosslinking with RNF4 (100 nM), while RINF4-RING (50 nM) crosslinks independently of polySUMO chains. [Figure 3-2] Refer to the explanation in Figure 3. [Figure 4-1]Figure 4: Activity-dependent profiling and activity-based proteomic analysis of c-Cbl E3 ligase activity in EGF-stimulated vs. unstimulated HEK293 T cells. a) Only c-Cbl (3 μM) pre-incubated with c-Src (1.5 μM) undergoes crosslinking with photo-ABP Bpa31 (5 μM). Crosslinking is not observed when Cbl Y371 (3 μM) (which cannot be phosphorylated at the activation site) is incubated with Src. Phosphorylation of Cbl results in decreased electrophoretic mobility. b) Transient overexpression of GFP-Cbl and c-Src in mammalian HEK293 cells. Extracts were treated with photo-ABP31 Bpa31 or F62A control probe (5 μM). IB shows immunoblot, and the primary antibody used for detection is adjacent (i.e., anti-Cbl). c) Immunoblot analysis of HEK293T extracts using either photo-ABP-UbBpa31 or photo-ABP-Bpa31 F62A control probe (10 μM). Blotting was performed against hexahistidine reporter tags present within ABPs. Samples were irradiated for 60 minutes or withheld from irradiation. d) Schematic diagram illustrating the activity-based proteomics workflow using biotinylated photo-ABP-Bpa31. e) Spectral count values ​​obtained from ABP-profiled HEK293T cells. Search results were filtered against the PFAM domain term "RING," and only RING E3s with >2 spectral count values ​​in any replication experiment were plotted. Cells were serum-starved and treated with or without EGF, and with or without UV irradiation. Error bars correspond to the standard error from two technically replicated LC-MS / MS analyses. [Figure 4-2] Refer to the explanation in Figure 4. [Figure 5]Activated RING: Structural superposition of the E2-Ub complex. Activated RING E3s bind to E2-Ub and induce a “closed” E2-Ub conformation that activates the thioester for aminolysis (NB (Note), in the presented crystal structure, the unstable thioester is replaced by an ester or isopeptide linkage by mutations of the E2 catalytic cysteine ​​to serine or lysine, respectively). The closed conformation is induced by the binding of RING to the interface of the hybrid E2-Ub. Particularly important, in the case of RNF4 and BIRC7, the Ub component is retained in the closed conformation by interaction with RING protomer 2. For Cbl-b, phosphorylated tyrosine moyety (pTyr) functions as a non-RING element and promotes the closed conformation. In the case of TRIM25, the presence of dimerized and non-RING glutamic acid (Glu) residues promotes the closed conformation. [Figure 6] This study involved the integration of the photocrosslinked amino acid p-benzoyl-L-phenylalanine (Bpa) into the proximal RING region of ubiquitin. Visualization was performed by SDS-PAGE analysis and Coomassi staining. As a positive control, the reference amino acid t-butyloxycarbonyl-L-lysine (BocK) was integrated into Ub at position 6. To facilitate purification, a C-terminal His tag was added to Ub, which was then removed by treatment with the deubiquitinating enzyme UCH-L3. For each mutation, samples were analyzed before and after UCH-L3 treatment. [Figure 7-1]Figure 7: Electrospray ionization mass spectra for ubiquitin Bpa mutations. The spectra correspond to Ub after cleavage of the C-terminal His-tag. Except for the amber mutation clone for position 6, Ub is expressed with the N-terminal MGS motif. This is the introduction of the DNA coding sequence for this accelerated cloning. For these alt-part clones, the N-terminal methionine is cleaved to varying degrees by cellular methionyl aminopeptidase. A) UbBpa6, observed mass = 8686 Da; expected = 8787.95 Da. B) UbBpa9(-Met), observed mass = 8857 Da; expected = 8859.15 Da. UbBpa9, observed mass = 8988 Da, expected = 8990.35 Da. C) UbBpa11(-Met), observed mass = 8830 Da; expected = 8832.08 Da. UbBpa11, observed mass = 8961 Da, expected = 8963.28 Da. D) UbBpa13 (-Met), observed mass = 8845 Da; expected = 8847.1 Da. E) UbBpa14 (-Met), observed mass = 8857 Da; expected = 8859.15 Da. UbBpa14, observed mass = 8988 Da, expected = 8990.35 Da. F) UbBpa31 (-Met), observed mass = 8830 Da; expected = 8832.12 Da. UbBpa31, observed mass = 8961 Da, expected = 8963.32 Da. G) UbBpa32 (-Met), observed mass = 8845 Da; expected = 8845.17 Da. UbBpa32, observed mass = 8976 Da, expected = 8976.37 Da. H) UbBpa34 (-Met), observed mass = 8830 Da; expected = 8831.14 Da. UbBpa34, observed mass = 8961 Da; expected = 8962.34 Da. I) UbBpa40 (-Met), observed mass = 8831 Da; expected = 8832.12 Da. UbBpa40, observed mass = 8962 Da, expected = 8963.32 Da. J) UbBpa64 (-Met), observed mass = 8830 Da; expected = 8831.14 Da. UbBpa64, observed mass = 8961 Da, expected = 8962.34 Da. K)UbBpa72(-Met), observed mass = 8803 Da; expected = 8804.07 Da. The peak observed at 8836 Da corresponds to an unidentified adduct. [Figure 7-2] See the explanation in Figure 7. [Figure 7-3] See the explanation in Figure 7. [Figure 7-4] See the explanation in Figure 7. [Figure 7-5] See the explanation in Figure 7. [Figure 7-6] See the explanation in Figure 7. [Figure 8] Representative enzymatic conjugation of UbBpa31 to E2 via stabilizing isopeptide linkage to yield photo-ABP-UbBpa31. Visualization by SDS-PAGE analysis and Coomassi staining (top). Probe product purified by size exclusion chromatography (bottom). [Figure 9] This is a typical LC-MS analysis of photo-ABP-Bpa31. The HPLC chromatogram was measured at 214 nm. Photo-ABP Bpa31(-Met), observed mass = 27769 Da; expected mass = 27777.67 Da. Photo-ABP Bpa31(-Met), observed mass = 27901 Da; expected mass = 27908.87 Da. [Figure 10] This study evaluated the RNF4-RING photocrosslinking efficiency when Bpa was incorporated at 10 different Ub positions within the isopeptide-linked E2-Ub conjugate. Significant incorporation was achieved only when Bpa was incorporated at position 31, giving the probe photo ABP-UbBpa31. The reference amino acid t-butyloxycarbonyl-L-lysine (BocK) and Bpa were incorporated at position 6, as this site is highly tolerant of the incorporation of non-native amino acids, and served as a control. [Figure 11] This is a dose-responsive photocrosslinking of RNF4-RING. The photocrosslinking efficiency of RNF4-RING responded to the increasing concentration of photo-ABP-UbBpa31. [Figure 12]This involves photocrosslinking of phosphorylated c-Cbl with photo-ABP-UbBpa31 and photo-ABP-UbBpa31 F62A control probes. Purified c-Cbl (3 μM) was pre-incubated with c-Src (1.5 μM) in the presence of ATP (5 mM) at 37°C for 45 minutes. The reaction mixture was then profiled with the specified probes (5 μM). Probe photo-ABP-Bpa31 underwent c-Cbl crosslinking, while photo-ABP-Bpa31F62A did not. [Figure 13] This study compares the photocrosslinking efficiency of Bpa integration at multiple locations within ubiquitin. Prior to probe analysis, c-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 3 μM). Only Bpa integration at positions 31 and 32 yields functional probes. [Figure 14] This is a dose-response analysis of Cbl crosslinking. C-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 μM) prior to probe analysis. No increase in Cbl labeling efficiency was observed as a result of increasing photo-ABP-UbBpa31 concentrations above 5 μM. [Figure 15] This is a Phostag SDS-PAGE analysis of c-Src treated c-Cbl. The decreased electrophoretic mobility of c-Cbl is observed only in the presence of ATP. Since the gel shift is quantitative, this indicates that phosphorylation must also be quantitative. However, the crosslinking efficiency by photo-ABP-UbBpa31 was not dose-responsive above 5 μM. This means that c-Cbl is phosphorylated at multiple sites, which is not inconsistent with the quasi-stoichiometric behavior in Y371. [Figure 16] Photocrosslinking of cellular Cbl remains strictly dependent on the presence of Y371. ¹¹C-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 μM) prior to probe analysis. Cbl labeling efficiency was observed as a result of photo-ABP-UbBpa31, which increases above 5 μM. This may be due to quasi-stoichiometric phosphorylation at Y371. [Figure 17-1]Figure 17: Biotin labeling of Cys-tagged UbBpa31 and enzymatic conjugation to E2. LC-MS characteristics of UbBpa31 expressed with the N-terminal MGCSSG-labeled motif (observed mass = 8947 Da; expected mass = 8948.2 Da) (top left). The cysteine-labeled motif was alkylated with EZ-Link Iodoacetyl-PEG2-Biotin (Thermofisher). The product was purified by preparative RP-HPLC and characterized by LC-MS (observed mass = 9360 Da; expected mass = 9362.72 Da) (top right). Refolded biotin-tagged UbBpa31 was enzymatically conjugated onto E2, and the probe was purified by size exclusion chromatography (bottom). [Figure 17-2] See the explanation in Figure 17. [Figure 18] This is an LC-MS analysis of biotin-photo-ABP-Bpa31. The HPLC chromatogram was measured at 214 nm. Photo-ABP-Bpa31(-Met), observed mass = 28301 Da; expected mass = 28308.27 Da. [Figure 19-1]Figure 19: Confirmation of receptor tyrosine kinase activation and evaluation of non-RING E3 photocrosslinking. a) EGF-dependent receptor activation is confirmed by immunoblotting for ERK1 / 2 phosphorylation. HEK293T cells were serum-starved and stimulated with recombinant EGF. Cells were treated with the proteasome inhibitor MG132 prior to stimulation. NB (Note), for proteomics experiments, cells were treated with MG132 and bafilomycin. b) Detection of other ubiquitin system components by activity-based proteomics using biotinylated photoABP-UbBpa31. Spectral count values ​​obtained from ABP-profiled HEK293T cells. Search results were filtered for the PFAM domain terms "HECT, IBR and zf-UBR", and only RING E3s with spectral count values ​​>2 in any replication experiment were plotted. Cells were in a serum-starved state and treated with or without EGF, and with or without UV irradiation. Error bars correspond to the standard error from two technical iterations. c) As above, however, DUBs were filtered using a combination of PFAM domain terminology and manual curation. [Figure 19-2] See the explanation in Figure 19. [Figure 20] This study confirms that UBE2N / Ubc13 photocrosslinking activity-based probes undergo covalent labeling of their homologous E3 TRAF6 in a UV-dependent manner. Experiments were also performed in the presence of the UBE2N substrate receptor (Ube2V2), which did not appear to affect the activity toward TRAF6. [Modes for carrying out the invention]

[0017] (Detailed description of the present invention) ABPs based on engineered E2-Ub conjugates in which unstable cysteine ​​thioesters are substituted with more stable conjugates, such as lysine isopeptides (Plechanovova, A. et al., 2012, op. cit.), are disclosed herein.

[0018] The activated ubiquitin molecule of the present invention is remarkably effective as an ABP when conjugated to an E2 conjugating enzyme. The ABPs of the present invention are effective in activity profiling of RING E3 enzymes, RING E1 enzymes and / or deubiquitinating enzymes in a variety of sample types. Next, the activated ubiquitin molecule, conjugate, use, and method of the present invention will be described in detail.

[0019] In the following discussion, many terms are referenced and, unless the context indicates otherwise, have the meanings set forth below. The nomenclature used herein to define compounds, and in particular molecules according to the present invention, is generally based on the IUPAC rules for chemical compounds, in particular the "IUPAC Compendium of Chemical Terminology (Gold Book)".

[0020] The term “comprising” or its variations shall be understood to mean the inclusion of the element, integer, or step, or group of elements, integers, or steps, and not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0021] The term "consisting" or its variations shall be understood to mean the inclusion of the stated element, complete or step, or group of elements, complete or steps, and the exclusion of any other element, complete or step, or group of elements, complete or steps.

[0022] The term "aryl" is well known in this art and refers to all monovalent groups formed by removing a hydrogen atom from an arene cyclic carbon. The term "arene" refers to monocyclic and polycyclic aromatic hydrocarbons.

[0023] The term "heteroaryl" refers to an aryl compound in which one or more methine (-C=) and / or vinylene (-CH=CH-) groups are replaced by trivalent or divalent heteroatoms, respectively, in a manner that maintains the continuous π-electron system characteristic of aromatic systems.

[0024] The term "alkyl" is well known in this technology and defines a monovalent group derived from an alkane by removing a hydrogen atom from any carbon atom, where the term "alkane" refers to the general formula CnH 2n+2 This formula is intended to define cyclic or acyclic branched or unbranched hydrocarbons having n, where n is an integer ≥ 1.

[0025] The term "hydrocarbyl" is well known in this art and defines all monovalent groups formed by removing a hydrogen atom from a hydrocarbon. The term "hydrocarbon" is equally well known and here refers to all aliphatic and aromatic compounds consisting only of carbon and hydrogen, including branched, unbranched, acyclic and cyclic alkanes, alkenes and alkynes. To avoid any doubt, cycloalkanes and cycloalkenes fall within the scope of this definition of hydrocarbon.

[0026] The term "halo" is well known in this technology and refers to halogen radicals that, when bonded to carbon radicals, form fluoride, chloride, bromide, or iodide compounds.

[0027] The activated ubiquitin molecule of the present invention contains the photocrosslinking agent Moiety in place of the glutamine residue at position 31 and / or the aspartic acid residue at position 32 of ubiquitin. Ubiquitin consists of 76 amino acids having the following sequence (SEQ ID: 1). [ka]

[0028] Substitution of the glutamine residue at position 31 and / or the aspartic acid residue at position 32 (underlined and bolded in the above sequence) with the photocrosslinking agent Moieti results in remarkably effective photocrosslinking between activated ubiquitin molecules and E2 conjugating enzymes (see below).

[0029] In some embodiments, the photocrosslinking agent Moieti is present in place of a glutamine residue at position 31 of ubiquitin or an aspartic acid residue at position 32. In some embodiments, the photocrosslinking agent Moieti is present in place of a glutamine residue at position 31 of ubiquitin.

[0030] The photocrosslinking agent Moiety may be any Moiety capable of substituting the glutamine residue at position 31 and / or the aspartic acid residue at position 32 of ubiquitin, and forming a linker between the ubiquitin molecule and the E2 conjugating enzyme upon irradiation with photons. Preferably, the activated ubiquitin molecule is stable in the absence of photons and is permitted to be stored under appropriate conditions before use. In some embodiments, the activated ubiquitin molecule is stable in the presence of natural light, and in further embodiments, it is stable in the presence of natural and indoor artificial lighting (i.e., typical household lighting, mainly consisting of visible light) used to enhance luminosity.

[0031] Here, "stable" is used to mean that the amount of chemical degradation of a material or substance that occurs over time is not so severe as to render the material or substance unusable for practical purposes. This means that a certain degree of chemical degradation of the material or substance is acceptable, ranging from negligible chemical degradation to a level of chemical degradation that allows the amount of undegraded material or substance to withstand practical use.

[0032] A person skilled in the art can assess what conditions are suitable for storing activated ubiquitin molecules. For example, if activated ubiquitin molecules are stable in the absence of photons but not in the presence of natural light, a skilled person will recognize that the molecules should be stored in the dark; and if activated ubiquitin molecules are unstable at room temperature, the molecules should be stored in a refrigerator.

[0033] In certain embodiments, the photocrosslinking agent Moieti is a non-native amino acid, i.e., a modified native amino acid that may be entirely or partially synthesized. Typically, the non-native amino acid is derived from the modification of a native amino acid that has a functional group capable of forming a linker between ubiquitin molecules and E2 conjugating enzymes upon photon irradiation.

[0034] In some embodiments, the non-natural amino acid comprises any one or more combinations selected from the group consisting of diarylketones, diazirines, arylazides, diaryl / heteroarylketones, diheteroarylketones, heteroarylazides, and 2-aryl-5-carboxytetrazole. In certain embodiments, the non-natural amino acid comprises one functional group selected from the group consisting of diarylketones, diazirines, arylazides, diaryl / heteroarylketones, diheteroarylketones, heteroarylazides, and 2-aryl-5-carboxytetrazole. When the non-natural amino acid comprises diarylketones, arylazides, and 2-aryl-5-carboxytetrazole, the aryl may be optionally substituted and selected from the group consisting of phenyl or naphthalenyl. When the non-natural amino acid comprises diaryl / heteroarylketones, diheteroarylketones, and heteroarylazides, the heteroaryl may be optionally substituted and selected from the group consisting of indolyl, imidazolyl, pyridyl, thiophenyl, and furanyl.

[0035] Aryl or heteroaryl is C 1-4 Hydrocarbyl, C 1-4 Alkyloxy, C1-4 One or more carbon atoms may be substituted with any one or combination selected from the group consisting of haloalkyl, hydroxy, and halo. In some embodiments, the halo is fluoro. In certain embodiments, the aryl or heteroaryl may be optionally substituted with any one or combination selected from the group consisting of methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethyl, hydroxy, and fluoro.

[0036] In some embodiments, the non-natural amino acid comprises any one or combination of functional groups selected from the group consisting of diarylketones, diazirines, and arylazides (Pham ND, Parker RB, Kohler JJ, Curr. Opin. Chem. Biol. (Current Opinion in Chemical Biology), (2013), 17, 1, 90-101; Kauer, JC, Erickson-Viitanen S., Wolfe HR, DeGrado WF, J. Biol. Chem. (Journal of Biological Chemistry), (1986), 261, 23, 10695-10700).

[0037] In certain embodiments, the non-natural amino acids include diaryl ketones. Often, the diaryl ketone is any one selected from the group consisting of benzophenone, methylbenzophenone, dimethylbenzophenone, methoxybenzophenone, dimethoxybenzophenone, tertbutoxybenzophenone, ditertbutoxybenzophenone, trifluoromethylbenzophenone, di(trifluoromethyl)benzophenone, tetrakis(trifluoromethyl)benzophenone, hydroxybenzophenone, dihydroxybenzophenone, trihydroxybenzophenone, fluorobenzophenone, difluorobenzophenone, and trifluorobenzophenone.

[0038] In certain embodiments, the non-natural amino acid includes benzophenone. Often, the non-natural amino acid is derived from any one of the group consisting of phenylalanine, tryptophan, and histidine. In specific embodiments, the non-natural amino acid is derived from phenylalanine.

[0039] In a specific embodiment, the photocrosslinking agent Moieti is p-benzoyl-L-phenylalanine.

[0040] The activated ubiquitin molecule of the present invention may be derived from ubiquitin from any eukaryote. However, typically, the ubiquitin in the present invention is derived from an animal, such as a mammal. In some embodiments, the activated ubiquitin molecule of the present invention is derived from a human.

[0041] The activated ubiquitin molecule of the present invention may include a reporter tag. The purpose of the reporter tag is to enable the analytical detection of activated ubiquitin. Therefore, any tag suitable for binding to ubiquitin may be employed for detection, for example, by fluorescence.

[0042] In some embodiments, the reporter tag comprises any one or combination selected from the group consisting of biotin, epitopes, and fluorophores. Epitopes are recognized by conventional antibodies that bind to specific epitopes. When the reporter tag contains an epitope, it is capable of binding to an antibody, and localization, purification, and molecular characterization of the activated ubiquitin molecule are permitted. Epitopes include Myc-tag, HA-tag, FLAG-tag, GST-tag, 6xHis, V5-tag, and OLLAS.

[0043] Fluorophores are fluorescent tags. When the reporter tag contains a fluorophore, the activated ubiquitin molecule of the present invention can be detected by fluorescence microscopy or with the naked eye. Fluorophores include fluorescein; green fluorescent protein, e.g., optimized S65T mutation, enhanced green fluorescent protein (EGFP), monomer A206K mutation, superfolder GFP (sfGFP), Emerald, TagBFP, mCerulean3, mCitrine / mVenus, tdTomato, mCherry, mApple, mKate2, and mNeptune; FLAsH-EDT2 and ReAsH-EDT2.

[0044] When a reporter tag contains biotin, it can bind to its native ligand, namely avidin, streptavidin, or neutravidin, to allow for the localization, purification, and molecular characterization of the activated ubiquitin molecule. The native ligand may be itself, a fluorescent probe containing any of the fluorophores listed above; an enzyme reporter, such as horseradish peroxidase or alkaline phosphatase; or bound to an anti-biotin antibody.

[0045] Detection of activated ubiquitin molecules, including reporter tags, may be performed via fluorescence microscopy (or visual observation), electron microscopy, enzyme-linked immunosorbent assays (ELISAs), and / or Western blotting.

[0046] In certain embodiments, the reporter tag is a biotin moiety, which may be covalently linked to the ubiquitin or activated ubiquitin molecule of the present invention. In some embodiments, the biotin moiety is covalently linked to the ubiquitin or activated ubiquitin molecule via a terminal cysteine-, serine-, threonine-, or tyrosine-containing motif. The terminal motif may be C-terminus or N-terminus, but is typically N-terminus. In certain embodiments, the terminal motif is an N-terminal cysteine-containing motif. In more specific embodiments, the terminal motif has the sequence MGCSSG (SEQ ID NO: 2). The biotin and epitope tags allow for the enrichment of cross-linked and activated RING E3s, enabling their identification and quantification of activity by mass spectrometry.

[0047] A second embodiment of the present invention comprises an activating molecule of the first embodiment conjugated to an E2 conjugating enzyme. During ubiquitination, ubiquitin is transferred to a catalytic cysteine ​​at E2, forming an unstable thioester-linked E2 intermediate (E2~Ub) before the subsequent transfer of ubiquitin to a lysine residue on the target protein.

[0048] To prevent the dissociation of the activated ubiquitin molecule from the E2 conjugating enzyme of the present invention, the catalytic cysteine ​​residue of the E2 enzyme may be substituted with a residue that forms a more stable linker, such as a lysine residue. Thus, in some embodiments, the E2 conjugating enzyme contains a lysine residue instead of a catalytic cysteine ​​residue. In certain embodiments, the catalytic cysteine ​​residue of the E2 conjugating enzyme is at position 85, i.e., the E2 conjugating enzyme contains the C85K mutation. In some embodiments, the catalytic cysteine ​​residue of the E2 conjugating enzyme is at position 87, i.e., the E2 conjugating enzyme contains the C87K mutation. Alternative methods to prevent the dissociation of ubiquitin from E2 may include the incorporation of unnatural lysine derivatives (i.e., diaminopropionic acid, diaminobutyric acid, and ornithine) having progressively shorter aliphatic side chains.

[0049] Some E2 conjugating enzymes, such as UBE2D3, contain additional serine residues capable of forming non-covalent bonds with ubiquitin. These interactions promote the association (self-association) of ubiquitin of one conjugate molecule with the E2 conjugating enzyme of another conjugate molecule. To suppress the self-association of conjugate molecules, the serine residues may be substituted with residues that do not associate with ubiquitin, such as arginine residues. In certain embodiments, the serine residue capable of forming non-covalent bonds with ubiquitin is located at position 22, i.e., the E2 conjugating enzyme contains the S22R mutation.

[0050] Some E2-conjugating enzymes, such as UBE2N, contain an additional native lysine residue capable of conjugating to ubiquitin, and thus ubiquitin conjugation is enhanced at the native residue rather than at or before the catalytic cysteine ​​residue (e.g., where cysteine ​​is replaced with a more stable linker, such as lysine). Self-association may also be enhanced. To suppress ubiquitin conjugation at the native lysine residue, the native lysine residue may be replaced with a residue unrelated to ubiquitin, such as an arginine residue. In certain embodiments, the native lysine residue is at position 92, i.e., the E2 conjugating enzyme contains the K92A mutation.

[0051] The E2 conjugating enzyme can be any E2 enzyme capable of binding to ubiquitin. However, typically, the E2 conjugating enzyme is UBE2D3. In some embodiments, the E2 conjugating enzyme is UBE2D3 or UBE2N.

[0052] In some embodiments, the E2 conjugating enzyme has an N-terminal histidine tag, such as a hexahistidine tag. These tags facilitate the purification of the conjugate molecules of the present invention and also facilitate the detection of the conjugate molecules via Western blot (also known as immunoblot) analysis.

[0053] The conserved (and activity-dependent) consensus interactions of the Ub components within closed E2-Ub, coupled with the increased free energy of binding for activated RING E3s, are utilized for the development of ABPs. The ABPs of this invention are effective in activity profiling of RING E3 ligases, RING E1 ligases, and deubiquitinating enzymes in a variety of sample types.

[0054] Accordingly, in a third embodiment, the present invention provides the use of a second embodiment of a conjugated molecule in a method for activity profiling of RING E3 enzymes, RING E1 enzymes and / or deubiquitinating enzymes. In one embodiment, the use of a second embodiment of a conjugated molecule is in a method for activity profiling of a RING E3 enzyme. In a further embodiment, the RING E3 enzyme is any one or a combination of RNF4, Cbl, e.g., c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), and RCR(1). For example, the RING E3 enzyme may be RNF4 or c-CBI. In some embodiments, the RING E3 enzyme is any one or a combination of RNF4, Cbl, e.g., c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), and RCR(1), TRAF6, TRAF2, and HLTF. For example, the RING E3 enzyme could be RNF4, c-CBI, or TRAF6.

[0055] The RING E3 enzyme is inactive in monomeric form and is dominant at endogenous concentrations. Activation of the RING E3 enzyme can be achieved by RING dimerization, leading to ligase activity of the RING E3 enzyme (Rojas-Fernandez, A. et al., 2014, op. cit.). Alternatively, the monomeric RING E3 enzyme can be activated by non-RING elements (NREs), see, for example, Dou H. et al., 2013, op. cit. Activation of the E3 enzyme leads to ubiquitination (see Figure 1a), i.e., the active RING E3 enzyme binds to E2-Ub (a conjugate containing the E2 conjugating enzyme and ubiquitin), inducing a closed E2-Ub three-dimensional structure.

[0056] The active RING E3 enzyme can bind to the conjugated molecule of the present invention via RING E3 or NRE. Upon irradiation of the resulting complex, the photocrosslinking agent moisture of the conjugated molecule can covalently bind to RING E3 or NRE (see Figure 1b). Therefore, the conjugated molecule of the present invention can act as a RING E3 ABP, where the active RING E3 enzyme can be detected and further characterized.

[0057] In a fourth aspect, the present invention provides a method for detecting interactions between a conjugate molecule of the second aspect and a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme, the method comprising contacting the conjugate molecule of the second aspect with the RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme, and detecting the formation of any new conjugate.

[0058] As used herein, "contact" refers to any means that allow the conjugate molecule of the present invention to interact with the RING E3 / RING E1 / deubiquitinating enzyme. Typically, contact occurs in an aqueous solution containing the conjugate molecule of the present invention and the RING E3 enzyme.

[0059] Upon irradiation, the photocrosslinking agent moisture of the conjugated molecule can covalently bond to the RING E3 / RING E1 / deubiquitinating enzyme. If the conjugated molecule of the present invention is unstable in the presence of photons, then the photocrosslinking agent moisture of the conjugated molecule can covalently bond to the enzyme by irradiation with photons of any frequency. However, if the conjugated molecule is stable in the presence of natural light, a higher frequency than natural light may be required to form a covalent bond between the activated ubiquitin molecule and the enzyme. Typically, UV light, i.e., light with wavelengths in the range of 10 to 400 nm, is used to covalently link the photocrosslinking agent moisture to the enzyme.

[0060] Experienced individuals recognize that the irradiation time required for covalent bond formation between the enzyme and the conjugated molecule depends on various factors. For example, irradiation time can be influenced by the concentration of the sample being irradiated (higher concentrations are thought to require longer irradiation times); the frequency of the light used to irradiate the sample (frequency matching the frequency absorbed by the photocrosslinking agent moisture is thought to require shorter irradiation times); and the power output of the irradiation source (higher power output is thought to result in shorter irradiation times). The conjugated molecule and enzyme of the present invention may be irradiated for 1 to 50 minutes. In some embodiments, the conjugated molecule and enzyme of the present invention are irradiated for 1 to 40 minutes. Typically, irradiation is between 1 and 30 minutes.

[0061] When the enzyme is a RING E3 enzyme, the conjugated molecule of the present invention can be used in any study where the detection of a RING E3 enzyme is useful. This includes studies of RING E3 enzyme regulation; discovery of novel RING E3 enzymes; screening of inhibitors; and stabilization of enzyme intermediates for selective profiling and / or structural studies of inhibitors.

[0062] Novel conjugates of the present invention can be detected using a variety of methods, including fluorescence microscopy (or visual observation), electron microscopy, enzyme-linked immunosorbent assays (ELISAs), gel electrophoresis, and / or Western blotting. Often, a combination of analytical methods is used to detect novel conjugates of the present invention. Experienced individuals will recognize that the method suitable for detecting novel conjugates of the present invention differs depending on whether a reporter tag is employed and the identity of any reporter tag. For example, gel electrophoresis can be used to separate novel conjugates from a sample mixture according to size and charge, and can be useful regardless of whether a reporter tag is employed. However, fluorescence microscopy can only be used to identify novel conjugates if the conjugated molecules of the present invention contain a fluorescent reporter tag.

[0063] The conjugate molecules of the present invention may interact with any active RING E3 enzyme; that is, the uses and methods of the present invention are not limited to any particular type of RING E3 enzyme. However, in some embodiments, the RING E3 enzyme is RNF4, Cbl, e.g., c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), and RCR(1) enzymes. In specific embodiments, the RING E3 enzyme is the c-CbI enzyme. In some embodiments, the RING E3 enzyme is any one or a combination of RNF4, Cbl, e.g., c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), and RCR(1), TRAF6, TRAF2, and HLTF. For example, the RING E3 enzyme may be RNF4, c-CBI, or TRAF6.

[0064] Any discussion herein relating to documents, laws, materials, apparatus, articles and other similar items shall not be construed as an acknowledgment that any or all of these items constitute part of the prior art foundation or were common general knowledge in the art relating to this disclosure as if they existed prior to the priority date of each claim of this application.

[0065] Those skilled in the art will recognize that numerous modifications and / or alterations to the invention described herein can be made as described without departing from the scope of the invention. Therefore, these embodiments should be considered for illustrative purposes only and are not restrictive, nor are they limited to the scope described herein. Those skilled in the art should understand that these embodiments may be read individually or in combination, and may be used in combination with any one or a combination of the features described herein.

[0066] The subject matter of each patent and non-patent document cited herein is incorporated here in its entirety by reference.

[0067] The present invention may be further understood in relation to the following non-limiting items:

[0068] 1. An activated ubiquitin molecule containing a photocrosslinking agent Moiety instead of a glutamine residue at position 31 and / or an aspartic acid residue at position 32 of ubiquitin.

[0069] 2. The photocrosslinking agent Moiety is present in the activated ubiquitin molecule of item 1, in place of the glutamine residue at position 31 or the aspartic acid residue at position 32 of ubiquitin.

[0070] 3. The photocrosslinking agent Moieti is the activated ubiquitin molecule of item 1, in place of the glutamine residue at position 31 of ubiquitin.

[0071] 4. The photocrosslinking agent Moieti is a non-natural amino acid, any one of the activated ubiquitin molecules from items 1 to 3.

[0072] 5. An activated ubiquitin molecule according to item 4, wherein the non-natural amino acids include any one or combination selected from the group consisting of diaryl ketones, diazilines, aryl azides, diaryl / heteroaryl ketones, diheteroaryl ketones, heteroaryl azides, and 2-aryl-5-carboxytetrazoles.

[0073] 6. An activated ubiquitin molecule according to item 4, wherein the non-natural amino acid contains one functional group selected from the group consisting of diarylketones, diazirines, arylazides, diaryl / heteroarylketones, diheteroarylketones, heteroarylazides, and 2-aryl-5-carboxytetrazoles.

[0074] 7. An activated ubiquitin molecule according to item 4, wherein the non-natural amino acid contains any one functional group selected from the group consisting of diarylketones, diazilines, arylazides, and 2-aryl-5-carboxytetrazoles.

[0075] 8. Non-natural amino acids include diaryl ketones, which are activated ubiquitin molecules as described in item 4.

[0076] 9. The aryl is optionally substituted and may be selected from the group consisting of phenyl or naphthalenyl; the heteroaryl is optionally substituted and may be selected from the group consisting of indolyl, imidazolyl, pyridyl, thiophenyl and furanyl, any one activated ubiquitin molecule from item 5 to 8.

[0077] 10. Aryl or heteroaryl is C 1-4 Hydrocarbyl, C 1-4 Alkyloxy, C 1-4 Any one activated ubiquitin molecule from items 5 to 9, which can be substituted with one or more carbon atoms by any one or combination selected from the group consisting of haloalkyl, hydroxy, and halo.

[0078] 11. Halo is fluoro, the activated ubiquitin molecule of item 10.

[0079] 12. Any one activated ubiquitin molecule from items 5 to 9, wherein the aryl or heteroaryl is optionally substituted with any one or combination selected from the group consisting of methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethyl, hydroxy, and fluoro.

[0080] 13. The activated ubiquitin molecule of item 4, comprising diaryl ketones selected from the group consisting of benzophenone, methylbenzophenone, dimethylbenzophenone, methoxybenzophenone, dimethoxybenzophenone, tert-butoxybenzophenone, ditert-butoxybenzophenone, trifluoromethylbenzophenone, di(trifluoromethyl)benzophenone, tetrakis(trifluoromethyl)benzophenone, hydroxybenzophenone, dihydroxybenzophenone, trihydroxybenzophenone, fluorobenzophenone, difluorobenzophenone, and trifluorobenzophenone, which are non-natural amino acids.

[0081] 14. Non-natural amino acids include benzophenone, the activated ubiquitin molecule of item 4.

[0082] 15. Any one activated ubiquitin molecule from item 4 to 14, derived from any one of the non-natural amino acids consisting of phenylalanine, tryptophan, and histidine.

[0083] 16. The non-natural amino acid is any one activated ubiquitin molecule from item 4 to 14, derived from phenylalanine.

[0084] 17. The photocrosslinking agent is p-benzoyl-L-phenylalanine, any one of the activated ubiquitin molecules from items 1 to 3.

[0085] 18. Ubiquitin is derived from animals; any one activated ubiquitin molecule from items 1 through 17.

[0086] 19. Ubiquitin is derived from mammals; any one activated ubiquitin molecule from items 1 through 17.

[0087] 20. Ubiquitin is derived from humans, and is any one activated ubiquitin molecule from item 1 through 17.

[0088] 21. Any one activated ubiquitin molecule from item 1 to 20, further including a reporter tag.

[0089] 22. An activated ubiquitin molecule according to item 21, wherein the reporter tag contains any one or combination selected from the group consisting of biotin, epitope, and fluorophore.

[0090] 23. The activated ubiquitin molecule of item 22: (i) The epitope is any one or combination selected from Myc-tag, HA-tag, FLAG-tag, GST-tag, 6xHis, V5-tag and OLLAS; and (ii) The fluorophore is any one or combination selected from fluorescein, optimized S65T mutation, enhanced green fluorescent protein (EGFP), monomer A206K mutation, superfolder GFP (sfGFP), Emerald, TagBFP, mCerulean3, mCitrine / mVenus, tdTomato, mCherry, mApple, mKate2 and mNeptune, FLAsH-EDT2 and ReAsH-EDT2.

[0091] 24. The reporter tag is biotin moiety, the activated ubiquitin molecule of item 21.

[0092] 25. The activated ubiquitin molecule of item 24, wherein the biotin moiety is covalently linked to the ubiquitin or activated ubiquitin molecule via a terminal cysteine-, serine-, threonine-, or tyrosine-containing motif.

[0093] 26. The activated ubiquitin molecule of item 25, with the terminal motif being the N-terminus.

[0094] 27. The activated ubiquitin molecule of item 25, whose terminal motif is an N-terminal cysteine-containing motif.

[0095] 28. Any one activated ubiquitin molecule from items 25 to 27, having the terminal motif sequence MGCSSG.

[0096] 29. A conjugate molecule containing an activated ubiquitin molecule conjugated to an E2 conjugating enzyme, following any one preceding term.

[0097] 30. E2 conjugating enzymes are conjugate molecules of item 29, which contain lysine residues instead of cysteine ​​residues related to the catalyst.

[0098] 31. E2 conjugating enzymes include the C85K or C87K mutations of the conjugate molecules in item 29.

[0099] 32. Any one conjugate molecule from items 29 to 31, wherein a serine or lysine residue capable of forming a non-covalent bond with ubiquitin is substituted with an arginine residue.

[0100] 33. The E2 conjugating enzyme is any one of the conjugate molecules from items 29 to 30, including the C85K and S22R mutations.

[0101] 34. The E2 conjugating enzyme is UBE2D3, any one of the conjugate molecules from items 29 to 33.

[0102] 35. The E2 conjugating enzyme is any one of the conjugate molecules from items 29 to 30, including the C87K and K92A mutations.

[0103] 36. The E2 conjugating enzyme is UBE2N, any one of the conjugate molecules from items 29 through 32 and 35.

[0104] 37. E2 conjugating enzyme is any one of the conjugate molecules from items 29 to 36 that has an N-terminal histidine tag.

[0105] 38. The E2 conjugating enzyme is any one of the conjugate molecules from items 29 to 36 that has a hexahistidine tag.

[0106] 39. Use of any one conjugated molecule (also called a conjugated molecule) from items 29 to 38 in a method for activity profiling of RING E3 enzymes, RING E1 enzymes and / or deubiquitinating enzymes.

[0107] 40. The RING E3 enzyme is any one or combination of RNF4, Cbl, e.g., c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), RCR(1), TRAF6, TRAF2, and HLTF, and the deubiquitinating enzyme is DUBs(31), as used in item 39.

[0108] 41. Activity profiling is of the RING E3 enzyme, used in accordance with section 39 or section 40.

[0109] 42. Use of paragraph 41 for the following: (i) Research on the regulation of RING E3 enzyme; (ii) Discovery of a novel RING E3 enzyme; (iii) Inhibitor screening; (iv) Inhibitor-selective profiling; and / or (v) Stabilization of enzyme intermediates for structural studies.

[0110] 43. A method for detecting an interaction between any one conjugate molecule from items 29 to 38 and a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme, the method comprising contacting the conjugate molecule with the RING E3 enzyme, a RING E1 enzyme and / or a deubiquitinating enzyme and detecting the formation of any new conjugate.

[0111] 44. The RING E3 enzyme is any one or combination of RNF4, Cbl, such as c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), RCR(1), TRAF6, TRAF2, and HLTF, and the deubiquitinating enzyme is DUBs(31), according to the method of item 43.

[0112] 45. The interaction is with the RING E3 enzyme, as described in section 43 or 44.

[0113] 46. ​​Any one of the methods from items 43 to 45, further comprising irradiating the conjugate molecule and the RING E3 / RING E1 / deubiquitinase so that the photocrosslinking agent moiety of the conjugate molecule covalently binds to the RING E3 / RING E1 / deubiquitinase.

[0114] 47. Irradiation is by UV light, as in the method of item 46.

[0115] 48. The method of item 47, wherein the UV light has a wavelength in the range of 200 to 400 nm.

[0116] 49. The RING E3 enzyme is RNF4, CbI, or TRAF6 enzyme, as per any one of the methods from section 45 to 48.

[0117] 50. The CbI enzyme is the c-CbI enzyme, as described in section 49.

[0118] The present invention is further illustrated by the following non-limiting examples. [Examples]

[0119] Demonstrate the activity-dependent profiling of two cancer-related RING E3s, RNF4 and c-Cbl, in response to their natural activating signals. RNF4 is activated by polySUMO chain-induced dimerization, while c-Cbl is activated by tyrosine phosphorylation. It is demonstrated that by combining biotin reporter-tagged conjugates with mass spectrometry, parallel measurements of the natural RING E3 activity of cells can be performed. Furthermore, cellular perturbation (e.g., growth factor stimulation) can be used to distinguish the cellular activation of individual RING E3s. The conjugate molecules of the present invention have the potential to advance E3 ligase research and the development of selective modulators for this enzyme class.

[0120] (Materials and Methods) (Details of Experimental Models and Subjects) H293T cells were obtained from ATCC. 293T is a human cell line derived from the HEK293 cell line, which expresses a mutant version of the SV40 large T antigen (RRID:CVCL_0063). The cells were cultured at 37 °C in a humidified incubator under a 5% CO2 atmosphere. Dulbecco's modified Eagle medium was used and supplemented with fetal bovine serum and L-glutamine.

[0121] In this study, BL21(DE3) and Escherichia coli BL21 Rosetta™(DE3) cells used for protein expression were cultured in 1 L flasks containing 1 L of LB medium supplemented with 100 mL -1 of ampicillin and 34 mL -1 of chloramphenicol (see STAR Methods - Expression of Recombinant Proteins for details).

[0122] (Details of the method) (Site-specific integration of the non-native amino acid pBpa into ubiquitin) The pEvol-Bpa plasmid was derived from pEVOL-pBoF (courtesy of P. Schultz, The Scripps Research Institute). To construct the pEVOL-Bpa plasmid, mutations for Bpa integration were introduced into both copies of the MjYRS gene (Young et al., 2010; Chin et al., 2002). BL21 cells (50 μL) were co-transformed with pET-Ubiquitin-6His-TAGx (where x is the Bpa integration site) and the pEvol-Bpa plasmid using heat shock, and harvested in 200 μL SOC medium at 37°C for 1 hour, and 100 μg mL -1 Ampicillin and 34 μg mL -1 It was used to inoculate 50 mL of Luria-Bertani (LB) containing chloramphenicol. Then, 10 mL of the overnight culture was used to inoculate 1 L of LB broth containing the same concentration of antibiotic. OD 600 The cells were grown until the pH was ~0.6, and the culture was divided into two 500 mL portions. One portion was supplemented with 1 mM p-benzoyl-L-phenylalanine (Bpa; Bachem), and the other was treated as a control without Bpa. The culture was incubated for 20 minutes (37°C, 200 rpm), or OD 600 The cultures were incubated until the pH reached 0.6–0.7, and protein expression was induced by adding 0.02% arabinose and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cultures were incubated for 5 hours (37°C, 200 rpm). Cells were harvested and transferred to 50 mL Falcon tubes in 10 mL of BugBuster. (R) Protein Extraction (Bug Buster Protein Extraction) (R)The solution was suspended in Merk Millipore reagent (*indicates registered trademark in the United States, etc.). The lysate was incubated for 20 minutes, then clarified by centrifugation before transferring to a 50 mL Falcon tube containing 1 mL Ni-NTA agarose beads, and incubated for 1 hour with gentle shaking. The resin was centrifuged (4°C, 1000 rpm) and washed with a washing buffer (20 mM Na2HPO4, pH 7.5, 25 mM imidazole). Finally, the protein was eluted with 200 μL of elution buffer (20 mM Na2HPO4, pH 7.5, 300 mM imidazole). 20 μL aliquots from the eluted fraction were mixed with an equal volume of 4X SDS-added loading buffer and loaded onto a 4-12% SDS-PAGE gel. Proteins were detected by segregation at 200 V for 30 minutes using MES buffer and Coomassie blue staining. Separate 20 μL of protein was analyzed by LC-MS. LC-MS was performed using an Agilent 1200 LC-MS system equipped with a Max-Light Cartridge flow cell coupled to a 6130 Quadrupole spectrometer. Unless otherwise stated, an Agilent ZORBAX 300SB-C3 5 μm, 2.1 × 150 mm column was used. The solvent system consisted of 0.05% trifluoroacetic acid in H2O as buffer A and 0.04% TFA acid in acetonitrile as buffer B. Protein UV absorption was monitored at 214 and 280 nm. MS acquisition was performed in positive ion mode, and the total protein mass was calculated by deconvolution within MS Chemstation software (Agilent Technologies). The fraction containing pBpa-integrated Ub was pooled concentrated using an Amicon Ultra-15 3kDa MWCO centrifugal filter (Millipore).The sample was desalted in 10 mM Tris-HCl pH 7.5 using a PD-10 column (GE Life Sciences). DTT (1 mM) was added to the sample, followed by the final concentration of 15 μg mL. -1 Hexahistidine tag cleavage was continued using UCH-L3 (Virdee et al., 2010). The samples were incubated at 37°C for 2 hours to remove the N-terminal His tag. Bpa-integrated Ub was further purified by semi-prepared HPLC, and the fraction was lyophilized to produce approximately 8-10 mg of Ub-pBpa.

[0123] (Expression of UBE2D3(S22R / C85K) recombinant protein) S22R and C85K were introduced into UBE2D3 using site-directed mutagenesis. Cells were osmotically treated at 37°C and 200 rpm. 600 The cells were grown until the volume reached 0.6-0.7. Protein expression was controlled by OD. 600 When the pH reached 0.6-0.7, induction was performed by adding IPTG (1 mM), and the cells were incubated at 37°C for 3 hours. The cells were harvested and resuspended in buffer (20 mM Na2HPO4, pH 7.5, 150 mM NaCl, 1 mM TCEP, complete protease inhibitor cocktail (EDTA-free, Roche)). Lysozyme was added (0.5 mg mL). -1The cells were incubated on ice for 30 minutes, followed by sonication. The clarified lysate containing His6-tagged UBE2D3 (S22R / C85K) was loaded onto Ni-NTA resin and washed with a buffer (20 mM pH 7.5, Na2HPO4, pH 7.5, 25 mM imidazole, 150 mM NaCl, 1 mM TCEP), followed by elution with an elution buffer (20 mM pH 7.5, Na2HPO4, pH 7.5, 300 mM imidazole, 150 mM NaCl, 1 mM TCEP). The samples were further purified by size exclusion chromatography using a HiLoad Superdex-75 16 / 60 column (GE Healthcare) with a running buffer (20 mM Na2HPO4, pH 7.5, 150 mM NaCl, 1 mM TCEP).

[0124] (Preparation of biotin-UbBpa31) Lyophilized UbBpa31 (10 mg) containing an N-terminal MGCSSG cysteine ​​motif was reconstituted in 1 mL of 10% DMSO / 90% 0.5 mM TCEP (aqueous solution) and incubated at 23°C for 45 minutes with gentle mixing. Subsequently, 5 molar equivalents of EZ-link iodo-acetyl PEG2-Biotin (Thermofisher) were added to reaction buffer (50 mM Na2HPO4, 150 mM NaCl, 0.5 mM TCEP). The reaction was incubated at 23°C for 1 hour with gentle shaking, and completion was monitored by LC-MS. The product was then purified by preparative HPLC at flow rate and lyophilized to produce biotin-UbBpa31 (6-8 mg).

[0125] (Preparation of isopeptide-linked photo-ABPs) To produce UBE2D3(S22R / C85K)-UbBpa, UBE2D3(S22R / C85K) (200 μM) was incubated with UbBpa (200 μM) and His6-Uba1 (1 μM) in a conjugation buffer (50 mM Tris, pH 10.0, 150 mM NaCl, 3 mM ATP, 5 mM MgCl2, 1 mM TCEP) at 35°C for 26 hours. The E2-UbBpa conjugate was applied to a HiLoad 16 / 60 Superdex 75 gel filtration column (GE Healthcare) (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP). The purified UBE2D3(S22R / C85K)-UbBpa conjugate was divided into 2 mg ml -1 The solution was concentrated and stored at -80°C. Biotin-optical ABP probes were prepared using the same procedure.

[0126] (Expression of recombinant RNF4 protein.) The cloning, expression, and purification of a linear fusion of two RNF4 RING domains and related mutants were previously described (Plechanovova et al., 2011). The fusion of the two RNF4 RING domains was expressed in E. coli Rosetta (DE3) cells (Novagen). Exposure was performed at 37°C and 200 rpm. 600 The cells were grown until the OD reached 0.6-0.7. 600 Once the pH reached 0.6-0.7, protein expression was induced by adding IPTG (1 mM), and the mixture was incubated overnight at 16°C and 200 rpm.

[0127] Cells were collected and resuspended in a lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 10 mM imidazole, 2 mM benzamidine, complete protease inhibitor cocktail (EDTA-free, Roche)), and cells were lysed by sonication. His6-MBP fusion proteins were purified by Ni-NTA (Qiagen) chromatography and subsequently cleaved with TEV protease at 4°C overnight. To remove any uncleaved fusion proteins, His6-tagged MBP, and His6-tagged TEV protease, the material was reduced against fresh Ni-NTA resin and then subjected to size exclusion chromatography on a HiLoad Superdex 75 16 / 60 column (GE Healthcare) (20 mM Tris, 150 mM NaCl, 1 mM TCEP, pH 7.5).

[0128] (Expression of c-Cbl and c-Cbl(Y371F) recombinant proteins) BL21(DE3) cells (50 μL) were transformed with the pGEX6P-1-Cbl plasmid, harvested in 200 μL SOC medium at 37°C for 1 hour, and 100 μg mL -1 The cells were used to inoculate 50 mL of Luria-Bertani (LB) containing ampicillin. Then, 10 mL of the overnight culture was used to inoculate LB broth containing the same concentration of antibiotic and 0.2 mM zinc chloride. The cells were then osmothered at 37°C and 200 rpm. 600 The culture was continued until the OD reached 0.6-0.7. 600When the pH reached 0.6-0.7, protein expression was induced by adding 1 mM IPTG, and the cells were left overnight at 16°C and 200 rpm. The cells were collected and resuspended in a buffer (50 mM Hepes, pH 7.5, 0.5 M NaCl, 1 mM TCEP), and lysed by sonication. The lysates were incubated with glutathione Sepharose beads for 1 hour with gentle shaking. The resin was centrifuged (4°C, 1000 rpm), washed with a buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP), and then cleaved with Rhinovirus 3C protease overnight at 4°C. The cleaved proteins were further purified by size exclusion chromatography using a HiLoad Superdex 200 16 / 600 column (GE Healthcare) (20 mM HEPES, 150 mM NaCl, 1 mM TCEP, pH 7.5).

[0129] (c-Cbl phosphorylation) Purified c-Cbl (3 μM) was phosphorylated by incubation at 37°C for 45 minutes with Src kinase (1.5 μM), 10 mM MgCl2, and 5 mM ATP. The sample (15 μl) was collected and thoroughly mixed with 4× LDS solution (ThermoFisher), then boiled before loading onto a 7.5% acrylamide phos-tag gel. Proteins were separated at 160 V for 60 minutes using MOPS buffer and analyzed using Coomassi staining and Western blotting.

[0130] Furthermore, ATP-dependent phosphorylation and photocrosslinking of c-Cbl with photo-ABP-UbBpa31 (5 μM) were analyzed using Coomassi staining. Samples (15 μl) were collected and thoroughly mixed with 4× LDS additive, then boiled at 95°C for 5 minutes before loading onto 4-12% SDS-PAGE gels with MOPS running buffer, and analyzed using Coomassi staining. The gels were further blotted and analyzed using Western blotting with anti-Cbl (1:5000 dilution) as the primary antibody and anti-mouse (1:10000 dilution) as the secondary antibody.

[0131] (UV irradiation conditions for photocrosslinking) Photocrosslinking (45 μL) was performed in a 24-well plate (Cryshem HR3-158, Hampton Research) using reaction buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP). The sample was divided into two parts. One part was irradiated on ice with 365 nm light for 10–30 minutes from a handled UV lamp (BLE-8T365, Spectroline) at a distance of 2 cm, while the other part was stored in the dark. Purified proteins, such as RNF4-RING (5–50 μM), c-Cbl (3 μM), and c-Cbl Y371F (3 μM), were photocrosslinked using the photo-ABP-UbBpa31 probe (5–50 μM) and irradiated with UV light. Samples were split by SDS-PAGE and visualized by Coomassi staining or immunoblotting. Control experiments were performed under the same conditions.

[0132] (Photocrosslinking in cell extracts) HEK293 cells were transfected with plasmids expressing GFP-Cbl, GST-Src, and GFP-Cbl. The cells were lysed in a lysis buffer (50 mM Na2HPO4, 10 mM Glycerophosphate, 50 mM Sodium Fluoride, 5 mM Sodium Pyrophosphate, 1 mM Sodium Vanadate, 0.25 M Sucrose, 50 mM NaCl, 0.2 mM PMSF, 1 mM Benzamidine, 10 μM TCEP, 1% NP-40). Probe light ABP-Bpa31 (25 μM) was mixed with the cell lysates and irradiated with UV (10 minutes) using the photocrosslinking procedure described in general methods. Samples were analyzed by 4-12% SDS-PAGE gel using MOPS running buffer (160V, 60 min), and visualized by immunoblotting with anti-Cbl (1:5000 dilution) as primary and anti-mouse (1:10000 dilution) as secondary antibody.

[0133] (Phos-tag(trademark) gel electrophoresis) To evaluate Src-mediated c-Cbl phosphorylation, we poured decomposition gels (7.5% acrylamide / bisacrylamide, 375 mM Tris-HCl pH 8.8, 0.1% sodium dodecyl sulfate (SDS), 100 μM MnCl2, 50 μM Phos-tag (trademark), 0.05% (w / v) ammonium persulfate (APS), 0.0625% (v / v) tetramethylethylenediamine (TEMED)) and stacking gels (4% acrylamide / bisacrylamide, 125 mM Tris-HCl pH 6.6, 0.1% SDS, 0.05% (w / v) APS, 0.1% (v / v) TEMED) into the gels, degassed with argon, and polymerized at room temperature for three hours. Cell extracts (50 μg) were boiled in LDS-sample buffer and supplemented with 10 mM MnCl2 before loading. Electrophoresis was performed at 70V through a concentrating gel and at 130V through a resolving gel using a running buffer (25mM Tris-HCl, 192mM Glycine, 0.1% SDS), followed by 1×20 minutes in a transfer buffer supplemented with 0.05% SDS, before staining with Coomassie dye or washing for 3×20 minutes in a transfer buffer supplemented with 10mM EDTA and 0.05% SDS to chelate manganese. Subsequently, the proteins were transferred to a 0.45 μm nitrocellulose membrane in the transfer buffer at 100V, 3 hours, and 4°C.

[0134] (Cell culture, transfection, and lysis) 293T cells were incubated in 10% (v / v) fetal bovine serum (FBS), 2.0 mM. L - Glutamine and antibiotics (100 units mL) -1 Penicillin, 0.1 mg mL -1 Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with streptomycin (37°C, 5% CO2). Cells were placed in 4 × 10⁶ 10⁶ dishes in 100 mm dishes. 6Seeds were seeded at the following density. Eighteen hours after seeding, cell transfection (2 μg DNA, empty vector (pcDNA (Thermo Fisher)), pcDNA and GST-Src, pcDNA and GFP-c-Cbl, or GST-Src and GFP-c-Cbl) was performed using 5 μL Fugene-6 (Promega) in 200 μL Eagle's Minimum Essential Medium (Opti-MEM). MG132 (25 μM) was added to the cells 90 minutes before harvest. Cells were rinsed with ice-cold PBS, collected, and extracted on ice for 30 minutes with ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 10 mM sodium 2-glycerophosphate, 50 mM sodium fluoride, 5.0 mM sodium pyrophosphate, 1.0 mM sodium orthovanadate, 0.27 M sucrose, 50 mM NaCl, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 1.0 mM benzamidine, 10 μM TCEP, 1% NP-40). The lysate was clarified by centrifugation at 4°C and 21,100 g for 20 minutes. The supernatant was collected, and the protein concentration was determined by the Bradford assay.

[0135] (Proteomics profiling based on the activity of EGF-stimulated HEK293 cells) 293T cells were placed in a 150 mM dish at a rate of 5 × 10⁶ 6 Seeds were sown at a density of 2.0 mM, and 10% (v / v) fetal bovine serum (FBS) was added. L - Glutamine and antibiotics (100 units mL) -1 Penicillin, 0.1 mg mL -1 Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with streptomycin (37°C, 5% CO2). The following day, the medium was replaced with DMEM lacking FBS. The next day, cells were treated with 20 μM MG132 and 200 nM bafilomycin for 6 hours at 37°C, followed by 100 ng mL of recombinant EGF.-1 The cells were treated at 37°C for 15 minutes with or without Thermo Fisher Scientific (PHG0311). The dishes were transferred to ice, washed, resuspended in ice-cold PBS, washed twice at 4°C, and the lysates were extracted in ice-cold buffer. 293T cells were treated with a biotinylation probe (biotin-photoABP-UbBpa31) (20 μM). The samples were divided and irradiated with UV for 1 hour or withhold UV. Biotin enrichment was then performed on streptavidin resin, followed by on-resin tryptic digestion and LC-MS / MS analysis and data processing as previously described (Pao et al., 2018, op. cit.).

[0136] (result) (Design and assembly of photocrosslinked ring ABPs) To establish potential locations for the incorporation of the photocrosslinking agent, we generated structural superpositions of the unraveled RING E3:E2~Ub cocrystal structure (Figure 5) (Dou et al., 2012b, 2013; Koliopoulos et al., 2016; Plechanovova et al., 2012, op. cit.). Remarkable conservation in binding modes was evident, and multiple residues within both Ub and E2 are located near the RING domain(s). To make the probe activity-dependent, we incorporated the photocrosslinking moiety into Ub, because this component binds only in the vicinity of active RING E3s. We selected 10 consensus sites within Ub that are close to activated RINGs (Figure 1c), and the evolved tyrosyl-tRNA synthetase-tRNA CUAThe photocrosslinked amino acid p-benzoyl L-phenylalanine (Bpa) was incorporated using pairs (Figure 1b, c) (Chin et al., 2002, op. cit.). Efficient incorporation was achieved at all sites, producing ~4-6 mg of protein per liter of culture medium. The Ub variants were purified to homogeneity and characterized by LC-MS (Figures 6 and 7). Next, all mutant Ub variants were enzymatically conjugated to promiscuous E2 UBE2D3 (Brzovic, PS et al. (2006). Mol Cell 21, 873-880), accompanied by an N-terminal hexahistidine tag (Figure 1d). In addition to facilitating purification, the latter part serves as a convenient reporter for immunoblot analysis. Conjugation to E2 is carried out by the E1 activating enzyme, and to form a more stable link between Ub and E2, the catalytic cysteine ​​is mutated to lysine, enabling stable isopeptide conjugation (Plechanovova et al., 2012, op. cit.) (Figure 2a). Importantly, structural analysis showed that the isopeptide is an acceptable structural mimetic of the native thioester (Koliopoulos et al., 2016; Plechanovova et al., 2012; Wright et al., 2016, op. cit.). Furthermore, we introduced the S22R mutation into the E2 component, which disrupts the non-covalent Ub binding site, leading to ABP self-association (Brzovic, PS, and Klevit, RE (2006). Cell Cycle, 5, 2867-2873). All E2-Ub varieties were purified to homogeneity by size exclusion chromatography so that they could be determined by SDS-PAGE and LC-MS analysis (Figures 2b-d and 8).

[0137] (Activity-dependent profiling of the dimer RING E3 RNF4) RING E3 RNF4 is inactive in monomeric form and is dominant at endogenous concentrations. Binding of polySUMO chains to SUMO Interacting Motifs (SIMs) within RNF4 increases the local concentration of RNF4, thereby enhancing homodimerization of the RING domain and activation of E3 ligase activity (Rojas-Fernandez et al., 2014, op. cit.). This leads to ubiquitination and degradation of SUMO-modified promyelocytic leukemia protein (PML) (Tatham, MH et al. (2008). Nat Cell Biol, 10, 538-546). Notably, therapeutic induction of this process leads to remission of acute promyelocytic leukemia in >90% of cases (Massaro, F., Molica, M., and Breccia, M. (2016). Int J Hematol Oncol (International Journal of Hematologic Oncology), 5, 105-118). An engineered version of RNF4 is designed to be constitutively active and consists of a full-length protein with an additional RING domain fused to the natural C-terminus via a flexible linker (RNF4-RING) (Figure 3a) (Plechanovova et al., 2011, op. cit.). To determine the optimal photocrosslinking agent position, we incubated all 10 Bpa mutant E2-Ub conjugates with RNF4-RING (Figure 9) and evaluated the crosslinking efficiency by UV irradiation (10 mins). Significant and dose-dependent crosslinking was observed only upon Bpa incorporation at position 31 (photo-ABP-UbBpa31) (Figures 3b and 10). Importantly, no crosslinking was observed when the RNF4-RING was incubated and UV irradiation was performed without conjugation of UbBpa31, indicating that photocrosslinking was dependent on E2-driven Ub approach, consistent with a natural mechanism. In particular, additional crosslinking products corresponding to the molecular weight were observed when two photo-ABP-UbBpa31 molecules were added (Figure 3b).Structural studies of the dimeric RING:E2~Ub complex revealed that both faces of the active RING dimer are involved in and activate separate E2~Ub conjugates (Figure 3c). The power of the fused RNF4-RING construct allows for the disruption of binding to a single E2~Ub molecule or both by introducing the M140A R181A double mutation into one or both RING domains (RNF4x-RING or RNF4x-RINGx, respectively) (Rojas-Fernandez et al., 2014, op. cit.). Consistent with this structurally elucidated bipartite mechanism of photo-ABP-UbBpa31 profiling, crosslinking of the second E2~UbBpa31 molecule was lost by RNF4x-RING and completely abolished by RNF4x-RINGx (Figure 3d). To further confirm activity-dependent photocrosslinking, we prepared mutant photo-ABP-UbBpa31 control probes. A portion of the conserved E2-RING interaction involves the E2 F62 residue (F63 in some model E2s), and mutations to alanine typically impair / abstain E3 binding (Weissman, AM (2001). Nat Rev Mol Cell Biol, 2, 169-178). This control probe would provide further information on whether the observed labeling is consistent with the natural E2-RING interaction and is therefore suitable for screening inhibitors that disrupt the natural interaction (Figure 3e). Inconsistent with the probe fitting in its natural manner, the photo-ABP-UbBpa31 F62A probe did not undergo RING crosslinking. This should also serve as a valuable control probe when agnostically profiling RING E3s (Figure 3f).

[0138] (ABP profiling of polySUMO chain-induced RNF4 activation) Cellular RNF4 is activated by supplementation of polySUMO chains via its SIM domain, thereby inducing dimerization. K of dimerization dThe saturation is ~180 Nm (Rojas-Fernandez et al., 2014, op. cit.), and therefore, by acting at concentrations below this value, we established a biochemical assay to evaluate whether photo-ABP-UbBpa31 could profile the intrinsic polySUMO chain-induced activation of RNF4 (Figure 3g). As expected, constitutively active RNF4-RING was insensitive to dilution and underwent crosslinking with photo-ABP-UbBpa31, while intrinsic RNF4 was not (Figure 3h). However, in the presence of linear amide-linked tetraSUMO (SUMOx4) fusion protein (10 μM), it recapitulates the intrinsic isopeptide-linked polySUMO chain activation properties (Tatham et al., 2008, op. cit.), and photo-ABP-UbBpa31 crosslinking was observed with efficiency comparable to that of RNF4-RING. Insightfully, the crosslinked band was observed upon the addition of a second photo-ABP-UbBpa31 molecule (Figure 3g). This suggests that spontaneously activated wild-type RNF4 retains its bipartite activity, and its associated processivity is putatively utilized in the cell. Taken together, the data so far demonstrate that photo-ABP-UbBpa31 undergoes activity-dependent crosslinking of spontaneously activated RING E3, which lacks a nucleophile for catalytic activity.

[0139] (Activity-dependent profiling of phosphorylation-induced RING E3 activation) Photo-ABP-UbBpa31 is tested by RING E3, which is activated via a distinct mechanism. The Cbl protein is a multi-domain and multifunctional RING E3 ligase consisting of three homologs: c-Cbl, Cbl-b, and Cbl-c (Lyle, CL, Belghasem, M., and Chitalia, VC (2019). Cells, 8). Most Cbl function is related to RING E3 activity and is involved in regulating angiogenesis. Abnormalities in Cbl activity have been associated with numerous cancers. Cbl is overexpressed in many thoracic cancer cells and tissues, and is also found to be downregulated in myeloproliferative neoplasms (MDS / MPNs) and non-small cell lung cancer (Kales, SC et al., (2010). Cancer Research, 70, 4789-4794; Tan, YH et al., (2010). PLoS One, 5, e8972). Therefore, regulating Cbl E3 activity is an attractive therapeutic strategy and has attracted considerable interest from pharmaceutical companies. The most common mutation seen clinically is at residue Y371. Y371 is phosphorylated by c-Src kinase, which leads to a structural change that presents a non-RING element, increasing affinity for E2~Ub and stimulating Cbl E3 activity (Butow et al., 2016; Dou et al., 2013, op. cit.). In fact, the affinity for Cbl phosphorylated at Y371 (c-Cbl pTyr371) increases the affinity for E2-Ub by approximately 30 times (Butow et al., 2016, op. cit.).

[0140] To evaluate whether photo-ABP-UbBpa31 can profile Src-dependent activation of c-Cbl E3 activity, we incubated recombinant Src with Cbl and Cbl Y371F, with the latter expected to be insensitive to phosphorylation-induced activation. Photo-ABP-UbBpa31 crosslinking was observed for c-Cbl in the presence of c-Src, but not for c-Cbl Y371F, nor was it observed in the absence of c-Src (Figure 4a). Labeling was again abolished by the photo-ABP-UbBpa31 F62A control probe (Figure 11) and was also ATP-dependent (Figure 12). Therefore, consistent with previous studies, phosphorylation at Y371 is clearly required for the activation of E3 activity (Dou et al., 2013, op. cit.). We also tested a panel of engineered E2-Ub conjugates with Bpa integration at different positions relative to c-Src activated c-Cbl and found some with partial overlap with the productive sites for RNF4 (Figure 13). The optimal Bpa position was 31, but unlike RNF4, crosslinking at position 32 (photo-ABP-UbBpa31) also occurred with similar efficiency. This is likely due to a reflection of the nuances between monomeric and dimeric activation mechanisms shown by these two RING E3s (Dou et al., 2013; Plechanovova et al., 2012, op. cit.). Interestingly, the photocrosslinking efficiency remained quasi-stoichiometric regardless of the concentration of photo-ABP-UbBpa31, suggesting the activation of a subpopulation of recombinant protein preparations (Figure 14). Phostag gel analysis indicated quantitative phosphorylation of Cbl (Figure 15), although Src is known to phosphorylate multiple sites within Cbl, and the degree of probe labeling may reflect quasi-stoichiometric phosphorylation at position Y371 (Dou, H. et al. (2012a). Nat Struct Mol Biol, 19, 184-192). We tested whether photocrosslinking efficiency improved by incubation with increased concentrations of Src, but found that photocrosslinking was suppressed as the concentration approached stoichiometric levels; presumably because Src competes with photo-ABP-UbBpa31 for Cbl binding (data not shown).

[0141] (Profiling of c-Cbl activation in human cell lines) To establish whether c-Cbl activation in human cell lines could be profiled, we transiently transfected human embryonic kidney cells (HEK293) with GST-tagged c-Src (GST-Src) along with GFP-tagged c-Cbl (GFP-Cbl) or GFP-tagged c-Cbl Y371F (GFP-Cbl Y371F). To prevent potential degradation of activated Cbl by autoubiquitination, we treated the cells with the proteasome inhibitor MG132 for 90 minutes prior to lysis. Activity-dependent crosslinking was strictly dependent on Src co-expression and the presence of Y371, but was not observed with the photo-E2~UbBpa31 F62A control probe (Figures 4b and 16). To give potential for future use in parallelized proteomic profiling of endogenous RING E3 ligases, we prepared biotinylated mutants of photo-ABP-UbBpa31. Bpa was incorporated into an N-terminally cysteine-tagged Ub and labeled with idodoacetyl-PEG2-biotin. Biotinylated UbBpa31 was then enzymatically conjugated with E2 via an isopeptide using the procedure for untagged Ub (Figure 17).

[0142] (Profiling endogenous RING E3 activation in response to growth factor stimulation) We then evaluated our ability to perform parallel profiling of endogenous RING E3 activation in response to physiological stimuli. Such experiments may allow us to attribute poorly understood RING E3s to regulatory functions across both physiological and pathophysiological processes. Deletion of ABP due to disordered crosslinking may reduce the coverage of RING E3s, which we initially tested by immunoblotting against a hexahistidine reporter tag (Figure 4c). Furthermore, crosslinking was substantially reduced by the photo-ABP-UbBpa31 F62A control probe, thereby suggesting that many of the crosslinked proteins are likely E3s (Figure 4c).

[0143] We prepared a biotinylated variant of photoABP-UbBpa31 to allow selective enrichment of cross-linking proteins from complex cell samples. Bpa was incorporated into N-terminally cysteine-tagged Ub and labeled with iodoacetyl-PEG2-biotin (Pao et al., 2018, op. cit.). Biotin-labeled UbBpa31 was then enzymatically conjugated to E2 via isopeptides using the procedure for untagged Ub (Figures 17 and 18). Next, we tested whether endogenous Cbl activation could be detected in response to EGF stimulation, which induces Cbl phosphorylation (Levkowitz et al., 1999; Levkowitz et al., 1998). HEK293T cells were stimulated with EGF, and to prevent potential degradation of activated RING E3s, we pre-treated them with the proteasome and lysosomal inhibitors MG132 and bafilomycin, respectively. In parallel experiments, EGF responsiveness was confirmed by immunoblotting of downstream mitogen-activated protein (MAP) kinase activation, which is a robust marker of EGF receptor activation (Traverse, S. et al., (1992). Biochem. J. (Biochemical Journal), 288(Pt 2), 351-355, Figure 19a).

[0144] The extracted proteome was incubated with biotinylated photo-ABP-UbBpa31 and concentrated against streptavidin resin (Pao et al., 2018, op. cit., Figure 4d). Identification of crosslinked proteins and their probe reactivity were inferred by data-dependent liquid chromatography-tandem mass spectrometry (LC-MS / MS) and spectral counting following streptavidin concentration (Pao et al., 2018, op. cit.). Twenty-five RING E3s were detected, including Cbl. The Cbl peptide was detected only in EGF- and UV-treated samples (Figure 4e). This suggests that the photocrosslinking probe can detect innate RING E3 activation at endogenous levels. Interestingly, there was a significant increase in spectral counts for two other RING E3s, Praja2 and TRIM11, which was EGF- and UV-dependent (Figure 4e). Since both of these E3s were involved in growth factor signaling, their detection may also reflect their activation or upregulation in response to EGF stimulation (Di, K. et al., Oncogene, 32, 5038-5047; Rinaldi, L. et al. (2016). Cell Death Dis. 7, e2230.).

[0145] We unexpectedly obtained UV-dependent enrichment of HECT(11), RBR(1), and RCR(1)E3s, as well as deubiquitinating enzymes (DUBs)(31) and E1 activating enzymes (Figures 19a and 19b). As a result, probe modifications of these additional ubiquitin system components were able to modulate their activity and alter the activation state, or stability, of the RING E3s under study. However, this does not seem likely to present any limitations beyond those associated with the adoption of cell extracts, where the majority of cellular processes would be halted.

[0146] (Production of photobridged ABP based on alternative E2) Furthermore, ABP based on a different E2 enzyme was synthesized. The enzyme UBE2N (also known as Ubc13) was used. The same strategy as above was employed to conjugate the Bpa31 mutant Ub molecule to the active site. The cysteine ​​(Cys87) related to the catalyst was mutated to lysine, enabling stable E1-mediated isopeptide conjugation. It is noteworthy that in other studies using wild-type ubiquitin, conjugation to a natural lysine residue (Lys92) adjacent to the active site was observed (Branigan et al., Nat Struct Mol Biol 22, 597-602). Thus, to ensure homogeneous modification, the UBE2N C87K K92A double mutant was adopted, as exemplified in the study by Branigan et al. The UBE2N probe functioned with the E3 ligase (TRAF6), which is known to be the physiological partner of the UBE2N E2 enzyme (Figure 20). The UBE2N-based probe was shown to allow for biotinylation and preparation of the ubiquitin molecule (Ubc13-biotin-UbBpa3), as described above for UBE2D3, and enables activity-based proteomic profiling of RING E3 ligase in cell extracts.

[0147] (Consideration) In short, we developed activity-based probes for the adapter-like activity of RING E3 ligases. We demonstrate how activity-dependent signaling and ABP-based readout in response to their intrinsic activation cues for RNF4, c-Cbl, and TRAF6 can provide further mechanistic insights. These tools enable direct assessment of RING E3 activity (E1, E2, or substrate-independent) in a variety of sample types. We also demonstrate parallel profiling of subsets of endogenous RING E3s in extracted proteomes and detect Cbl activation in response to growth factor stimulation. As such, this technique should prove useful in research on RING E3 regulatory biology, target discovery, biomarker applications, and modulator discovery. The detection of only a subset of RING E3s in our LC-MS / MS experiments may reflect that many are inactive or exceed the detection limits of our current experimental conditions. Another possibility is that many E3s do not function with the E2 enzymes they utilize (photo-ABP-UbBpa31 is based on (UBE2D3)). However, engineered isopeptide conjugation strategies have demonstrated the stabilization of unstable thioesters with E2s branching off from UBE2D3, e.g., UBE2N (Branigan et al., 2015; Ordureau et al., 2015). Therefore, our advanced modular probe production strategy should be easily applicable to other E2s simply by using distinct recombinant E2 building blocks. This would potentially provide a broader RING E3 applicability and also offer insights into the cellular E2-E3 interaction network.

Claims

1. A conjugate molecule comprising an activated ubiquitin molecule conjugated to an E2 conjugating enzyme capable of binding to the RING E3 enzyme, wherein the activated ubiquitin molecule comprises a photocrosslinking agent Moiety instead of the glutamine residue at position 31 and / or the aspartic acid residue at position 32 of the ubiquitin molecule of SEQ ID NO:

1.

2. The conjugate molecule according to claim 1, wherein the ubiquitin is human ubiquitin.

3. The photocrosslinking agent Moieti is a conjugate molecule according to claim 1 or 2, derived from a modified native amino acid.

4. The conjugate molecule according to claim 3, wherein the modified native amino acid is p-benzoyl-L-phenylalanine.

5. The activated ubiquitin molecule further comprises a reporter tag, as described in any one of claims 1 to 4.

6. Reporter tags are: (i) Biotin Moyeti, or other reporters that enable analytical detection: or (ii) Epitope tag or fluorophore The conjugate molecule according to claim 5.

7. The conjugate molecule according to claim 5 or 6, wherein the biotinmoy / reporter tag is covalently attached to the ubiquitin molecule through a linker molecule attached to the N-terminus of ubiquitin.

8. The conjugate molecule according to any one of claims 1 to 7, wherein the E2 conjugating enzyme is a mutant enzyme containing a C85K or C87K mutation and / or an S22R or K92A mutation.

9. The conjugate molecule according to any one of claims 1 to 8, wherein the E2 conjugating enzyme is UBE2D3 or UBE2N.

10. below: (i) N-terminal His-tag group: or (ii) Hexahistidine tag A conjugate molecule according to any one of claims 1 to 9, further comprising:

11. A method for activity profiling of the RING E3 enzyme, comprising the use of a conjugate molecule according to any one of claims 1 to 10, wherein the activated ubiquitin molecule of the conjugate comprises a photocrosslinking agent Moiety in place of the glutamine residue at position 31.

12. A method for activity profiling of the RING E3 enzyme, comprising the use of a conjugate molecule according to any one of claims 1 to 10, wherein the activated ubiquitin molecule of the conjugate comprises a photocrosslinking agent Moiety in place of the aspartic acid residue at position 32.

13. Uses according to claim 11 for (i) studying RING E3 enzyme regulation, (ii) discovering novel RING E3 enzymes, (iii) inhibitor screening, (iv) inhibitor-selective profiling; and / or (v) stabilizing enzyme intermediates for structural studies.

14. A method for detecting an interaction between a conjugate molecule and a RING E3 enzyme according to any one of claims 1 to 10, comprising contacting the conjugate molecule with the RING E3 enzyme, wherein the activated ubiquitin molecule of the conjugate comprises a photocrosslinking agent Moiety in place of a glutamine residue at position 31, and detecting the formation of any new conjugate.

15. A method for detecting an interaction between a conjugate molecule and a RING E3 enzyme according to any one of claims 1 to 10, comprising contacting the conjugate molecule with the RING E3 enzyme, wherein the activated ubiquitin molecule of the conjugate comprises a photocrosslinking agent Moiety in place of an aspartic acid residue at position 32, and detecting the formation of any new conjugate.

16. The method according to claim 14, wherein the RING E3 enzyme is any one or a combination of RNF4, Cbl, Praja2, TRIM11, TRAF6, TRAF2, and HLTF.

17. The method according to claim 14 or 15, wherein the interaction is between the conjugate molecule and the c-CblRING E3 enzyme.

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