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Characterization of Full-Length, Recombinant AMSH/STAM, USP25, and USP9x Carsten Schwerdtfeger, Ivan Tomasic, Nate Russell, Bradley Brasher, Anthony Mauriello, Greg Tuffy, Thamara DeSilva and Francesco Melandri Boston Biochem Inc., Cambridge, MA 02139 Attachment of polyubiquitin to substrate proteins generates important biological signaling cues that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling cascades. An important feature of protein ubiquitination is that it is reversible. Substrate- anchored chains may be edited or removed from proteins by specialized proteases called deubiquitinating enzymes (DUBs). Currently, there are 80-90 DUBs identified in humans and many have been identified as potential drugable targets because of their involvement in various disease states. Deubiquitinase activity is often modulated by multiple parameters, including 1) specificity for a protein substrate(s) to which polyubiquitin chains are conjugated, 2) protein cofactor(s) that may be required for DUB activation, or 3) preference for polyubiquitin linkage- types. Thus, understanding the mechanisms, kinetics, and substrate preferences for deubiquitinases is of great interest, from both academic and clinical viewpoints. Kinetic Analysis of USP25 with Fluorogenic Ubiquitin Substrates A B A B Figure 10: USP9x hydrolysis of Ub-AMC and Ub-Rh110. A: Reactions containing 1nM USP9x were initiated by the addition of Ub-AMC at final concentrations of 50nM – 8μM. Reactions were carried out in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC. Results are shown in Table 3. B: USP25 reactions detailed in 10A were repeated, substituting Ub-Rh110 (shown above) or K63-linked di-Ub FRET (not shown) for Ub-AMC substrate. Results are shown in Table 3. K m (μM) K cat (s -1 ) k cat /K m (M -1 s -1 ) Ub-AMC 9.4 0.37 3.9 x 10 4 Ub-Rh110 12.4 0.30 2.4 x 10 4 K63 Di-Ub FRET 7.5 0.11 1.4 x 10 4 K m [μM] K cat [s -1 ] k cat /K m [M -1 s -1 ] Ub-AMC 0.7 0.80 1.1 x 10 6 Ub-Rh110 1.9 1.14 6.1 x 10 5 K63 Di-Ub FRET 3.8 0.97 2.5 x 10 5 Table 2. Tabulated Steady State Kinetic Parameters of USP25 Table 3. Tabulated Steady State Kinetic Parameters of USP9x Figure 7: USP25 hydrolysis of various linkages of di-ubiquitin. 2μg of K6, K11, K27, K29, K33, K48, K63, and linear di-Ub were incubated with 20nM USP25 in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min. Control reactions contained di-Ub substrate but no USP25. Reactions were separated using 10-20% SDS-PAGE and visualized by Coomassie staining. Figure 11: USP9x hydrolysis of various linkages of di-ubiquitin. 2μg of K6, K11, K27, K29, K33, K48, K63, and linear di-Ub were incubated with 1nM USP9x in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min. Control reactions contained di-Ub substrate but no USP9x. Reactions were separated using 10-20% SDS-PAGE and visualized by Coomassie staining. Figure 9. Sumoylation of USP25 and its effect on deubiquitinase activity. A. Sumoylation reactions containing 400nM SUMO E1, 4μM Ubc9, 500nM PIAS2α, 800nM USP25, 25uM SUMO3, and 10mM Mg-ATP were incubated at 30°C for indicated times, then terminated for SDS-PAGE analysis. In subsequent experiments complete sumoylation of USP25 was achieved (data not shown), and this served as the source of USP25 used in the deubiquitinase assays described in 9B. B. Deubiquitinase were set up with 100nM sumoylated or non-sumoylated USP25 using either 250nM Ub-AMC or K63-Di-Ub-FRET substrates at 25°C. Initial velocities were calculated for each of the four reactions. For each substrate type, the initial velocity of the non-sumoylated USP25 (control) was defined as 100%, then the velocity of the sumoylated USP25 was plotted relative to that control. Sumoylated USP25 displayed a 25% reduction in initial velocity compared to non-sumoylated enzyme in reactions monitored by Ub-AMC. In contrast, sumoylated USP25 activity against di-ubiquitin was reduced nearly 75% relative to the unmodified enzyme. Figure 8: USP25 hydrolysis of Ub-AMC in the presence of non-hydrolyzable di-Ub. 10nM USP25 was pre-incubated for 60min at 37ºC with 0-2μM of K6, K11, K29, K33, K48, K63, and 76-76 DCA-linked non-hydrolyzable Di-Ub chains. Assays were initiated with the addition of 250nM Ub-AMC substrate in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC. A. Representative dose-response curve for reactions run with K63-linked DCA di-Ub. B. Percentage of remaining initial activity (vs. uninhibited control reaction) of USP25 in the presence of non-hydrolyzable DCA di-Ub of various linkages. Figure 12: USP9x hydrolysis of Ub-AMC in the presence of non-hydrolyzable di-Ub. 1nM USP9x was pre-incubated for 60min at 37ºC with 0-2μM of K6, K11, K29, K33, K48, K63, and 76-76 DCA-linked non-hydrolyzable Di-Ub chains. Assays were initiated with the addition of 250nM Ub-AMC substrate in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC. A. Representative dose-response curve for reactions run with K63-linked DCA di-Ub. B. Percentage of remaining initial activity (vs. uninhibited control reaction) of USP9x in the presence of non-hydrolyzable DCA di-Ub of various linkages. . A A A B Ubiquitin-Fluor’s (C-terminal cleavage releases fluorescent product) A Di-Ubiquitin Chains (Isopeptide bond cleavage) Di-Ubiquitin FRET (Isopeptide bond cleavage and release of quenched Fluorophore) Non-hydrolyzable DCA linked Di-Ubiquitin Chains (Competitive DUB inhibitors ) Fluor DUB Pro-Fluor Ub - Leu 73 – Arg 74 – Gly 75 – Gly 76 DUB Lys Ub - Leu 73 – Arg 74 – Gly 75 – Gly 76 Ub Ub Lys Ub DUB DUB Ub - Leu 73 – Arg 74 – Gly 75 – Cys 76 AMC Ub - Leu 73 – Arg 74 – Gly 75 – Gly 76 Cys Ub DUB X DCA Inhibition of USP25 with Non-Hydrolyzable DCA-Linked Di-Ubiquitin Effect of Sumoylation on USP25 Activity QSY Lys Ub - Leu 73 – Arg 74 – Gly 75 – Gly 76 Ub DUB DUB FL Lys Ub QSY Ub FL Figure 1: Tools for studying deubiquitinating enzymes. Several substrates and substrate analogs are useful for characterizing deconjugating enzymes in vitro, using kinetic or gel-based assays. A. Fluorogenic C-terminal derivatives are useful for kinetic studies since the release of the fluorophore (AMC, AFC, or R110) results in a signal that is directly proportional to activity. B. Di-ubiquitin substrates are linked via native isopeptide bonds and disassembled in vitro for gel- based end-point assays. C. Fluorophore labeled di-ubiquitin FRET substrates are linked via isopeptide bonds and disassembled in vitro by DUBS to study chain disassembly in plate-based real-time assays. D. Non-hydrolyzable dichloroacetone-linked (DCA) di-ubiquitin chains are competitive inhibitors of some DUBs and may be used to investigate chain specificity of the enzymes. Di-Ub Mono-Ub 14kDa 21kDa 6kDa K6 K11 K27 K29 K33 K48 K63 linear ; MW + – + – + – + – + – + – + – + – 14kDa 21kDa 6kDa K6 K11 K27 K29 K33 K48 K63 linear ; MW + – + – + – + – + – + – + – + – K i = 124nM K i = 853nM USP25-SUMO3 n 116kDa 200kDa MW 0 10 20 30 40 : Time (min) SUMO 3 USP25 66kDa 35kDa 31kDa 14kDa 55kDa 21kDa 6kDa PIAS2α Introduction Substrates for Analyzing DUB Activity USP25 Hydrolysis of Di-Ubiquitin Chains Kinetic Analysis of USP9x with Fluorogenic Ubiquitin Substrates USP9x Hydrolysis of Di-Ubiquitin Chains Inhibition of USP9x with Non-Hydrolyzable DCA-Linked Di-Ubiquitin Summary B C D Biology of AMSH, USP25, and USP9x AMSH is a JAMM-class metalloprotease that specifically cleaves K63-linked polyubiquitin chains. This DUB is activated by its partner STAM at the endosome, where its activity opposes ubiquitin-dependent sorting of receptors to lysosomes. AMSH plays important roles in cell growth, and IL-2, GM-CSF, and BMP (bone morphogenetic protein) signaling pathways. USP25 hydrolyzes ubiquitin-conjugated substrates and may be involved in the processing of newly synthesized ubiquitin. This DUB is reported to hydrolyze both K48- and K63-linked polyubiquitin. A muscle-specific USP25 isoform may have a role in the regulation of muscular differentiation and function. Sumoylation in the vicinity of the tandem UIM domains is reported to diminish USP25 hydrolysis of polyubiquitin chains. USP9x is an essential component of TGFβ/BMP signaling cascade. USP9x biology is likely to be complex, as increased expression of the DUB correlates with increased MCL1 protein—a driving force in human follicular lymphoma and diffuse large B-cell lymphomas, whereas decreased expression of USP9x cooperates with K-ras mutations to accelerate aggressive pancreatic tumors in mice. This DUB is reported to specifically hydrolyze K29- and K33-linked polyubiquitins chains, as well as numerous K48-polyubiquitinated substrates. Domain Structure of AMSH, USP25, and USP9x JAMM/MPN+ MIT AMSH (424aa) USP25 (1087aa) UBL USP USP9x (2547aa) Domains: UBA UBL UIM USP JAMM/MPN+ MIT Ubiquitin Associated Ubiquitin-Like Microtubule-Interacting & Trafficking Ubiquitin Interacting Motif Ubiquitin-Specific Proteases Domain Metalloenzyme catalytic domain SIM SUMO Interacting Motif Site of poly-sumoylation in USP25 Ψ UBA UIM UIM USP SIM Ψ Figure 2: AMSH/STAM hydrolysis of various linkages of di-ubiquitin. 2μg of K6, K11, K27, K29, K33, K48, K63, and linear Di-Ub were incubated with 100nM AMSH and 600nM STAM in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC for 60min. Control reactions contained di-Ub substrate but no AMSH/STAM. Reactions were separated using 10-20% SDS-PAGE and visualized by Coomassie staining. K6 K11 K27 K29 K33 K48 K63 linear ; MW + – + – + – + – + – + – + – + – 14kDa 21kDa 6kDa AMSH/STAM Hydrolysis of Di-Ubiquitin Chains AMSH/STAM Hydrolysis of Fluorogenic Ubiquitin Substrates K63 Di-Ub FRET K48 Di-Ub FRET K11 Di-Ub FRET Ub-AMC RFU Reaction Time (seconds) Figure 3: AMSH/STAM processing of mono- and di-ubiquitin fluorogenic substrates. 200nM AMSH was pre-incubated with 3μM STAM for 30min at 37ºC. Assays were then initiated by the addition of 0.5μM Ub-AMC, or 0.5μM K11-, K48-, or K63-Di-Ub FRET substrate. All reactions were conducted in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC. Figure 4: Dose Response analysis of STAM stimulating AMSH activity. 200nM AMSH was pre-incubated with 0 - 6.4μM STAM for 30min at 37ºC, then assays were initiated with the addition of 0.5μM K63 Di-Ub FRET substrate in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC. Half-maximal activation of AMSH occurred at 400nM STAM, and >90% activation was achieved at 3μM STAM. (Inset: Reaction progress curves for AMSH in the presence of increasing concentrations of STAM) Kinetics of AMSH Activation by STAM STAM1 (μM) V initial (nM s -1 ) Reaction Time (s) RFU K63 Di-Ub FRET (μM) Figure 5: AMSH activity assay in the absence or presence of STAM activating protein. A: 200nM AMSH was pre-incubated for 30min at 37ºC. Assay was then initiated by the addition of K63-linked Di-Ub FRET substrate at final concentrations of 0.10μM-13μM. Reactions were carried out in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 37ºC. Results are shown in Table 1. B: 200 nM AMSH plus 3μM STAM were pre-incubated for 30min at 37ºC, then reactions were conducted as described in 5A. Results are shown in Table 1. B K m (μM K63 Di-Ub) K cat (s -1 ) k cat /K m (M -1 s -1 ) AMSH 16.5 7.65 x 10 -3 0.47 AMSH + STAM 1 2.4 1.42 x 10 -2 5.91 Kinetic Analysis of AMSH, and AMSH/STAM with Fluorogenic Di-ubiquitin Substrate V initial (nM s -1 ) A Table 1. Tabulated Steady State Kinetic Parameters of AMSH and AMSH/STAM K63 Di-Ub FRET (μM) V initial (nM s -1 ) Figure 6: USP25 hydrolysis of Ub-AMC and Ub-Rh110. A: Reactions containing 10nM USP25 were initiated by the addition of Ub-AMC at final concentrations of 50nM – 8μM. Reactions were carried out in 50mM HEPES pH8, 100mM NaCl and 2mM DTT at 25ºC. Results are shown in Table 2. B: USP25 reactions detailed in 6A were repeated, substituting Ub-Rh110 (shown above) or K63-linked di-Ub FRET (not shown) for Ub-AMC substrate. Results are shown in Table 2. V initial (nM s -1 ) V initial (nM s -1 ) Ub-AMC (μM) Ub-Rh110 (μM) V initial (RFU s -1 ) K63-linked DCA Di-Ub (nM) Activity at 1μM Inhibitor (%) B DCA Di-Ubiquitin Linkage Activation 1/2 = 400nM USP25 Activity (%) Ub-AMC K63-Di-Ub FRET V initial (RFU s -1 ) K63-linked DCA Di-Ub (nM) B Activity at 1μM Inhibitor (%) DCA Di-Ubiquitin Linkage AMSH USP25 USP9x Di-ubiquitin chain specificity in SDS-PAGE assay K63 only K11, K33, K48, K63 K6, K11, K29, K33, K48, K63 Utilizes mono-Ub fluorogenic substrates (Ub-AMC)? No Yes Km: 20.9 μM kcat: 0.7 s -1 kcat/Km: 2.6 x10 4 M -1 s -1 Yes Km: 0.7 μM kcat: 0.7 s -1 kcat/Km: 1.1 x10 6 M -1 s -1 Utilizes di-Ub FRET substrates? K63 only Km: 16.5 μM kcat: 7.7x10 -3 s -1 kcat/Km: 4.7x10 -1 M -1 s -1 K63 Km: 7.5 μM kcat: 1.1 x10 -1 s -1 kcat/Km: 1.4 x10 4 M -1 s -1 K48 not determined K63 Km: 3.8 μM kcat: 9.8 x10 -1 s -1 kcat/Km: 2.5 x10 5 M -1 s -1 K11, K48 not determined Inhibited by DCA- linked di-Ub? (> 50% inhibition at 1μM DCA di-Ub) No Yes K33, K48, K63, 76-76 Yes K29, K48, K63, 76-76 Inhibited by sumoylation? N/D Yes V init with Ub-AMC ↓25% V init with K63 di-Ub FRET ↓75% N/D Activated by STAM Yes Km: 2.4 μM kcat: 1.4 x10 -2 s -1 kcat/Km: 5.9 M -1 s -1 (kcat/Km ↑ 13-fold) N/D N/D V initial (nM s -1 ) V initial (nM s -1 ) Ub-AMC (μM) Ub-Rh110 (μM) Di-Ub Mono-Ub Di-Ub Mono-Ub
