Active-based probe

Abstract

The present invention relates to the development of a novel photocrosslinking-based probe (ABP) and its use. Specifically, a ubiquitin-filled E2 conjugating enzyme was engineered and found to be an effective ABP for RING ubiquitin E1 and E3 ligases as well as deubiquitination enzymes.

Description

Technology Field

[0001] The present invention relates to the development of a novel photocrosslinking-based probe (ABP) and its use. Specifically, a ubiquitin-filled E2 conjugating enzyme was engineered and found to be an effective ABP for RING ubiquitin E1 and E3 ligases as well as deubiquitination enzymes. Background Technology

[0002] Activity-based protein profiling is a highly useful technique for promoting enzyme biology research and therapeutic development. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (pathological)physiological processes. The largest subtype is RING E3, which has over 600 members. RING E3 possesses adapter-like activity that can be targeted by various regulatory mechanisms and has become an attractive drug target. To the best of the knowledge, no activity-based probe (ABP) exists to measure RING E3 activity.

[0003] Ubiquitination is a fundamental post-translational modification that regulates normal cell physiology, and dysfunction of it can lead to the onset of disease (Rape, M. (2018). Nat Rev Mol Cell Biol 19, 59-70). Ubiquitination is carried out by an enzyme cascade comprising the sequential activity of ubiquitin E1 activation (E1), ubiquitin E2 conjugation (E2), and ubiquitin E3 ligase (E3) (Hershko, A. and Ciechanover, A. (1998). Annu Rev Biochem67, 425-479.). Ubiquitin (Ub) is covalently transferred from the catalytic cysteine ​​of E1 to the catalytic cysteine ​​of E2 to form a thioester-linked E2 intermediate (E2–Ub). It is known that hundreds of E3s exist to recruit E2–Ub and ubiquitine specific substrates. The divergence of the E3 mechanism is divided into two general classes. Among them, "Cys E3s," which number about 50, utilize catalytic cysteine ​​to form a covalent thioester-linked intermediate with Ub prior to substrate modification (KC Pao et al. Nature , 2018, 556, 381-385; Scheffner, M., Nuber, U., & Huibregtse, J. M. (1995). Nature 373, 81-83; Wenzel, DM et al. (2011), Nature 474, 105-108).

[0004] However, the largest class of E3s is the adapter-like E3, which has over 600 distinct forms (Deshaies, RJ and Joazeiro, CA (2009). Annu Rev Biochem Adapter-like E3s lack catalytic nucleophiles and catalyze the direct transfer of Ub from E2 to Ub to the substrate. This adapter-like activity is utilized by the multi-subunit Cullin-RING E3 and about 350 single polypeptides RING E3 (hereinafter simply referred to as RING E3). The latter may exist as monomers, homodimers, or heterodimers (Metzger, MB et al. (2014). Biochim Biophys Acta 1843, 47-60). Activity regulation is a particularly important aspect of E3 biology for ensuring cellular homeostasis and adaptive signaling. Since dysregulation can lead to disease development, RING E3 has become an attractive therapeutic target (Burgess, A. et al. (2016). Front OncolHowever, the cellular roles and regulatory mechanisms of most RING E3s are not well understood. Additionally, RING E3s have recently been found to be compatible with targeted protein degradation strategies (e.g., PROTAC methodologies) (Naito, M., Ohoka, N., and Shibata, N. (2019). Drug Discov Today Technol , 31, 35-42; Spradlin, JN et al. (2019). Nat Chem Biol 15, 747-755; Ward, CC et al. (2019). ACS Chem Biol.). To further utilize this potential, a tool is needed to determine which RING E3 is active in a clinical setting.

[0005] A characteristic of adapter-like E3 is that when active, it shifts the dynamic E2–Ub structural ensemble into distinct groups in which the E2–Ub junction folds back or adopts a “closed” structure (Dou, H. et al. (2012b). Nat Struct Mol Biol , 19, 876-883; Plechanovova, A. et al. (2012). Nature 489, 115-120.; Pruneda, JN et al. (2012). Mol Cell , 47, 933-942; Pruneda, JN et al. (2011). Biochemistry 50, 1624-1633; see Fig. 1a). This structure activates thioester bonds within E2–Ub for nucleophilic attack and is necessary for efficient aminolysis activity. RING E3 activity can be regulated, and transition to an activated state is achieved by E3 binding to the Ub component to acquire structural features that promote the induction of a closed structure. For example, RING E3s such as RNF4 and BIRC7 are activated by RING domain dimerization in which the tail region of the second RING protomer binds to the Ub component (Dou, H. et al. 2012b; Plechanovova, A. et al. 2012, see above). Dimerization can be regulated by cellular signaling, and in the case of RNF4, this occurs by binding to the poly-SUMO chain (Rojas-Fernandez, A. et al. (2014). Mol Cell, 53, 880-892).

[0006] For the activation of monomer RING E3, so-called non-RING elements were shown to play a role in binding Ub components, and in the case of Cbl-b and c-Cbl, this is a phosphorylated tyrosine residue (Dou, H. et al. (2013). Nat Struct Mol Biol, 20, 982-986). Phosphorylation is performed by kinase c-Src in response to growth factor stimulation, and Cbl activation induces ubiquitination and degradation of receptor and non-receptor tyrosine kinases. RING E3, which requires dimerization and the presence of non-RING elements, has also been reported (Koliopoulos, MG et al. (2016). EMBO J, 35, 1204-1218). Additional RING E3 activation mechanisms exist, including allosteric binding of cofactors or ligands (DaRosa, PA et al. (2015). Nature , 517, 223-226; Dickson, C. et al. (2018). Elife , 7; Duda, DM et al. (2012). Mol Cell , 47, 371-382; Wright, J.D., Mace, P.D., and Day, C.L. (2016). Nat Struct Mol Biol , 23, 45-52). Numerous crystal structures of E2–Ub coupled to the activation ring E3, elucidating highly conserved binding modes, have been interpreted (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, see citation above). Importantly, the matching region of the Ub component in the closed E2–Ub conjugate is close to the active ring. Furthermore, biophysical analysis demonstrates that the active ring E3 studied so far may have higher binding free energies for E2–Ub than the inactive form (Berndsen, CE et al. (2013). Nat Chem Biol . 9, 154-156.; Buetow, L. et al. (2016). BMC Biol . 14, 76).

[0007] Activity-based probes (ABPs) are powerful chemical tools for performing activity-dependent covalent labeling of enzyme family members (Hewings, DS et al. (2017). FEBS J ., 284, 1555-1576; Niphakis, MJ, and Cravatt, BF (2014). Annu Rev Biochem ., 83, 341-377). This enables the stabilization of enzyme intermediates for (i) enzyme regulation studies, (ii) discovery of new enzyme classes, (iii) inhibitor screening, (iv) inhibitor selectivity profiling, and (v) structural studies (Hu, M. et al. (2002). Cell, 111, 1041-1054). Our research team and other researchers developed an ABP for Cys E3 distributed to analyze the E3 activation mechanism and discover a completely new class of E3s (Love, KR et al. (2009). 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. (Camb). , 55, 7109-7112).

[0008] To the best of the inventor's knowledge, there is currently no ABP for evaluating RING E3 ligase activity. The present invention aims to solve this by providing an ABP for evaluating RING E3 ligase.

[0009] Without being bound by theory, the inventors believe that the conserved (and activity-dependent) matching interactions of the Ub components within the closed E2~Ub, combined with the enhanced free energy of the binding to the active RING E3, can be utilized for the development of ABP.

[0010] An ABP based on a engineered ubiquitin molecule is disclosed herein. An activated ubiquitin molecule containing a photocrosslinking agent moiety in place of the glutamine residue at position 31 and / or the aspartate residue at position 32 of the ubiquitin was found to be surprisingly effective as an ABP when conjugated to an E2 conjugation enzyme. Such ABPs are effective for profiling activity of RING E3 ligase in various sample types and may be useful for studies, for example, RING E3 regulation, target discovery, biomarker application, regulatory discovery, and structural studies.

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

[0012] In a second embodiment, the present invention provides a conjugated molecule comprising an activating molecule of the first embodiment conjugated to an E2 conjugated enzyme.

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

[0014] In a fourth embodiment, the present invention provides a method for detecting interactions between a conjugate molecule of the second embodiment and a RING E3 enzyme, a RING E1 enzyme, and / or a deubiquitination enzyme, the method comprising the steps of contacting the conjugate molecule of the second embodiment with a RING E3 enzyme, a RING E1 enzyme, and / or a deubiquitination enzyme, and detecting the formation of any new conjugate. For example, by adding a reporter group capable of enriching to the conjugate, such as biotin, the activated RING E3 can be enriched in 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 all embodiments of said aspect. For example, any reference to a first aspect of the present invention includes the first aspect and all embodiments of the first aspect. Brief explanation of the drawing

[0016] Fig. 1: Strategy and synthetic reaction scheme for the generation of photocrosslinked ABP for RING E3 ligase. a) Binding of the activated RING E3 induces a closed structure of the otherwise structurally dynamic E2–Ub conjugate. Activation can be achieved by RING dimerization, whereas monomeric RING E3 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) The appropriate incorporation of p-benzoyl-L-phenylalanine (Bpa) crosslinking amino acid into the stabilized E2–Ub conjugate acts as an ABP for RING E3 activity. c) Crystal structure of E2–Ub in a complex with the activated dimeric RING E3 (RNF4). Ten amino acid sites within the Ub adjacent to the activated E3 were tested for Bpa incorporation. Q31 was found to be optimal. d) Synthetic reaction scheme for photocrosslinked ABP. Fig. 2: Assembly and characterization of photoABP and biotin-tagged photoABP-UbBpa31 probes. a) SDS-PAGE analysis of a representative enzymatic conjugation of E2 of UbBpa31 (UBE2D3 C85K S22K double mutant) using ubiquitin E1 activator. Asterisks correspond to the estimated diubiquitin species. b) Representative purified fraction of the probe product after size exclusion chromatography (SEC). c) RP-HPLC chromatogram for purified photoABP-UbBpa31. d) Resolved mass spectrum for photoABP-UbBpa31. Observed mass = 27901 Da, Observed mass (-Met) = 27771 Da. Predicted mass = 27908.87 Da, Predicted mass (-Met) = 2777.67 Da. Fig. 3: Activity-dependent profiling of RNF4 E3 ligase activity. a) Constitutively active RNF-RING fusion protein can productively bind to two E2–Ub conjugates. b) Probe photoABP-UbBpa31 (40 μM) undergoes two cross-linking reactions with RNF4-RING (10 μM). c) Binding of one or both E2–Ub conjugates can be interrupted by the M140A R181A double mutation introduced into one or both of the RING domains of RNF-RING. d) Probe photoABP-UbBpa31 (40 μM) cross-linking is attenuated or abolished depending on whether one or both of the RING domains are mutated. e) Introduction of the F62A mutation into the E2 component must eliminate E3 binding. f) Cross-linking is abolished with photoABP-UbBpa31 F62A (probe concentration 5 μM). g) For dimerization K d At concentrations below this level, RNF4 is inactivated. The binding of the poly-SUMO chain induces dimerization and E3 ligase activity. h) photoABP-UbBpa31 (5 μM) proceeds with poly-SUMO chain (10 μM)-dependent crosslinking of natural RNF4 (100 nM), whereas RINF4-RING (50 nM) crosslinks independently of the poly-SUMO chain. Fig. 4: Activity-dependent profiling of c-Cbl E3 ligase activity and activity-based proteomic analysis of EGF-stimulated versus unstimulated HEK293T cells. a) Only c-Cbl (3 μM) pre-cultured with c-Src (1.5 μM) underwent photoABP Bpa31 (5 μM) cross-linking. No cross-linking was observed when Cbl Y371 (3 μM) (which cannot be phosphorylated at the active site) was cultured with Src. Phosphorylation of Cbl reduced electrophoretic mobility. b) Transient overexpression of GFP-Cbl and c-Src in mammalian HEK293 cells. Extracts were treated with photoABP31 Bpa31 or F62A control probes (5 μM). IB represents the immunoblot, and the primary antibody used for detection is adjacent to it (i.e., anti-Cbl). c) Immunoblot analysis of HEK293T extracts using photoABP-UbBpa31 or photoABP-Bpa31 F62A control probe (10 μM). Blotting was performed for hexahistidine reporter tags present in ABP. Samples were irradiated or withheld for 60 minutes. d) Schematic diagram illustrating the activity-based proteomic workflow using biotinylated photoABP-Bpa31. e) Spectral counts obtained from ABP-profiled HEK293T cells. Search results were filtered for the PFAM domain term "RING," and only RING E3s with more than two spectral counts in all replication experiments were plotted. Cells were serum-deprived, treated with or without EGF, and with or without UV irradiation. Error bars correspond to the standard error of the two technical replication LC-MS / MS analyses. Fig. 5: Structural overlap of the activated RING:E2~Ub complex. The activated RING E3 binds to E2~Ub and induces a "closed" E2~Ub structure that activates the thioester for aminolysis (NB, in the presented crystal structure, the unstable thioester was replaced by serine or lysine via ester or isopeptide bonds, respectively, due to mutations in the E2 catalyst). The closed structure is induced by binding the RING to the complex E2~Ub interface. Of particular importance is that, in the case of RNF4 and BIRC7, the Ub component is maintained in the closed structure by interaction with RING protomer 2. In the case of Cbl-b, the phosphotyrosine moiety (pTyr) acts as a non-RING element and promotes the closed structure. In the case of TRIM25, dimerization and the presence of non-RING glutamate (Glu) residues promote the closed structure. Fig. 6: Incorporation of the ring-adjacent site of ubiquitin of the photocrosslinked amino acid p-benzoyl-L-phenylalanine (Bpa). SDS-PAGE analysis and visualization by Coomassie staining. As a positive control, the reference amino acid t-butyloxycarbonyl-L-lysine (BocK) was incorporated into Ub at position 6. For easy purification, a C-terminal His tag was added to Ub, which was subsequently removed by treatment with the deubiquitase UCH-L3. For each mutation, the results before and after UCH-L3 treatment were analyzed. Fig. 7: Electron spray ionization mass spectra for ubiquitin Bpa mutants. The spectra correspond to Ub after cleavage of the C-terminal His-tag. Except for the amber mutant clone for position 6, Ub is expressed as an N-terminal MGS motif. The introduction of the DNA coding sequence for this facilitated cloning. In the case of these latter clones, N-terminal methionine is cleaved to varying degrees by cellular methionyl aminopeptidase. a) UbBpa6, observed mass = 8686 Da; predicted mass = 8787.95 Da. b) UbBpa9 (-Met), observed mass = 8857 Da; predicted mass = 8859.15 Da. UbBpa9, observed mass = 8988 Da; predicted mass = 8990.35 Da. c) UbBpa11 (-Met), observed mass = 8830 Da; Predicted mass = 8832.08 Da. UbBpa11, Observed mass = 8961 Da; Predicted mass = 8963.28 Da. d) UbBpa13 (-Met), Observed mass = 8845 Da; Predicted mass = 8847.1 Da. e) UbBpa14 (-Met), Observed mass = 8857 Da; Predicted mass = 8859.15 Da. UbBpa14, Observed mass = 8988 Da; Predicted mass = 8990.35 Da. f) UbBpa31 (-Met), Observed mass = 8830 Da; Predicted mass = 8832.12 Da. UbBpa31, Observed mass = 8961 Da; Predicted mass = 8963.32 Da. g) UbBpa32 (-Met), Observed mass = 8845 Da; Predicted mass = 8845.17 Da. UbBpa32, observed mass = 8976 Da; predicted mass = 8976.37 Da. h) UbBpa34 (-Met), observed mass = 8830 Da; predicted mass = 8831.14 Da. UbBpa34, observed mass = 8961 Da; predicted mass = 8962.34 Da. i) UbBpa40 (-Met), observed mass = 8831 Da; predicted mass = 8832.12 Da.UbBpa40, observed mass = 8962 Da; predicted mass = 8963.32 Da. j) UbBpa64 (-Met), observed mass = 8830 Da; predicted mass = 8831.14 Da. UbBpa64, observed mass = 8961 Da; predicted mass = 8962.34 Da. k) UbBpa64 (-Met), observed mass = 8803 Da; predicted mass = 8804.07 Da. The peak observed at 8836 Da corresponds to an unidentified adduct. Fig. 8: Representative enzymatic conjugation of UbBpa31 to E2 via stabilized isopeptide binding to provide photoABP-UbBpa31. SDS-PAGE analysis and visualization by Coomassie staining (top). Probe product purified by size exclusion chromatography (bottom). Fig. 9: Representative LC-MS analysis for photoABP-Bpa31. HPLC chromatogram measured at 214 nm. PhotoABP Bpa31 (-Met), observed mass = 27769 Da; predicted mass = 27777.67 Da. PhotoABP Bpa31 (-Met), observed mass = 27901 Da; predicted mass = 27908.87 Da. Fig. 10: Evaluation of RNF4-RING photocrosslinking efficiency for the incorporation of Bpa at 10 different Ub positions within an isopeptide-linked E2–Ub conjugate. Significant incorporation was achieved only when Bpa was incorporated at position 31, which provides the probe photoABP-UbBpa31. The reference amino acid t-butyloxycarbonyl-L-lysine (BocK) and Bpa were incorporated at position 6, as this site allows for the incorporation of non-natural amino acids and was used as a control. Fig. 11: Dose-responsive photocrosslinking of RNF4-RING. The photocrosslinking efficiency of RNF4-RING responds to an increase in the concentration of photoABP-UbBpa31. Fig. 12: Photocrosslinking of phosphorylated c-Cbl with photoABP-UbBpa31 and photoABP-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 probe (5 μM). Probe photoABP-Bpa31 proceeds with c-Cbl crosslinking, but photoABP-Bpa31F62A does not. Fig. 13: Comparison of photocrosslinking efficiency for Bpa incorporation at various locations within ubiquitin. c-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 3 μM) prior to probe analysis. Functional probes were provided by incorporating Bpa only at locations 31 and 32. Fig. 14: Dose-response analysis of Cbl crosslinking. C-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 μM) prior to probe analysis. When the photoABP-UbBpa31 concentration was increased to 5 μM or higher, no increase in Cbl labeling efficiency was observed. Fig. 15: Phostag SDS-PAGE analysis of c-Src-treated c-Cbl. Reduced electrophoretic mobility of c-Cbl was observed only in the presence of ATP; since gel migration is quantitative, this indicates that phosphorylation must be highly quantitative. However, the crosslinking efficiency using photoABP-UbBpa31 did not show a dose-response at concentrations exceeding 5 μM. This is consistent with the fact that c-Cbl is phosphorylated at multiple sites but stoichiometrically at Y371. Fig. 16: Photocrosslinking of cellular Cbl is strictly dependent on the presence of Y371. C-Cbl (3 μM) was phosphorylated by incubation with c-Src (1.5 μM) prior to probe analysis. When the photoABP-UbBpa31 concentration was increased above 5 μM, no increase in Cbl labeling efficiency was observed. This may be due to stoichiometric phosphorylation at Y371. Fig. 17: Biotin labeling of Cys-tagged UbBpa31 and enzymatic conjugation to E2. LC-MS characterization of UbBpa31 represented by the N-terminal MGCSSG labeling motif (Observed mass = 8947 Da; Predicted mass = 8948.2 Da) (Top left). The cysteine ​​labeling 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; Predicted mass = 9362.72 Da) (Top right). Refolded biotin-tagged UbBpa31 was enzymatically conjugated to E2, and the probe was purified by size exclusion chromatography (Bottom). Fig. 18: LC-MS analysis of biotin-photoABP-Bpa31. HPLC chromatogram measured at 214 nm. photoABP Bpa31(-Met), observed mass = 28301 Da; predicted mass = 28308.27 Da. Fig. 19: Confirmation of receptor tyrosine kinase activation and evaluation of non-RING E3 photocrosslinking. a) EGF-dependent receptor activation was confirmed by immunoblotting for ERK1 / 2 phosphorylation. HEK293T cells were serum-depleted and stimulated with recombinant EGF. Cells were treated with the proteasome inhibitor MG132 prior to stimulation. Note: For proteomic experiments, cells were treated with MG132 and valpyromycin. b) Detection of other ubiquitin system components by activity-based proteomics using biotinylated photoABP-UbBpa31. Spectral counts obtained from ABP-profiled HEK293T cells. Search results were filtered for the PFAM domain terms "HECT, IBR, and zf-UBR," and only RING E3 with more than 2 spectral counts in all replication experiments was plotted. Cells were serum-deficient, treated with or without EGF, and with or without UV irradiation. Error bars correspond to the standard error of two technical replications. c) As previously described, but DUB was filtered using a combination of PFAM domain terms and manual curation. Fig. 20: Confirmation of the covalent labeling of its homolog E3 TRAF6 in a UV-dependent manner by the UBE2N / Ubc13 photocrosslinking-active-based probe. Experiments were also performed in the presence of the UBE2N substrate receptor (Ube2V2). Its presence does not appear to affect activity toward TRAF6. Specific details for implementing the invention

[0017] An ABP based on a engineered E2-Ub conjugate (Plechanovova, A. et al., 2012, see above) in which an unstable cysteine ​​thioester is replaced with a more stable linkage chemistry such as lysine isopeptide is disclosed herein.

[0018] The activating ubiquitin molecule of the present invention is surprisingly effective as an ABP when conjugated to an E2 conjugating enzyme. The ABP of the present invention is effective for activity profiling of ring E3 enzymes, ring E1 enzymes, and / or deubiquitination enzymes in various sample types. The activating ubiquitin molecule, conjugate, uses, and methods of the present invention are now described in detail.

[0019] In the following discussion, references to a number of terms having the meanings provided below are provided, unless the context indicates otherwise. The nomenclature used herein to define compounds, particularly 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 “including” or variations thereof should be understood to mean the inclusion of the mentioned element, integer or step, or group of element, integer or step, but not the exclusion of any other element, integer or step, or group of element, integer or step.

[0021] The term “consisting of” or variations thereof implies the inclusion of the mentioned element, integer, or step, or group of element, integer, or step, and should be understood as excluding any other element, integer, or step, or group of element, integer, or step.

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

[0023] The term "heteroaryl" defines an aryl 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 characteristics of the aromatic system.

[0024] The term "alkyl" is well known in the art and defines a monovalent group derived from an alkane by removing a hydrogen atom from any carbon atom, wherein the term "alkane" is C n H 2n+2 It is intended to define cyclic or non-cyclic branched or unbranched hydrocarbons having the chemical formula (n is an integer greater than or equal to 1).

[0025] The term "hydrocarbyl" is well known in the art and defines all monovalent groups formed by removing a hydrogen atom from a hydrocarbon. The term "hydrocarbon" is well known in the art and, as used herein, refers to all aliphatic and aromatic compounds composed solely of carbon and hydrogen, including branched, unbranched, acyclic, and cyclic alkanes, alkenes, and alkynes. For clarity, cycloalkanes and cycloalkenes are included within the scope of the definition of hydrocarbon as used herein.

[0026] The term "halo" is well known in the art and defines a halogen radical that forms fluoride, chloride, bromide, or iodide compounds when bonded to a carbon radical.

[0027] The activated ubiquitin molecule of the present invention comprises a photocrosslinking agent moiety in place of the glutamine residue at position 31 and / or the aspartate residue at position 32 of ubiquitin. The ubiquitin consists of 76 amino acids having the following sequence (SEQ No. 1):

[0028] MQIFVKTLTGKTITLEVEPSDTIENVKAKI QD KEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG

[0029] Replacing the glutamine residue at position 31 and / or the aspartate residue at position 32 (underlined in bold in the sequence above) with a photocrosslinking moiety results in surprisingly effective photocrosslinking between the activated ubiquitin molecule and the E2 junction enzyme (see below).

[0030] In some embodiments, the photocrosslinking agent moiety replaces the glutamine residue at position 31 of ubiquitin or the aspartate residue at position 32. In some embodiments, the photocrosslinking agent moiety replaces the glutamine residue at position 31 of ubiquitin.

[0031] The photocrosslinking agent moiety may be any moiety capable of replacing the glutamine residue at position 31 and / or the aspartate residue at position 32 of ubiquitin and forming a linker between the ubiquitin molecule and the E2 junction enzyme upon photon irradiation. Preferably, the activated ubiquitin molecule is stable in the absence of photons so that it can 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, is stable in the presence of natural and indoor artificial lighting (i.e., general household lighting consisting mainly of visible light) used to increase visibility.

[0032] "Stable" is used herein to mean that the amount of chemical degradation of a substance or material occurring over time is not severe enough to render the substance or material unusable. This allows for some degree of chemical degradation of the substance or material, ranging from negligible chemical degradation to a level of chemical degradation where the amount of undegraded substance or material is usable.

[0033] A person skilled in the art can assess what conditions are suitable for the storage of activated ubiquitin molecules. For example, if an activated ubiquitin molecule is stable in the presence of photons but not in the presence of natural light, a person skilled in the art will recognize that the molecule must be stored in a dark place, and if an activated ubiquitin molecule is unstable at room temperature, the molecule must be stored in a refrigerator.

[0034] In a specific embodiment, the photocrosslinking agent moiety is a non-natural amino acid, i.e., a modified natural amino acid that can be synthesized wholly or partially. Generally, the non-natural amino acid is derived from a modification of a natural amino acid that uses a functional group capable of forming a linker between a ubiquitin molecule and an E2 conjugating enzyme upon photon irradiation.

[0035] In some embodiments, the non-natural amino acid comprises any one or a combination selected from the group consisting of diaryl ketone, diazirin, aryl azide, diaryl / heteroaryl ketone, diheteroaryl ketone, heteroaryl azide, and 2-aryl-5-carboxytetrazole. In some embodiments, the non-natural amino acid comprises one type of functional group selected from the group consisting of diaryl ketone, diazirin, aryl azide, diaryl / heteroaryl ketone, diheteroaryl ketone, heteroaryl azide, and 2-aryl-5-carboxytetrazole. Where the non-natural amino acid comprises diaryl ketone, aryl azide, and 2-aryl-5-carboxytetrazole, the aryl may be optionally substituted and selected from the group consisting of phenyl or naphthalenyl. When non-natural amino acids include diaryl / heteroaryl ketones, diheteroaryl ketones and heteroaryl azides, the heteroaryl is optionally substituted and may be selected from the group consisting of indolyl, imidazolyl, pyridyl, thiophenyl, and furanyl.

[0036] Aryl or heteroaryls have one or more carbon atoms, C 1-4 Hydrocarville, C 1-4 Alkyloxy, C 1-4It may be substituted with any one or a combination selected from the group consisting of haloalkyl, hydroxy, and halo. In some embodiments, halo is fluoro. In certain embodiments, the aryl or heteroaryl may be optionally substituted with any one or a combination selected from the group consisting of methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethyl, hydroxy, and fluoro.

[0037] In some embodiments, the non-natural amino acid comprises any one or a combination of functional groups selected from the group consisting of diaryl ketones, diazirins, and aryl azides (Pham ND, Parker RB, Kohler JJ, Curr. Opin. Chem. Biol. , (2013), 17, 1, 90-101; Kauer, J.C., Erickson-Viitanen S., Wolfe H.R., DeGrado W.F., J. Biol. Chem. , (1986), 261, 23, 10695-10700).

[0038] In certain embodiments, the non-natural amino acid comprises a diaryl ketone. 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.

[0039] In certain embodiments, the non-natural amino acid comprises benzophenone. Often, the non-natural amino acid is derived from any one of the group consisting of phenylalanine, tryptophan, and histidine. In certain embodiments, the non-natural amino acid is derived from phenylalanine.

[0040] In a specific embodiment, the photocrosslinking agent moiety is pIt is benzoyl-L-phenylalanine.

[0041] The activating ubiquitin molecule of the present invention may be derived from ubiquitin from any eukaryotic organism. However, typically, the ubiquitin of the present invention is derived from animals, e.g., mammals. In some embodiments, the activating ubiquitin molecule of the present invention is derived from humans.

[0042] 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 the activated ubiquitin. Accordingly, any tag suitable for binding to ubiquitin, for example, for detection by fluorescence, may be used.

[0043] In some embodiments, the reporter tag comprises any one or a combination selected from the group consisting of biotin, an epitope, and a fluorescent dye. The epitope is recognized by a common antibody that binds to a specific epitope. If the reporter tag comprises an epitope, it can bind to an antibody and allow for the localization, purification, and molecular characterization of an activated ubiquitin molecule. Epitopes include Myc-tag, HA-tag, FLAG-tag, GST-tag, 6xHis, V5-tag, and OLLAS.

[0044] A fluorophore is a fluorescent tag. When a reporter tag contains a fluorophore, the activated ubiquitin molecule of the present invention can be detected by fluorescence microscopy or by the naked eye. The fluorophore includes fluorescein; green fluorescent proteins, e.g., optimized S65T mutant, enhanced green fluorescent protein (EGFP), monomer A206K mutant, superfolder GFP (sfGFP), Emerald, TagBFP, mCerulean3, mCitrine / mVenus, tdTomato, mCherry, mApple, mKate2, and mNeptune; FLAsH-EDT2 and ReAsH-EDT2.

[0045] If the reporter tag contains biotin, it can bind to its natural ligand, namely avidin, streptabin, or nutravidin, and allow for the localization, purification, and molecular characterization of an activated ubiquitin molecule. The natural ligand itself may bind to any of the fluorescent dyes listed above; an enzyme reporter such as horseradish peroxidase or alkaline phosphatase; or a fluorescent probe containing an anti-biotin antibody.

[0046] Detection of activated ubiquitin molecules containing reporter tags can be performed via fluorescence microscopy (or visual inspection), electron microscopy, enzyme-linked immunosorbent assay (ELISA), and / or Western blot.

[0047] In certain embodiments, the reporter tag is a biotin moiety. This 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 through a terminal cysteine-, serine-, threonine-, or tyrosine-containing motif. The terminal motif may be C- or N-terminal, but is generally N-terminal. In certain embodiments, the terminal motif is an N-terminal cysteine-containing motif. In further specific embodiments, the terminal motif has the sequence MGCSSG (SEQ No. 2). The biotin and epitope tags enable the performance of identification and activity quantification via mass spectrometry by enhancing the cross-linked activated RING E3.

[0048] The conjugate molecule of the second embodiment of the present invention comprises the activator molecule of the first embodiment conjugated to an E2 conjugation enzyme. During ubiquitination, ubiquitin is transferred to the catalytic cysteine ​​of E2 and forms an unstable thioester-linked E2 intermediate (E2~Ub) prior to the sequential transfer of ubiquitin to a lysine residue on the target protein.

[0049] To prevent dissociation of the activated ubiquitin molecule of the present invention from the E2 conjugation enzyme, the catalytic cysteine ​​residue of the E2 enzyme may be replaced with a residue that forms a more stable linker, such as a lysine residue. Thus, in some embodiments, the E2 conjugation enzyme comprises a lysine residue instead of a catalytic cysteine ​​residue. In a specific embodiment, the catalytic cysteine ​​residue of the E2 conjugation enzyme is at position 85. That is, the E2 conjugation enzyme comprises a C85K mutation. In some embodiments, the catalytic cysteine ​​residue of the E2 conjugation enzyme is at position 87. That is, the E2 conjugation enzyme comprises a C87K mutation. An alternative method to prevent dissociation of ubiquitin from E2 may include the incorporation of non-natural lysine derivatives having progressively shorter aliphatic side chains (i.e., diaminopropionic acid, diaminobutyric acid, and ornithine).

[0050] Some E2 junction enzymes, such as UBE2D3, additionally contain a serine residue capable of forming a non-covalent bond to ubiquitin. This interaction promotes the binding of ubiquitin from one junction molecule to the E2 junction enzyme of another junction molecule (self-binding). To inhibit the self-binding of the junction molecule, the serine residue can be replaced with a residue that does not bind to ubiquitin, such as an arginine residue. In a specific embodiment, the serine residue capable of forming a non-covalent bond to ubiquitin is located at position 22. That is, the E2 junction enzyme contains the S22R mutation.

[0051] Some E2 conjugation enzymes, such as UBE2N, additionally include a natural lysine residue capable of conjugating to ubiquitin, thereby promoting the conjugation of ubiquitin at the natural residue rather than at the location of the catalytic cysteine ​​residue or the previous location of the catalytic cysteine ​​residue (e.g., when cysteine ​​is replaced with a more stable linker such as lysine). Self-conjugation may also be promoted. To inhibit the conjugation of ubiquitin at the natural lysine residue, the natural lysine residue may be replaced with a residue that does not bind to ubiquitin, such as an arginine residue. In a specific embodiment, the natural lysine residue is at position 92. That is, the E2 conjugation enzyme contains the K92A mutation.

[0052] The E2 conjugation enzyme may be any E2 enzyme capable of binding to ubiquitin. However, the E2 conjugation enzyme is generally UBE2D3. In some embodiments, the E2 conjugation enzyme is UBE2D3 or UBE2N.

[0053] In some embodiments, the E2 conjugation enzyme possesses an N-terminal histidine tag, such as a hexahistidine tag. These tags not only facilitate the purification of the conjugate molecules of the present invention but also assist in the detection of the conjugate molecules through Western blot (also known as immunoblot) analysis.

[0054] The conserved (and activity-dependent) matching interactions of the Ub component within closed E2~Ub, combined with the enhanced free energy of binding to the activating RING E3, can be utilized for the development of ABP. The ABP of the present invention is effective for activity profiling of RING E3 ligase, RING E1 ligase, and / or deubiquitination enzymes in various sample types.

[0055] Accordingly, in a third embodiment, the present invention provides a use of the conjugated molecule of the second embodiment in a method for profiling the activity of a RING E3 enzyme, a RING E1 enzyme, and / or a deubiquitination enzyme. In one embodiment, the use of the conjugated molecule of the second embodiment is in a method for profiling the activity of a RING E3 enzyme. In further 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). 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), RCR (1), TRAF6, TRAF2, and HLTF. For example, the RING E3 enzyme can be RNF4, c-CBI, or TRAF6.

[0056] The RING E3 enzyme is inactive in the monomeric state and predominates at endogenous concentrations. Activation of the RING E3 enzyme can be achieved by RING dimerization, which leads to ligase activity of the RING E3 enzyme (see Rojas-Fernandez, A. et al., 2014, cf.). Alternatively, the monomeric RING E3 enzyme can be activated by a non-RING element (NRE) (e.g., see Dou H. et al., 2013, cf.). Activation of the E3 enzyme induces ubiquitination (see Fig. 1a). That is, the active RING E3 enzyme binds to E2-Ub (a conjugate containing the E2 conjugate enzyme and ubiquitin) to induce a closed E2-Ub structure.

[0057] The active RING E3 enzyme can bind to the conjugated molecule of the present invention via RING E3 or NRE. When the resulting complex is irradiated, the photocrosslinking agent moiety of the conjugated molecule can be covalently bonded to RING E3 or NRE (see FIG. 1b). Thus, the conjugated molecule of the present invention can act as a RING E3 ABP, and the active RING E3 enzyme can be detected and further characterized.

[0058] In a fourth embodiment, the present invention provides a method for detecting an interaction between a conjugate molecule of the second embodiment and a RING E3 enzyme, a RING E1 enzyme, and / or a deubiquitination enzyme, the method comprising the steps of contacting the conjugate molecule of the second embodiment with a RING E3 enzyme, a RING E1 enzyme, and / or a deubiquitination enzyme, and detecting any new conjugate formation.

[0059] As used herein, "contact" refers to any means of causing the conjugate molecule of the present invention to interact with a RING E3 / RING E1 / deubiquitination enzyme. Generally, the contacting step takes place in an aqueous solution containing the conjugate molecule of the present invention and a RING E3 enzyme.

[0060] Upon irradiation, the photocrosslinker moiety of the conjugated molecule can be covalently bonded to the RING E3 / RING E1 / deubiquitination enzyme. If the conjugated molecule of the present invention is unstable in the presence of photons, the photocrosslinker moiety of the conjugated molecule can be covalently bonded to the enzyme when irradiated with photons of any frequency. However, if the conjugated molecule is stable in the presence of natural light, a frequency higher than that of natural light may be required to form a covalent bond between the activated ubiquitin molecule and the enzyme. Generally, UV light, i.e., light having a wavelength within the range of 10 to 400 nm, is used to covalently link the photocrosslinker moiety to the enzyme.

[0061] Those skilled in the art will recognize that the irradiation time required to form a covalent bond between the enzyme and the conjugated molecule depends on various factors. For example, the irradiation time may be influenced by the concentration of the sample to be irradiated (more concentrated samples may require longer irradiation times); the frequency of the light used to irradiate the sample (frequency matching absorbed by the photocrosslinking agent moiety may require shorter irradiation times); and the power of the radiation source (higher power may result in shorter irradiation times). The conjugated molecule and enzyme of the present invention may be irradiated for 1 minute to 50 minutes. In some embodiments, the conjugated molecule and enzyme of the present invention are irradiated for 1 minute to 40 minutes. Generally, irradiation is performed for 1 minute to 30 minutes.

[0062] Where the enzyme is a RING E3 enzyme, the conjugated molecule of the present invention may be used in any study where RING E3 enzyme detection is useful. This includes studies of RING E3 enzyme regulation, discovery of novel RING E3 enzymes, inhibitor screening, inhibitor selectivity profiling; and / or studies of stabilization of enzyme intermediates for structural studies.

[0063] Novel conjugates of the method of the present invention can be detected using various methods including fluorescence microscopy (or visual inspection), electron microscopy, enzyme-linked immunosorbent assay (ELISA), gel electrophoresis, and / or Western blot. Often, a combination of analytical methods is used to detect novel conjugates of the present invention. Those skilled in the art will recognize that the method suitable for detecting novel conjugates of the present invention varies depending on whether a reporter tag is used and the characteristics of any reporter tag. For example, gel electrophoresis can be used to separate novel conjugates from a sample mixture based on the size and charge of the sample and may be useful regardless of whether a reporter tag is used. However, fluorescence microscopy can be used to identify novel conjugates only when the conjugated molecule of the present invention contains a fluorescent reporter tag.

[0064] The conjugate molecule of the present invention may interact with any active RING E3 enzyme. That is, the use and method of the present invention are not limited to any specific 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) enzyme. In certain embodiments, the RING E3 enzyme is a 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), RCR (1), TRAF6, TRAF2, and HLTF. For example, the RING E3 enzyme may be RNF4, c-CbI, or TRAF6.

[0065] Because all of the discussions of this application regarding documents, acts, materials, devices, articles, etc. existed prior to the priority date of each claim of this application, it should not be construed that some or all of these matters formed part of the prior art basis or were ordinary general knowledge in the relevant field.

[0066] Those skilled in the art will understand that various modifications and / or variations may be made to the invention as described herein without departing from the scope of the invention as described. Accordingly, the embodiments described herein should be considered for illustrative purposes only, are not limiting, and are not restricted to the scope described in the embodiments. Those skilled in the art should understand that the embodiments described herein may be read alone or in combination, and may be combined with any one or combination of the features described herein.

[0067] The contents of each patent and non-patent reference cited herein are incorporated herein by reference in their entirety.

[0068] The present invention may be further understood in connection with the following non-limiting section.

[0069] 1. An activated ubiquitin molecule comprising a photocrosslinking agent moiety instead of a glutamine residue at position 31 and / or an aspartate residue at position 32 of ubiquitin.

[0070] 2. In Section 1, the photocrosslinking agent moiety is an activated ubiquitin molecule that replaces the glutamine residue at position 31 or the aspartate residue at position 32 of ubiquitin.

[0071] 3. In Section 1, the photocrosslinking agent moiety is an activated ubiquitin molecule that replaces the glutamine residue at position 31 of ubiquitin.

[0072] 4. In any one of Sections 1 to 3, the photocrosslinking agent moiety is an activated ubiquitin molecule that is a non-natural amino acid.

[0073] 5. In Section 4, the non-natural amino acid comprises any one or a combination selected from the group consisting of diaryl ketone, diazirin, aryl azide, diaryl / heteroaryl ketone, diheteroaryl ketone, heteroaryl azide and 2-aryl-5-carboxytetrazole, an activated ubiquitin molecule.

[0074] 6. In Section 4, the non-natural amino acid comprises an activated ubiquitin molecule comprising one type selected from the group consisting of diaryl ketone, diazirin, aryl azide, diaryl / heteroaryl ketone, diheteroaryl ketone, heteroaryl azide, and 2-aryl-5-carboxytetrazole.

[0075] 7. In Section 4, the non-natural amino acid is an activated ubiquitin molecule comprising any one functional group selected from the group consisting of diaryl ketone, diazirin, aryl azide, and 2-aryl-5-carboxytetrazole.

[0076] 8. In Section 4, the non-natural amino acid is an activated ubiquitin molecule containing a diaryl ketone.

[0077] 9. An activated ubiquitin molecule, wherein, in any one of Sections 5 to 8, the aryl is optionally substituted and may be selected from the group consisting of phenyl or naphthalenyl; and the heteroaryl is optionally substituted and may be selected from the group consisting of indolyl, imidazolyl, pyridyl, thiophenyl and furanyl.

[0078] 10. In any one of Sections 5 through 9, the aryl or heteroaryl is C at one or more carbon atoms 1-4 Hydrocarville, C 1-4 Alkyloxy, C 1-4 An activated ubiquitin molecule that can be substituted with any one or combination selected from the group consisting of haloalkyl, hydroxyl, and halo.

[0079] 11. In Section 10, halo is a fluoroin, an activated ubiquitin molecule.

[0080] 12. An activated ubiquitin molecule, wherein the aryl or heteroaryl is optionally substituted with any one or a combination selected from the group consisting of methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, trifluoromethyl, hydroxy, and fluoro.

[0081] 13. In Section 4, the non-natural amino acid comprises an activated ubiquitin molecule comprising a diaryl ketone 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.

[0082] 14. In Section 4, the non-natural amino acid is an activated ubiquitin molecule containing benzophenone.

[0083] 15. In any one of Sections 4 through 14, the non-natural amino acid is an activated ubiquitin molecule derived from any one of the group consisting of phenylalanine, tryptophan, and histidine.

[0084] 16. In any one of Sections 4 through 14, the non-natural amino acid is an activated ubiquitin molecule derived from phenylalanine.

[0085] 17. In any one of Sections 1 to 3, the photocrosslinking agent moiety is p - Activated ubiquitin molecule, which is benzoyl-L-phenylalanine.

[0086] 18. In any one of Sections 1 through 17, ubiquitin is an active ubiquitin molecule derived from an animal.

[0087] 19. In any one of Sections 1 through 17, ubiquitin is an activated ubiquitin molecule derived from mammals.

[0088] 20. In any one of Sections 1 through 17, ubiquitin is an activated ubiquitin molecule derived from a human.

[0089] 21. An activated ubiquitin molecule, wherein, in any one of Sections 1 through 20, it further comprises a reporter tag.

[0090] 22. In Section 21, the reporter tag comprises any one or a combination selected from the group consisting of biotin, an epitope, and a fluorescent dye, an activated ubiquitin molecule.

[0091] 23. In Section 22,

[0092] (i) The epitope is any one or a combination selected from Myc-tag, HA-tag, FLAG-tag, GST-tag, 6xHis, V5-tag and OLLAS;

[0093] (ii) The fluorophore is any one or a combination selected from fluorescein, optimized S65T mutant, enhanced green fluorescent protein (EGFP), monomer A206K mutant, superfolder GFP (sfGFP), Emerald, TagBFP, mCerulean3, mCitrine / mVenus, tdTomato, mCherry, mApple, mKate2 and mNeptune; and FLAsH-EDT2 and ReAsH-EDT2, an activating ubiquitin molecule.

[0094] 24. In Section 21, the reporter tag is a biotin moiety, an activated ubiquitin molecule.

[0095] 25. In Section 24, the biotin moiety is covalently linked to the ubiquitin or activating ubiquitin molecule through a terminal cysteine-, serine-, threonine-, or tyrosine-containing motif, activating ubiquitin molecule.

[0096] 26. In Section 25, the terminal motif is an activated ubiquitin molecule that is N-terminus.

[0097] 27. In Section 25, the terminal motif is an activated ubiquitin molecule, which is an N-terminal cysteine-containing motif.

[0098] 28. In any one of Sections 25 to 27, the terminal motif is an activated ubiquitin molecule having the sequence MGCSSG.

[0099] 29. A conjugate molecule comprising an activated ubicutin molecule according to any one of Sections 1 to 28 conjugated to an E2 conjugation enzyme.

[0100] 30. In Section 29, the E2 conjugation enzyme is a conjugated molecule containing a lysine residue instead of a catalytic cysteine ​​residue.

[0101] 31. In Section 29, the E2 conjugation enzyme is a conjugate molecule containing a C85K or C87K mutation.

[0102] 32. A conjugate molecule in which, in any one of Sections 29 to 31, a serine or lysine residue capable of forming a non-covalent bond to ubiquitin is replaced by an arginine residue.

[0103] 33. In either Section 29 or Section 30, the E2 conjugation enzyme is a conjugate molecule comprising C85K and S22R mutations.

[0104] 34. A conjugate molecule in which, in any one of Sections 29 through 33, the E2 conjugation enzyme is UBE2D3.

[0105] 35. In either Section 29 or Section 30, the E2 conjugation enzyme is a conjugate molecule comprising C87K and K92A mutations.

[0106] 36. A conjugate molecule in which, in any one of Sections 29 through 32 and Section 35, the E2 conjugation enzyme is UBE2N.

[0107] 37. In any one of Sections 29 through 36, the E2 conjugating enzyme is a conjugate molecule having an N-terminal histidine tag.

[0108] 38. In any one of Sections 29 through 36, the E2 conjugation enzyme is a conjugate molecule having a hexahistidine tag.

[0109] 39. Use of any one of the conjugate molecules of Sections 29 to 38 in a method for profiling the activity of RING E3 enzyme, RING E1 enzyme and / or deubiquitination enzyme.

[0110] 40. In Section 39, 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 deubiquitination enzyme is DUB (31).

[0111] 41. Uses in which the activity profiling in Section 39 or 40 is for the RING E3 enzyme.

[0112] 42. (i) Research on RING E3 enzyme regulation,

[0113] (ii) Discovery of a novel RING E3 enzyme,

[0114] (iii) Inhibitor screening,

[0115] (iv) Inhibitor selectivity profiling;

[0116] and / or (v) the use of Section 41 for stabilizing enzyme intermediates for structural studies.

[0117] 43. A method for detecting an interaction between a conjugate molecule of any one of Sections 29 to 38 and a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitination enzyme, the method comprising the steps of contacting the conjugate molecule with a RING E3 enzyme, a RING E1 enzyme and / or a deubiquitination enzyme, and detecting the formation of any new conjugate.

[0118] 44. In Section 43, the RING E3 enzyme is any one or a combination of RNF4, Cbl, e.g. c-Cbl, Praja2, TRIM11, HECT(11), RBR(1), RCR(1), TRAF6, TRAF2 and HLTF, and the deubiquitination enzyme is DUB(31).

[0119] 45. Method in Section 43 or 44, wherein the interaction is an interaction with the RING E3 enzyme.

[0120] 46. ​​A method comprising, in any one of Sections 43 to 45, further comprising the step of irradiating the conjugate molecule and the RING E3 / RING E1 / deubiquitination enzyme such that the photocrosslinking agent moiety of the conjugate molecule is covalently bonded to the aforementioned RING E3 / RING E1 / deubiquitination enzyme.

[0121] 47. In Section 46, the investigation is a method using UV light.

[0122] 48. In Section 47, the UV light has a wavelength within the range of 200 to 400 nm.

[0123] 49. A method in any one of Sections 45 through 48, wherein the RING E3 enzyme is an RNF4, CbI, or TRAF6 enzyme.

[0124] 50. In Section 49, the CbI enzyme is a c-CbI enzyme.

[0125] The present invention is further described by the following non-limiting embodiments.

[0126] Examples

[0127] Activity-dependent profiling of two cancer-associated RING E3, RNF4, and c-Cbl was demonstrated in response to their intrinsic activation signals. RNF4 is activated by poly-SUMO chain-induced dimerization, whereas c-Cbl is activated by tyrosine phosphorylation. It was demonstrated that parallel measurements of intrinsic cellular RING E3 activity can be performed by combining biotin reporter-tagged conjugates with mass spectrometry. Furthermore, distinct cellular activations of RING E3 can be identified upon cell perturbation (e.g., growth factor stimulation). The conjugate molecules of the present invention have the potential to advance E3 ligase research and the development of selective regulators for this class of enzymes.

[0128] Materials and Methods

[0129] Experimental Model and Subject Details

[0130] H293T cells were obtained from ATCC. 293T is a human cell line derived from the HEK293 cell line and expresses a mutated version of the SV40 large T antigen (RRID:CVCL_0063). Cells were cultured in a humidified incubator under a 5% CO2 atmosphere at 37°C. Dulbecco's modified Eagle medium was used and supplemented with fetal bovine serum and L-glutamine.

[0131] In this study, BL21(DE3) and E. coli used for protein expression ( Escherichia coli ) BL21 Rosetta TM (DE3) cells were 100 mL each -1 ampicillin and 34 mL -1 It was grown in a 1 L flask containing 1 L LB medium supplemented with chloramphenicol (for details, refer to STAR methods - expression of recombinant proteins).

[0132] Detailed method

[0133] p Site-specific incorporation of Bpa non-natural amino acids into ubiquitin

[0134] The pEvol-Bpa plasmid was derived from pEVOL-pBoF (provided by P. Schultz, The Scripps Research Institute). Mutations for Bpa incorporation were used to create the plasmid pEVOL-Bpa. Mj It was introduced into two copies of the YRS gene (Young et al., 2010; Chin et al., 2002). BL21 cells (50 μL) were co-transformed with pET-ubiquitin-6His-tag x (where x is the Bpa incorporation site) and pEvol-Bpa plasmids using heat shock, recovered in 200 μL SOC medium for 1 hour at 37°C, and 100 μg / mL -1 Ampicillin and 34 μg / mL -1 50 mL of Luria-Bertani (LB) containing chloramphenicol was used for inoculation. Subsequently, 10 mL was incubated overnight and inoculated into 1 L LB broth containing the same concentration of antibiotics. OD 600 The cells were grown until this reached approximately 0.6, and the culture was divided into two 500 mL portions. One portion was 1 mM p - It was supplemented with benzoyl-L-phenylalanine (Bpa; Bachem), and the other portion served as a control with Bpa withheld. The culture was for 20 minutes (37℃, 200 rpm), or OD 600 Incubate until this reaches 0.6 to 0.7, and add 0.02% arabinose and 1 mM isopropyl β Protein expression was induced by adding -D-1-thiogalactopyranoside (IPTG). The culture was incubated for 5 hours (37°C, 200 rpm). Cells were harvested and placed in a 10 mL BugBuster ®After suspending in protein extraction (Merck Millipore) reagent, the solution was transferred to a 50 mL Falcon tube. The lysate was incubated for 20 minutes, then purified by centrifugation and incubated for 1 hour with gentle shaking before transferring to a 50 mL Falcon tube containing 1 mL of Ni-NTA agarose beads. The resin was centrifuged (4°C, 1000 rpm) and washed with wash buffer (20 mM Na2HPO4, pH 7.5, 25 mM imidazole). Finally, the protein was washed with 200 μL of elution buffer (20 mM Na2HPO4, The product was eluted with pH 7.5, 300 mM imidazole. A 20 μL aliquot of the eluted fraction was mixed with an equal amount of 4X SDS loading buffer and loaded onto a 4 to 12% SDS-PAGE gel. Proteins were separated using MES buffer at 200 V for 30 minutes and detected using Coomassie blue staining. A 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 connected to a 6130 Quadrupole spectrometer. Unless otherwise specified, an Agilent ZORBAX 300SB-C3 5 μm, 2.1 x 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 absorbance was monitored at 214 and 280 nm. MS acquisition was performed in cation mode, and total protein mass was calculated by deconvolution within MS Chemstation software (Agilent Technologies). pThe fraction containing Bpa-incorporated Ub was pooled and concentrated using an Amicon Ultra-15 3 kDa MWCO centrifugal filter device (Millipore). The samples were desalted with 10 mM Tris-HCl pH 7.5 using a PD-10 column (GE Life Sciences). After adding DTT (1 mM) to the samples, 15 μg mL was added using UCH-L3 (Virdee et al., 2010). -1 Hexahistidine tag cleavage was performed at the final concentration. The sample was incubated at 37°C for 2 hours to remove the N-terminal His tag. Bpa-incorporated Ub was further purified by semi-preparative HPLC, and the fraction was lyophilized to yield approximately 8 to 10 mg of Ub- p Bpa was obtained.

[0135] Expression of UBE2D3(S22R / C85K) recombinant protein

[0136] S22R and C85K were introduced into UBE2D3 using site-directed mutagenesis. OD at 37°C, 200 rpm. 600 The cells were grown until this reached 0.6 to 0.7. OD 600 When this reached 0.6 to 0.7, IPTG (1 mM) was added to induce protein expression, and the cells were incubated at 37°C for 3 hours. 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 -1Cells were incubated on ice for 30 minutes and then sonicated. The purified lysate containing His6-tagged UBE2D3 (S22R / C85K) was loaded onto Ni-NTA resin, washed with buffer (20 mM pH 7.5, Na2HPO4, pH 7.5, 25 mM imidazole, 150 mM NaCl, 1 mM TCEP), and eluted with elution buffer (20 mM pH 7.5, Na2HPO4, pH 7.5, 300 mM imidazole, 150 mM NaCl, 1 mM TCEP). The sample was further purified by size exclusion chromatography using a HiLoad Superdex-75 16 / 60 column (GE Healthcare) with run buffer (20 mM Na2HPO4, pH 7.5, 150 mM NaCl, 1 mM TCEP).

[0137] Preparation of Biotin-UbBpa31

[0138] Lyophilized UbBpa31 (10 mg) containing an N-terminal MGCSSG cysteine-containing 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, after which 5 molar equivalents of EZ-linked iodo-acetyl PEG2-biotin (Thermofisher) were added in reaction buffer (50 mM Na2HPO4, 150 mM NaCl, 0.5 mM TCEP). The reaction mixture was incubated at 23°C for 1 hour with gentle shaking and monitored by LC-MS until completion. Subsequently, the product was purified by aliquot HPLC at random flow rates and lyophilized to obtain biotin-UbBpa31 (6 to 8 mg).

[0139] Preparation of isopeptide-linked photoABP

[0140] To generate UBE2D3(S22R / C85K)-UbBpa, UBE2D3(S22R / C85K) (200 μM) was incubated with UbBpa (200 μM) and His6-Uba1 (1 μM) in 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 prepared at a concentration of 2 mg / ml -1 It was concentrated and stored at -80°C. The biotin-photoABP probe was prepared using the same procedure.

[0141] Expression of recombinant RNF4 protein

[0142] The cloning, expression, and purification of a linear fusion of two RNF4 ring domains and related mutations have been previously described (Plechanovova et al., 2011). The fusion of the two RNF4 ring domains was expressed in E. coli Rosetta (DE3) cells (Novagen). OD was 37°C and 200 rpm. 600 The cells were grown until this reached 0.6 to 0.7. OD 600 When this reached 0.6 to 0.7, IPTG (1 mM) was added to induce protein expression, and the mixture was incubated at 16°C at 200 rpm.

[0143] Cells were harvested and resuspended in 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 the cells were lysed by sonication. His6-MBP-fusion proteins were purified by Ni-NTA (Qiagen) chromatography and then cleaved with TEV protease overnight at 4°C. To remove uncleaved fusion proteins, His6-tagged MBP, and His6-tagged TEV protease, the material was depleted against fresh Ni-NTA resin, and then size exclusion chromatography was performed using a HiLoad Superdex 75 16 / 60 column (GE Healthcare) (20 mM Tris, 150 mM NaCl, 1 mM TCEP, pH 7.5).

[0144] Expression of c-Cbl and c-Cbl(Y371F) recombinant proteins

[0145] BL21(DE3) cells (50 μL) were transformed with the pGEX6P-1-Cbl plasmid and recovered in 200 μL of SOC medium at 37°C for 1 hour, then 100 μg mL -1 It was inoculated into 50 mL of Luria-Bertani (LB) containing ampicillin. Subsequently, 10 mL was incubated overnight and inoculated into LB broth containing the same concentration of antibiotic and 0.2 mM zinc chloride. OD at 37°C, 200 rpm 600 The cells were grown until this reached 0.6 to 0.7. OD 600When the value reached 0.6 to 0.7, 1 mM IPTG was added to induce protein expression, and the cells were incubated overnight at 16°C at 200 rpm. Cells were harvested, resuspended in buffer (50 mM Hepes, pH 7.5, 0.5 M NaCl, 1 mM TCEP), and lysed by sonication. The lysate was incubated with glutathione cephalosporose beads for 1 hour while gently shaking. The resin was centrifuged (4°C, 1000 rpm), washed with 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 protein was 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).

[0146] c-Cbl phosphorylation

[0147] Purified c-Cbl (3 μM) was phosphorylated by incubation with Src kinase (1.5 μM), 10 mM MgCl2, and 5 mM ATP at 37°C for 45 minutes. A sample (15 μl) was collected and thoroughly mixed with 4X LDS loading buffer (ThermoFisher), then boiled before loading onto a 7.5% acrylamide phosph-tag gel. Proteins were separated in MOPS buffer at 160 V for 60 minutes and analyzed using Coomassie staining and Western blot.

[0148] In addition, ATP-dependent phosphorylation and photocrosslinking of c-Cbl and photoABP-UbBpa31 (5 μM) were analyzed using Coomassie staining. Samples (15 μl) were collected and thoroughly mixed with 4X LDS loading buffer, then boiled at 95°C for 5 minutes before loading onto a 4 to 12% SDS-PAGE gel using MOPS running buffer, and analyzed using Coomassie staining. Additionally, the gels were blotted and analyzed using Western blot with anti-Cbl (1:5000 dilution) as the primary antibody and anti-mouse (1:10000 dilution) as the secondary antibody.

[0149] UV irradiation conditions for photocrosslinking

[0150] Photocrosslinking reactions (45 μL) were performed in 24-well plates (Cryshem HR3-158, Hampton Research) in reaction buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM TCEP). The samples were divided into two portions. One portion was irradiated at 365 nm for 10 to 30 minutes on ice at a distance of 2 cm from a engineered UV lamp (BLE-8T365, Spectroline), and the other portion was preserved in the dark. For purified proteins such as RNF4-RING (5 to 50 μM), c-Cbl (3 μM), and c-Cbl Y371F (3 μM), photocrosslinking reactions were performed with photoABP-UbBpa31 probes (5 to 50 μM) and irradiated with UV light. Samples were analyzed by SDS-PAGE and visualized by Coomassie staining or immunoblotting. Control experiments were performed under the same conditions.

[0151] Photocrosslinking of cell extracts

[0152] HEK293 cells were transfected with plasmids expressing GFP-Cbl, GST-Src, and GFP-Cbl. Cells were lysed in 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 photoABP-Bpa31 (25 μM) was mixed with the cell lysates, and UV was irradiated (10 min) using the photocrosslinking procedure described in the general methods. Samples were analyzed on a 4 to 12% SDS-PAGE gel using MOPS run buffer (160 V, 60 min) and visualized by immunoblotting using anti-Cbl (1:5000 dilution) as the primary antibody and anti-mouse (1:10000 dilution) as the secondary antibody.

[0153] Phos-tag TM Gel electrophoresis

[0154] To evaluate Src-mediated c-Cbl phosphorylation, a degradation gel (7.5% acrylamide / bis-acrylamide, 375 mM Tris-HCl pH 8.8, 0.1% sodium dodecyl sulfate (SDS), 100 μM MnCl2, 50 μM Phos-tag) was prepared. TM0.05% (w / v) ammonium persulfate (APS), 0.0625% (v / v) tetramethylethylenediamine (TEMED), and stacking gel (4% acrylamide / bis-acrylamide, 125 mM Tris-HCl pH 6.6, 0.1% SDS, 0.05% (w / v) APS, 0.1% (v / v) TEMED) were poured, degassed with argon, and polymerized at room temperature for 3 hours. Cell extract (50 μg) was boiled in LDS-sample buffer and replenished with 10 mM MnCl2 before loading. Electrophoresis was performed at 70 V through a stacking gel and at 130 V through a run buffer (25 mM Tris-HCl, 192 mM glycine, 0.1% SDS), followed by washing for 3 x 20 minutes in a transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol) supplemented with 10 mM EDTA and 0.05% SDS to stain with Coomassie dye or to chelate manganese, followed by washing for 1 x 20 minutes in a transfer buffer supplemented with 0.05% SDS. Subsequently, the proteins were transferred to a 0.45 μm nitrocellulose membrane in a transfer buffer at 100 V, 4°C, and left for 3 hours.

[0155] Cell culture, transfection, and lysis

[0156] 293T cells with 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 modified Eagle medium (DMEM) supplemented with streptomycin (37°C, 5% CO2). Cells were placed in 4 x 100 mm dishes. 6Seeding was performed at a density of [value]. 18 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 of Fugene-6 (Promega) in 200 μL of Eagle's Minimum Essential Medium (Opti-MEM). 90 minutes before harvest, MG132 (25 μM) was added to the cells. Cells were rinsed and collected with ice-cold PBS, and extracted 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 purified by centrifugation at 21°C and 100 g for 20 minutes. The supernatant was collected, and protein concentration was determined by Bradford assay.

[0157] Protein profiling based on the activity of EGF-stimulated HEK293 cells

[0158] 293T cells 5 x 10 6 Cells were seeded in 150 mm dishes at a density and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum (FBS), 2.0 mM L-glutamine, and antibiotics (100 units mL-1 penicillin, 0.1 mg mL-1 streptomycin) (37°C, 5% CO2). The next day, the medium was replaced with DMEM without FBS. The following day, cells were treated with 20 μM MG132 and 200 nM bafilomycin for 6 hours at 37°C, followed by 100 ngmL recombinant EGF. -1The plates were treated at 37°C for 15 minutes with or without the addition of (Thermo Fisher Scientific, PHG0311). The plates were transferred to the ice, washed, resuspended in ice-cold PBS, washed twice at 4°C, and the lysate was extracted in ice-cold lysis buffer. 293T cells were treated with the biotinization probe (biotin-photoABP-UbBpa31) (20 μM). Samples were divided and irradiated with UV for 1 hour or withheld from UV. Subsequently, biotin enrichment was performed on the streptavidin resin as described above, followed by dendritic trypsin digestion, LC-MS / MS analysis, and data processing (Pao et al., 2018, see above).

[0159] result

[0160] Design and Assembly of RING ABP

[0161] To establish potential sites for photocrosslinker incorporation, structural superpositions were generated for the interpreted RING E3:E2–Ub co-crystal structure (Fig. 5) (Dou et al., 2012b, 2013; Koliopoulos et al., 2016; Plechanovova et al., 2012, see above). Notable conservation in the binding mode was evident, and multiple residues within both Ub and E2 are adjacent to the RING domain(s). To confer activity dependence on the probe, a photocrosslinking moiety was incorporated into Ub, as this component binds only in close proximity to the active RING E3. Ten consensus sites within Ub adjacent to the active RING (Fig. 1c) were selected, and the evolved Methanocoldococcus Janashi tyrosyl-Trna synthase-Trna CUA Photocrosslinking amino acids using pairs pBenzoyl-L-phenylalanine (Bpa) was incorporated (Figs. 1b and 1c) (Chin et al., 2002, see above). Efficient incorporation was achieved at all sites producing approximately 4 to 6 mg of protein per liter of culture medium. Ub mutants were homogeneously purified and characterized by LC-MS (Figs. 6 and 7). Subsequently, all mutant Ub variants were identified as indiscriminate E2 UBE2D3 possessing an N-terminal hexahistidine tag (Brzovic, PS et al. (2006). Mol Cell 21 It was enzymatically conjugated to (873-880) (Fig. 1d). In addition to facilitating purification, the latter serves as a convenient reporter for immunoblot analysis. Conjugation to E2 was performed with an E1 activating enzyme, and catalytic cysteine ​​was mutated to lysine to form a more stable link between Ub and E2, enabling stable isopeptide conjugation (Plechanovova et al., 2012, see above) (Fig. 2a). Importantly, structural analysis indicated that the isopeptide is an acceptable structural mimic of a natural thioester (Koliopoulos et al., 2016; Plechanovova et al., 2012; Wright et al., 2016, see above). Additionally, an S22R mutation was introduced within the E2 component to disrupt the non-covalent Ub binding site, which could lead to ABP self-binding (Brzovic, PS, and Klevit, RE (2006). Cell Cycle , 5, 2867-2873). All E2~Ub variants were homogeneously purified by size exclusion chromatography as determined by SDS-PAGE and LC-MS analysis (Figs. 2b to 2d and Fig. 8).

[0162] Activity-dependent profiling of dimeric RING E3 RNF4

[0163] RING E3 RNF4 is inactive in the dominant monomeric state at endogenous concentrations. The binding of a poly-SUMO chain to the SUMO interaction motif (SIM) within RNF4 enhances the local concentration of RNF4, promoting RING domain homomerization and the activation of E3 ligase activity (Rojas-Fernandez et al., 2014, see citation above). This leads to the ubiquitination and degradation of SUMO-modified promyelocytic leukemia protein (PML) (Tatham, MH et al. (2008). Nat Cell Biol , 10, 538-546). Surprisingly, therapeutic induction of this process leads to remission of acute promyelocytic leukemia in more than 90% of cases (Massaro, F., Molica, M., and Breccia, M. (2016). Int J Hematol Oncol,5, 105-118). A engineered version of constitutively active RNF4 was designed to consist of a full-length protein having an additional RING domain fused to the natural C-terminus via a flexible linker (RNF4-RING) (Fig. 3a) (Plechanovova et al., 2011, see above). To determine the optimal photocrosslinker site, all 10 Bpa mutant E2–Ub conjugates containing RNF4-RING were incubated (Fig. 9), and the crosslinking efficiency upon UV irradiation (10 min) was evaluated. Significant and dose-responsive crosslinking was observed only upon Bpa incorporation at site 31 (photoABP-UbBpa31) (Figs. 3b and 10). Importantly, no crosslinking was observed when RNF4-RING was incubated and UV was irradiated onto unconjugated UbBpa31, indicating that photocrosslinking relies on E2-induced Ub proximity consistent with natural mechanisms. In particular, additional crosslinking products corresponding to the molecular weight of the addition of two photoABP-UbBpa31 molecules were observed (Fig. 3b). Structural studies of the dimeric RING:E2–Ub complex demonstrated that both sides of the active RING dimer bind to and activate the separate E2–Ub conjugate (Fig. 3c). The fused RNF4-RING structure can disrupt binding to a single E2–Ub molecule or both by introducing the M140A R181A double mutant into one or both of the RING domains (RNF4x-RING or RNF4x-RINGx, respectively) (Rojas-Fernandez et al., 2014, see citation above). Consistent with photoABP-UbBpa31 profiling this structurally described binary mechanism, the crosslinking of the 2nd E2~UbBpa31 molecule was lost to RNF4x-RING and completely discarded to RNF4x-RINGx (Fig. 3d). To further confirm activity-dependent photocrosslinking, a mutant photoABP-UbBpa31 control probe was prepared.Some of the conserved E2-RING interactions involve E2 F62 residues (F63 in some E2 models), and mutations to alanine generally impair / discard E3 binding (Weissman, AM (2001). Nat Rev Mol Cell Biol (, 2, 169-178). These control probes further indicate whether the observed labeling is consistent with natural E2-RING interactions, and thus are suitable for screening inhibitors that interfere with natural interactions (Fig. 3e). In accordance with the probe being bound in a natural manner, the photoABP-UbBpa31 F62A probe did not undergo RING cross-linking. This should also serve as a useful control probe when agnostically profiling RING E3 (Fig. 3f).

[0164] ABP profiling of poly-SUMO chain-induced RNF4 activation

[0165] Cellular RNF4 induces dimerization by being recruited to and activated by the poly-SUMO chain through its SIM domain. Dimerization K d Since the value is approximately 180 Nm (see Rojas-Fernandez et al., 2014, above), we established a biochemical assay to evaluate whether photoABP-UbBpa31 could profile the poly-SUMO chain-induced activation of natural RNF4 by working at concentrations below this value (Fig. 3g). As expected, constitutively active RNF4-RING was insensitive to dilution and proceeded with photoABP-UbBpa31 crosslinking, whereas natural RNF4 was not (Fig. 3h). However, in the presence of a linear amide-linked tetra-SUMO (SUMOx4) fusion protein (10 μM) (see Tatham et al., 2008, above), which outlines the activation characteristics of natural isopeptide-linked poly-SUMO chains, photoABP-UbBpa31 crosslinking was observed to exhibit efficiency comparable to that of RNF4-RING. Significantly, a cross-linking band was observed upon the addition of a second photoABP-UbBpa31 molecule (Fig. 3g). This suggests that naturally activated wild-type RNF4 maintains its bipolar activity and that its associated progression can be utilized in cells. Taken together, the data to date demonstrate that photoABP-UbBpa31 promotes activity-dependent cross-linking of naturally activated RING E3, which lacks a catalytic nucleophile.

[0166] Activity-dependent profiling phosphorylation-induced RING E3 activation

[0167] PhotoABP-UbBpa31 was tested as a RING E3 activated through a distinct mechanism. The Cbl protein is a multidomain and multifunctional RING E3 ligase consisting of three homologs: c-Cbl, Cbl-b, and Cbl-c (Lyle, CL, Belghasem, M., and Chitalia, VC (2019). CellsMost of Cbl's functions are associated with RING E3 activity and include the regulation of angiogenesis. Abnormalities in Cbl activity are associated with various cancers. Cbl is overexpressed in many breast cancer cells and tissues and has been found to be downregulated in myeloproliferative neoplasms (MDS / MPN) and non-small cell lung cancer (Kales, SC et al., (2010). Cancer Res, 70, 4789-4794; Tan, YH et al., (2010). PLoS One, 5, e8972). Therefore, modulation of Cbl E3 activity is an attractive therapeutic strategy and is attracting significant interest from pharmaceutical companies. The most common mutation observed in clinical practice is located at residue Y371. Y371 is a target for phosphorylation by c-Src kinase, which induces structural changes that represent a non-RING element, enhance affinity for E2–Ub, and stimulate Cbl E3 activity (Buetow et al., 2016; Dou et al., 2013, see citation). Indeed, the affinity of Cbl phosphorylated at Y371 (c-Cbl pTyr371) enhances E2–Ub affinity by approximately 30-fold (Buetow et al., 2016, see citation).

[0168] To evaluate whether photoABP-UbBpa31 could profile the Src-dependent activation of c-Cbl E3 activity, recombinant Src was incubated with Cbl and Cbl Y371F, the latter expected to be refractory to phosphorylation-induced activation. PhotoABP-UbBpa31 crosslinking was observed in c-Cbl in the presence of c-Src but not in c-Cbl Y371F, nor was it observed when c-Src was withheld (Fig. 4a). The labeling was discarded with the photoABP-UbBpa31 F62A control probe (Fig. 11) and was also ATP-dependent (Fig. 12). Thus, consistent with previous studies, phosphorylation at Y371 is particularly required for the activation of E3 activity (Dou et al., 2013, see citation above). In addition, engineered E2–Ub conjugate panels with Bpa incorporation at different positions for c-Src-activated c-Cbl were tested, and it was found that the generation sites partially overlapped with the generation sites for RNF4 (Fig. 13). The optimal Bpa site was 31, but unlike RNF4, site 32 (photoABP-UbBpa31) was also cross-linked with similar efficiency. This may reflect subtle differences between the monomeric and dimeric activation mechanisms exhibited by these two RING E3s (Dou et al., 2013; Plechanovova et al., 2012, see citation above). Interestingly, the photocross-linking efficiency remained below stoichiometric levels regardless of the photoABP-UbBpa31 concentration, suggesting that a subgroup of the recombinant protein formulations is active (Fig. 14). Phostag gel analysis indicated that Cbl was quantitatively phosphorylated (Fig. 15), but Src is known to phosphorylate multiple sites within Cbl, and the degree of probe labeling may indicate stoichiometric phosphorylation at position Y371 (Dou, H. et al. (2012a). Nat Struct Mol BiolWe tested whether incubation with high concentrations of Src could improve photocrosslinking efficiency, but found that photocrosslinking was inhibited as the concentration approached stoichiometry, which appears to be because Src competes with photoABP-UbBpa31 for Cbl bonds (data not shown).

[0169] c-Cbl activation profiling in human cell lines

[0170] To determine whether c-Cbl activation could be profiled in human cell lines, human embryonic kidney cells (HEK293) were transiently transfected with GST-tagged c-Src (GST-Src) in combination with GFP-tagged c-Cbl (GFP-Cbl) or GFP-tagged c-Cbl Y371F (GFP-Cbl Y371F). To prevent potential degradation of the activated Cbl due to autoubiquitination, cells were treated 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 absent in the photoE2~UbBpa31 F62A control probe (Figs. 4b and 16). To provide potential for future use in the parallel proteome profiling of endogenous RING E3 ligase, a biotinylated variant of photoABP-UbBpa31 was prepared. Bpa was incorporated into N-terminal cysteine-tagged Ub and labeled with iodoacetyl-PEG2-biotin. Subsequently, biotin labeled as UbBpa31 was enzymatically conjugated to E2 via an isopeptide using a procedure for untagged Ub (Fig. 17).

[0171] Profiling of Endogenous RING E3 Activation in Response to Growth Factor Stimulation

[0172] Next, we evaluated the ability to perform parallel profiling of endogenous RING E3 activation in response to physiological stimuli. These experiments could potentially attribute the regulatory function of the poorly understood RING E3 across the spectrum of physiological and pathophysiological processes. Since depletion of ABP due to indiscriminate crosslinking can impair RING E3 coverage, we initially tested this with immunoblotting for a hexahistidine reporter tag (Fig. 4c). Furthermore, crosslinking was substantially reduced with the photoABP-UbBpa31 F62A control probe, suggesting that the majority of the crosslinked proteins are likely E3 (Fig. 4c).

[0173] A biotinylated variant of photoABP-UbBpa31 was prepared to enable the selective enrichment of cross-linked proteins from complex cell samples. Bpa was incorporated into N-terminal cysteine-tagged Ub and labeled with iodoacetyl-PEG2-biotin (Pao et al., 2018, see above). Subsequently, biotin labeled with UbBpa31 was enzymatically conjugated to E2 via isopeptide using a procedure for untagged Ub (Figs. 17 and 18). Next, it was tested whether endogenous Cbl activation could be detected in response to EGF stimulation inducing Cbl phosphorylation (Levkowitz et al., 1999; Levkowitz et al., 1998). HEK293T cells were stimulated with EGF and pre-treated with the proteasome and lysosomal inhibitors MG132 and bafilomycin, respectively, to prevent potential degradation of the activating ring E3. Parallel experiments confirmed EGF responsiveness by immunoblotting downstream mitogen-activated protein (MAP) kinase activation, a powerful marker of EGF receptor activation (Traverse, S. et al. (1992). Biochem. J ., 288(Pt 2), 351-355, Fig. 19a).

[0174] The extracted proteome was incubated with biotinylated photoABP-UbBpa31 and enriched for streptavidin resin (Pao et al., 2018, see above; Fig. 4d). The identification of cross-linked proteins and their probe reactivity were inferred by data-dependent liquid chromatography tandem mass spectrometry (LC-MS / MS) and spectral calculations following streptavidin enrichment (Pao et al., 2018, see above). Twenty-five RING E3s, including Cbl, were detected. The Cbl peptide was detected only in samples treated with EGF and UV (Fig. 4e). This suggests that photocross-linked probes can detect natural RING E3 activation at an endogenous level. Interestingly, the spectral coefficients for two other RING E3s, Praja2 and TRIM11, increased significantly and were dependent on EGF and UV (Fig. 4e). Since both of these E3s are associated with growth factor signaling, their detection can also indicate their activation or upregulation in response to EGF stimulation (Di, K. et al. (2013). Oncogene , 32, 5038-5047; Rinaldi, L. et al. (2016). Cell Death Dis. 7, e2230.).

[0175] Unexpectedly, UV-dependent enrichments of HECT (11), RBR (1), and RCR (1) E3, as well as deubiquitination enzyme (DUB) (31) and E1 activating enzyme were obtained (Figs. 19a and 19b). Consequently, probe modifications of these additional ubiquitin system components can modulate their activity and alter the activation state or stability of RING E3 under investigation. However, this will not impose any limitations beyond those associated with the use of cell extracts in which most cellular processes are inhibited.

[0176] Generation of alternative E2-based photocrosslinked ABP

[0177] ABP based on different E2 enzymes was also synthesized. The enzyme UBE2N (also known as Ubc13) was used. A strategy similar to the one described above was used to conjugate the Bpa31 mutant Ub molecule to the active site. Catalytic cysteine ​​(Cys87) was mutated to lysine, which enables stable E1-mediated isopeptide conjugation. It should be noted that in other works using wild-type ubiquitin, conjugation to a natural lysine residue near the active site was observed (Lys92) (Branigan et al. Nat Struct Mol Biol 22, 597-602). Therefore, to ensure homogeneous modification, the UBE2N C87K K92A double mutant was used as exemplified in the study by Branigan et al. The UBE2N probe was functional with the E3 ligase (TRAF6), known as the physiological partner of the UBE2N E2 enzyme (Fig. 20). As previously described for UBE2D3, a UBE2N-based probe biotinized with ubiquitin molecules was also prepared (Ubc13-Biotin-UbBpa3), which was shown to enable activity-based proteomic profiling of the RING E3 ligase in cell extracts.

[0178] Discussion

[0179] In summary, we developed an activity-based probe for the adapter-like activity of RING E3 ligase. We demonstrate how activity-dependent signals for RNF4, c-Cbl, and TRAF6 in response to natural activation signals, and how ABP-based readouts can provide better mechanistic insights. These tools enable the direct assessment of RING E3 activity in various sample types (independent of E1, E2, or substrates). Furthermore, we demonstrated parallel profiling of a subset of endogenous RING E3 in the extracted proteome and detected the activation of Cbl in response to growth factor stimulation. Therefore, this technique should find utility in RING E3 regulatory biology, target discovery, biomarker applications, and regulatory discovery studies. The detection of only a subset of RING E3 in the LC-MS / MS experiments of this invention may reflect that many are inactive or exceed the detection limits of current experimental conditions. Another possibility is that many E3s do not function with the E2 enzymes used (photoABP-UbBpa31 is based on UBE2D3). However, engineered isopeptide conjugation strategies for stabilizing unstable thioesters have been demonstrated with E2s branching from UBE2D3, such as UBE2N (Branigan et al., 2015; Ordureau et al., 2015). Therefore, the highly modular probe generation strategy of the present invention can be easily applied to other E2s simply by using distinct recombinant E2 building blocks. This potentially grants broader RING E3 coverage and provides insights into the cellular E2-E3 interaction network.

Claims

Claim 1 A conjugate molecule comprising an activated ubiquitin molecule of SEQ ID NO. 1 conjugated to an E2 conjugate enzyme, wherein the activated ubiquitin molecule of SEQ ID NO. 1 is modified into a photocrosslinking agent moiety by incorporating a non-natural amino acid in place of a glutamine residue at position 31 and / or an aspartic acid residue at position 32 of the ubiquitin, and the non-natural amino acid is a natural amino acid modified into a functional group capable of forming a linker upon photon irradiation. Claim 2 In paragraph 1, ubiquitin is a conjugate molecule that is human ubiquitin. Claim 3 In claim 1 or 2, the photocrosslinking agent moiety is instead of an aspartic acid / glutamine residue p - A conjugate molecule obtained by incorporating benzoyl-L-phenylalanine. Claim 4 A conjugate molecule according to claim 1, further comprising a reporter tag. Claim 5 In paragraph 4, the reporter tag is a conjugate molecule that is a biotin moiety, an epitope tag, or a fluorescent group. Claim 6 In claim 4 or 5, the reporter tag is a conjugate molecule that is covalently attached to a ubiquitin molecule through a linker molecule attached to the N-terminus of the ubiquitin. Claim 7 In claim 1, the E2 conjugation enzyme is a conjugate molecule that is a mutant enzyme comprising a C85K or C87K mutation and / or an S22R or K92A mutation. Claim 8 In claim 1, the E2 conjugation enzyme is a conjugate molecule that is UBE2D3 or UBE2N. Claim 9 A conjugate molecule according to claim 1, further comprising an N-terminal His-tag group. Claim 10 In claim 9, a conjugate molecule in which the N-terminal His-tag group is a hexahistidine tag. Claim 11 A method for detecting an interaction between a conjugate molecule of claim 1 and a RING E3 enzyme, HECT E3 enzyme, RBR ER3 enzyme, RCR E3 enzyme, E1 enzyme and / or deubiquitination enzyme, the method comprising the steps of contacting the conjugate molecule with the RING E3 enzyme, HECT E3 enzyme, RBR ER3 enzyme, RCR E3 enzyme, E1 enzyme and / or deubiquitination enzyme, and detecting the formation of a new conjugate. Claim 12 In paragraph 11, the RING E3 enzyme is any one or a combination of RNF4, BRAP, RN126, TIF1B, LONF2, HLTF, BRE1A, MIB1, RN114, RN185, BRCA1, BRE1B, TRI11, PJA2, CBL, RAD18, RNF10, TRI18, RN168, PCGF6, RING1, UBR1, TRIM1, LTN1, PJA1, and RN166; the HECT E3 enzyme is any one or a combination of UBR5, HECD1, HUWE1, HECD4, HERC1, TRIPC, UBE3C, HERC2, UBE3A, and ITCH; the RBR E3 enzyme is ARI2; the RCR E3 enzyme is MYCB2; the E1 enzyme is UBA1; and / or The above deubiquitination enzyme is any one or a combination of UBP5, UBP47, UBP24, USP9X, UBP4, UBP15, UBP19, OTUB1, OTUD4, UCHL1, OTUD3, OTU6A, UBP7, UBP14, UBP33, UBP16, UBP36, UBP32, UBP10, UCHL5, USP9Y, UCHL3, UBP6, UBP11, OTU7B, UBP34, OTU6B, ZUFSP, OTUB2, and OTUD5; a method. Claim 13 A method according to claim 11 or 12, wherein the interaction is an interaction with the RING E3 enzyme. Claim 14 delete Claim 15 delete Claim 16 delete

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