Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Griffin, Percy; Sexton, Ashley; MacNeill, Lauren; Iizuka, Yoshie; Lee, Michael K.; Bazzaro, Martina

doi:10.3791/52784

Cited by 3 publications

(4 citation statements)

References 11 publications

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“…An established method for assessing broad DUB selectivity in cells is to convert UbVs to HA-tagged ABPs and treat lysates followed by blotting for HA. ,, To develop ABPs for UCHL1, HA-UbVs containing a C-terminal intein sequence were expressed to allow for attachment of vinylmethyl ester (VME) and propargylamine (PRG) groups to the C-terminus of the HA-UbVs. These groups form covalent adducts with the active site cysteine of DUBs, allowing for assessment of reactivity of our UbVs utilizing Western blot molecular weight shift assays with cell lysates.…”

Section: Resultsmentioning

confidence: 99%

“…DUB engagement assays were performed according to previously published protocols with minor changes. 40 Cell pellets were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 5 mM MgCl 2 , 5 mM DTT, 2 mM ATP, 0.5% NP-40, and 10% glycerol (herein termed cell lysis buffer) for 30 min on ice. Every 10 min, the incubating cells were vortexed for 10 s to ensure homogeneous lysis.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases

et al. 2020

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Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases

et al. 2020

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“…It is also difficult to determine whether aumdubin-mediated tumor suppression in vivo is due to its DUB inhibition activity. Although DUB activity can be measured from tissue by using the Ub-derived active-site-directed probes [ 40 ], it is not clear if lysis procedure would displace the DUB inhibitor from its targets. Although we found that aumdubin induced accumulation of ubiquitinated proteins and speculated that this is caused by DUB inhibition, we still lack direct evidence of aumdubin in DUB inhibition in vivo.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Bax-dependent apoptosis by mitochondrial deubiquitinase USP30

Ding

Yang

et al. 2021

Cell Death Discov.

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Deubiquitinates (DUBs) have been suggested as novel promising targets for cancer therapies. Accumulating experimental evidence suggests that some metal compounds have the potential to induce cancer cell death via inhibition of DUBs. We previously reported that auranofin, a gold(I)-containing agent used for the treatment of rheumatoid arthritis in clinics, can induce cell death by inhibiting proteasomal DUBs in a series of cancer cell lines. Unfortunately, currently available gold compounds are not potent in inhibiting DUBs. Here, we report that: (i) aumdubin, a synthetic derivative of auranofin, exhibited stronger DUB-inhibiting and apoptosis-inducing activities than auranofin in lung cancer cells; (ii) aumdubin shows high affinity for mitochondrial DUB USP30; (iii) aumdubin induces apoptosis by increasing the ubiquitination and mitochondrial location of Bax protein; and (iv) USP30 inhibition may contribute to Bax-dependent apoptosis induced by aumdubin in lung cancer cells. These results suggest that gold(I)-containing agent aumdubin induces Bax-dependent apoptosis partly through inhibiting the mitochondrial DUB USP30, which could open new avenues for lung cancer therapy.

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“…DUB engagement assays were performed according to previously published protocols. [ 20 , 34 ] Cell pellets were lysed in 50 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 5 mM MgCl 2 , 5 mM DTT, 2 mM ATP, 0.5% NP-40, and 10% glycerol (herein referred to as cell lysis buffer) for 30 min on ice. Every 10 min the incubating cells were vortexed for 10 s to ensure homogeneous lysis.…”

Section: Methodsmentioning

confidence: 99%

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

2022

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There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

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Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

Cited by 3 publications

References 11 publications

Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases

Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases

Regulation of Bax-dependent apoptosis by mitochondrial deubiquitinase USP30

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

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