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DUB PRODUCTS & SERVICES 2013

DUBs in Cell Signaling • DUB Profiling Services • Ubiquitin Chains

Deubiquitinating Enzymes (DUBs) in Cell Signaling

Deubiquitinating Enzymes (DUBs) are key regulators of cellular pathways by reversing Ubiquitin-mediated events. Recent studies have revealed that DUBs are dynamic enzymes that partner with various interacting proteins to facilitate both substrate selection and DUB activity. This regulation in the Ubiquitin proteasome system is underscored by the increasing evidence that many DUBs are part of Ubiquitin ligase complexes, which enables DUBs to regulate the activity of many cellular pathways and processes, including: • Cell Signaling • Endocytosis • NF-kB Signaling • DNA Damage • Cell Proliferation • Growth Factor Signaling • Viral Replication • Oxidative Stress Signaling • Apoptosis

Boston Biochem has the best DUB research portfolio providing the most trusted products and services supported by the best technical expertise available. We are your source for DUB related research.

CAT # DUB Enzymes Process CAT # DUB Enzymes (continued) Process

E-320 Isopeptidase T/USP5 Catabolism NEW E-570 His6-USP28 Cell proliferation

E-324 Isopeptidase T/USP5 Short Form Catabolism NEW E-519 His6-USP7 Apoptosis

E-325 UCH-L3 Cancer NEW E-520 His6-USP8 Cell signaling

E-340 UCH-L1 Cell proliferation E-800 His6-NEDP1/SENP8

E-341 His6-Ataxin-3 Cell death CAT # DUB Substrates Description

E-344 His6-A20/TNFAIP3 Catalytic Domain Apoptosis U-550 Ubiquitin-AMC Most referenced

E-345 His6-BAP1 Tumor suppressor U-551 Ubiquitin-AFC Spectral shift E-504 USP2 Catalytic Domain Myogenesis U-555 Ubiquitin-Rhodamine 110 Industry standard

E-506 His6-USP2 Catalytic Domain Myogenesis NEW U-556 Ubiquitin-Aminoluciferin Sensitivity

E-518B His6-USP7 Catalytic Domain DNA repair U-558 Ubiquitin-Lys-TAMRA Polarization

E-519 His6-USP7 DNA repair NEW UF-210 Di-Ub (K48-linked) FRET TAMRA Pos1

E-520 His6-USP8 Ras protein signaling UF-221 Di-UB (K48-linked) FRET EDANS Pos2 Isopeptide Bond

E-522 His6-Otubain-1 DNA repair UF-310 Di-Ub (K63-linked) FRET TAMRA Pos1 Isopeptide bond E-544 USP14 Inhibitor of ERAD UF-320 Di-Ub (K-63 linked) FRET TAMRA Isopeptide Bond E-546 USP25-His ER degradation NEW 6 NEW UF-440 Di-Ub (K11-linked) FRET Isopeptide bond NEW E-548B AMSH Endocytosis S-100 Z-Leu-Arg-Gly-Gly-AMC NEW E-549 GST-AMSH Endocytosis CAT # DUB Inhibitors Enzyme NEW E-550 STAM-1 AMSH activator U-201 Ubiquitin Aldehyde PAN DUB

NEW E-552 USP9x-His6 Cancer U-202 Ubiquitin-Vinyl Sulfone PAN DUB

NEW E-554 His6-Otubain-2 ERAD U-203 Ubiquitin-Vinyl Methyl Ester PAN DUB

NEW E-556 His6-CYLD NF-kB signaling U-211 HA-Ubiuitin-Vinyl Sulfone DUB probe NEW E-558 FAM105B/OTULIN Inflammation NEW U-212 HA-Ubiquitin-Vinyl Sulfone DUB probe NEW E-560 ZRANB1/Trabid Wnt signaling U-214 Ubiquitin-propargylamide PAN DUB NEW E-562 OTUD7B/Cezanne Inflammation 2647 NSC 632839 Hydrochloride Cell perm USP9x

NEW E-564 His6-USP1 DNA damage response 3998 LDN 57444 Cell perm UCH-L1

NEW E-566 His6-UAF1 USP1 activator 4088 IU1 Cell perm USP14

NEW E-568 His6-USP1/UAF1 DNA damage response 4285 HBX 41108 Cell perm USP7

www.bostonbiochem.com DUB Profiling Services Boston Biochem offers customized in vitro profiling of your de-ubiquitinating enzyme (DUB), DUB substrate, and/or DUB activator or inhibitor. We can characterize your known DUB or confirm your putative DUB by testing it against all of the classic DUB substrates and inhibitors currently available. Our scientists will assay the activity of your DUB with any or all of our available substrates and inhibitors or test your inhibitor, substrate, or activator against the most comprehensive list of DUBs available.

Ubiquitin specificity can be determined by assaying for cross hydrolysis or inhibition using our extensive list of ubiquitin-like modifier (UBL) deconjugating enzyme substrates and inhibitors. We can also determine the optimal antibody for your DUB detection requirements, while checking for any cross reactivity utilizing our extensive range of poly- and monoclonal antibodies. If applicable, we can customize the appropriate components and information as a kit for you to test your enzyme or inhibitor in-house. Summary • Selectivity and specificity of DUB substrates • Test activation or interaction of your protein • Most comprehensive substrates available to with known DUBs monitor activity of all DUBs • Customizable DUB panel • Potency of compound vs. existing DUB panel • Most comprehensive substrates available to • Single concentration or IC50 determination monitor activity of all DUBs

Characterization of your DUB Enzyme Substrate Specificity Figure 1: AMSH/STAM processing of mono- and diubiquitin fluorogenic substrates. Not all DUBs hydrolyze the same substrates in vitro. Some prefer small ubiquitin C-terminal leaving groups (Ubiqui-Fluors) while K63 Di-Ub FRET others will only hydrolyze the real isopeptide bonds in ubiquitin K48 Di-Ub FRET chains. A group exists that can do both at various efficiencies K11 Di-Ub FRET and those efficiencies can be evaluated and quantitated. Of the Ub-AMC DUBs that hydrolyze chains, they can also have linkage specific activity. We can quickly and efficiently match your DUB to the RFU appropriate substrate and if multiple substrates are viable, we can determine which is optimal for your research application or assay format. Other ubiquitin-like substrates for the study of NEDPs, SENPs, etc. can be tested for cross hydrolysis with your enzyme for complete characterization.

Inhibitor Specificity Reac1on Time (seconds) Another important confirmation of a DUB enzyme is the ability Figure XX: AMSH/STAM processing of mono-­‐ and diubiquiIn fluorogenic substrates. to block activity with classic DUB inhibitors. Once an optimal in200nM AMSH was pre-­‐incubated with 3µM STAM for 30 minutes at 37°C. Assays were then ini1ated by the addi1on of 0.5 µM Ub-­‐ AMC, or Figure 0.5 µM 2: Active K11-­‐, K48-­‐, titration or of K63-­‐diubiqui1n UCHL3 by Ubiquitin-aldehyde. FRET substrate. AMSH/STAM efficiently hydrolyzed K63-­‐linked diubiqui1n substrate, vitro substrate is chosen and assay format is established, we willbut did not hydrolyze any of the other substrates including Ub-­‐AMC. test our known DUB inhibitors versus your enzyme for further 1.2 UCHL3 = 3.0 nM characterization. Other ubiquitin-like inhibitors for the study of UCHL3 = 6.2 nM NEDPs, SENPs, etc. can be tested for cross inhibition with your 1.0 enzyme for completion of characterization. 0.8 Antibody Specificity 0.6 x intercept = 3.0 Boston Biochem has over 80 DUB specific poly- and mono- 0.4 x intercept = 6.2 clonal antibodies for the detection of UCHs, USPs, OTUs, MJDs, Ini1al Velocity (RFU/sec) 0.2 JAB1s, SENPs, NEDP, and APG4B. Using any or all of our available antibodies we can determine what antibody works best for 0.0 your detection application whether a simple western, IP, or ELISA. The issue of cross reactivity is always part of antibody 0 10 20 30 40 50 characterization and once your optimal application antibody is [Ubiqui1n-­‐aldehyde] nM identified, we can test it against our panel of DUBs to identify Conclusion: X-intercepts demonstrate Ubiquitin-aldehyde is a any cross reactivity. viable active site titrant for UCHL3. Figure XX: InhibiIon of UCHL3 by UbiquiIn-­‐aldehyde. 1 nM (green circles, solid line) or 2 nM (blue circles, broken line) UCHL3 was pre-­‐incubated with various dilu1ons of Ubiqui1n-­‐ aldehyde (“Ub-­‐H”) prior to addi1on of Ubiqui1n-­‐AMC substrate. Ini1al reac1on veloci1es (RFU/second) were ploced versus the dilu1on www.bostonbiochem.com factor of Ub-­‐H to obtain an X-­‐intercept. The X-­‐intercept is the value of the dilu1on factor that when mul1plied by enzyme concentra1on yields the concentra1on of the original stock solu1on of Ub-­‐H. Characterization of your Inhibitor, Activator or Substrate

Figure 3: IC Curve for AMSH/STAM Treated with Inhibitor 50 Compound BBI-427 Boston Biochem has expertise that allows you to test your

protein, peptide or small molecule inhibitor with any or all 100 100 80 of our available DUBs. Critical to this analysis is the use of 60 90

RFU 40

appropriate substrates and assay formats for each DUB to 80 20 70 0 generate interpretable data. We have assays up and running 0 150 300 450 600 for every DUB in our portfolio. 60 seconds 50

Inhibition 40

If you are studying a DUB that is not in our portfolio, we will % 30 % Inhibi1on work with you to develop the appropriate assay to properly 20 assess inhibitory activity. All inhibition assay formats will be 10 0 confirmed with control DUB inhibitors. -10 -20 0.01 0.1 1 10 100 uM Compound [BBI-­‐427] µM

Activator Figure XX: IC50 Curve for AMSH/STAM Treated with Compound BBI-­‐427 100 nM AMSH and 1.5 µM STAM were pre-­‐incubated for 30 minutes at 37°C with various concentra1ons of BBI-­‐427 in 1% DMSO (final). Assays Test your small molecule, peptide, or protein for the ability to were increase ini1ated hydrolysis with the rates addi1on of any of 0.4 or all µM of our K63 diubiqui1n DUBs versus FRET the substrate in Reac1on Buffer. Ini1al veloci1es were calculated from progress curves (inset) for all reac1ons, then % inhibi1on was calculated using DMSO-­‐only, and no-­‐enzyme controls. Finally, concentra1on of BBI-­‐427 appropriate substrates. was plo]ed versus % inhibi1on to calculate an IC50 value. Figure 4: Reaction Progress Curves for AMSH with Increasing Figure 5: Dose Response analysis of STAM stimulating Concentrations of STAM Activator Protein. AMSH activity. ) -­‐1 ( nM s ini#al RFU V RFU

Reac1on Time (s)

Reac1on Time (s) STAM1 (μM) Figure XX: Dose Response analysis of STAM sImulaIng AMSH acIvity. 200 nM AMSH was pre-­‐incubated with 0 -­‐ 6.4 µM STAM for 30 minutes at 37°C, then assays were ini1ated with the addi1on of 0.5µM K63 diubiqui1n FRET substrate in Reac1on Buffer. Half-­‐maximal ac1va1on of AMSH occurred at 400 nM STAM, and >90% ac1va1on was achieved at Substrate 3 µM STAM. (Inset: Reac1on progress curves for AMSH in the presence of increasing concentra1ons of STAM) Boston Biochem will determine what DUBs hydrolyze your substrate via appropriate assay. If there are multiple DUBs that show activity against your substrate we will determine efficiencies and rates for the positively identified DUBs. Figure 6: AMSH/STAM and USP9x hydrolysis of various FigureRepresentative 7: UCHL3 catalyzed hydrolysisAssay of Data Ubiqui-Fluor linkages of diubiquitin. substrates: kinetic constants.

K6 K11 K27 K29 K33 K48 K63 linear ; MW + – + – + – + – + – + – + – + – Ubiqui-Fluor Antibody Detection 26S & 19S DUBs 21 kDa

14 kDa Di-Ub

6 kDa Mono-Ub

K6 K11 K27 K29 K33 K48 K63 linear ; MW + – + – + – + – + – + – + – + –

21 kDa

Di-Ub 14 kDa

6 kDa Mono-Ub

Figure XX: AMSH/STAM and USP9x hydrolysis of various linkages of diubiquiIn. 2µg of K6, K11, K27, K29, K33, K48, K63, and linear diubiqui1n were incubated with 100 nM AMSH and 600 STAM (upper panel) or 1 USP9x (bo]om panel) in Reac1on Buffer at 37°C for 60 minutes. Control reac1ons contained diubiqui1n substrate but no deubiqui1nase. Reac1ons were separated using 10-­‐20% SDS-­‐PAGE and visualized by Coomassie staining. AMSH/STAM hydrolyzed only K63-­‐linked diubiqui1n, whereas USP9x hydrolyzed all linkages other than linear and K27-­‐linked diubiqui1n. www.bostonbiochem.com

Ub chain disassembly Linkage Specific Chains POH1

Ub-AMC Hydrolysis

● Characterization of your Inhibitor, Activator or Substrate Inhibitor

Activator

Substrate

® Ubiquitin Chains Native Ubiquitin Chains Ubiquitin (Ub) is a highly conserved globular 76-residue eukaryotic protein Figure 8: Purified, K63-linked polyubiquitin chains of found in the cytoplasm and nucleus of cells. Ubiquitin exists both as a monomer length 2-5. Treating the chains with USP5 results in complete disassembly into mono-ubiquitin. and as isopeptide-linked polymers known as poly-ubiquitin chains. Di Tri Tetra Penta : Chain length - + - + - + - + : USP5 Unanchored (free c-terminus) polyubiquitin chains have been identified as MW potent and essential components in signaling pathways with functions just now 97 66 starting to be discovered. 55

36 • All Lysine Linkages: K6-, K11-, K27-, K29-, K33-, K48-, K63-, and Linear 31

• Various Lengths: Poly-ubiquitin chains from di (2) to octa (8) 21 • Custom Ubiquitin Chains: Chains can be formed with different 14 linkages and lengths 8 • Bulk Quantities

DUB-Resistant, Non-Hydrolyzable (NH) Chains Purified, K63-­‐linked polyubiqui?n chains of length 2-­‐5. Trea?ng the chains with USP5 results in complete disassembly into mono-­‐ The identification of pathways and cellular processes that utilize free polyubiquitin chains ubiqui?n. are difficult to identify due to their transient nature. To facilitate research in the area of unanchored polyubiquitin chains, we have developed a series of non-hydrolyzable (NH) polyubiquitin chain reagents. We offer chains of various linkages (linear, K11, K48, and K63) and lengths (di-, tri-, tetra, etc.) with two types of DUB-resistant linkages (NH and DCA). These chains are available free or covalently coupled to agarose for affinity pull downs. • DUB Resistant Chains • Various Linkages • UBA/UIM Binding Studies RIGI/MAVS Antiviral Signaling Pathway • Signal Transduction Studies • Stable in Crude Lysates • Free Chains • Agarose Bound for Affinity Applications Non-Hydrolyzable, Isopeptide-linked Chains These are chains that contain bona fide isopeptide bonds. An modification on the C-terminus of ubiquitin makes these chains DUB-resistant. Available as free or agarose bound chains. • K11-linked • K48-linked E.J. Bennett and J.W. Harper, Cell 141, April 16, 2010 • K63-linked • Linear chains DCA-linked Chains Dichloroacetone (DCA) forms of di-ubiquitin (Ub2) are chemically linked via a DCA linker using cysteine introduced into the ubiquitin sequence at the appropriate residues to mimic a native isopeptide linkage. DCA linkage is stable and cannot be processed by deubiquitinases. Available as free or agarose bound chains. • K6-linked • K48-linked • K11-linked • K63-linked • K29-linked • K76-linked chains • K33-linked

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