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High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity.

ABSTRACT: Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (associated molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent down-regulation of activated cell surface receptors via the endolysosomal degradative pathway. Therefore, small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently, there are no known selective inhibitors of AMSH or high-throughput compatible assays for their identification. We report the development and optimization of a novel fluorescence resonance energy transfer (FRET)-based add-and-read AMSH DUB assay in a 384-well format. In this format, the optimal temperature for a high-throughput screen (HTS) was determined to be 30°C, the assay tolerates 5% dimethyl sulfoxide (DMSO), and it has a Z-score of 0.71, indicating HTS compatibility. The assay was used to show that AMSH selectively cleaves Lys63-linked diubiquitin over Lys48- and Lys11-linked diubiquitin. The IC50 value of the nonspecific small molecule DUB inhibitor N-ethylmaleimide was 16.2±3.2 ?M and can be used as a qualitative positive control for the screen. We conclude that this assay is high-throughput compatible and can be used to identify novel small molecule inhibitors of AMSH.

SUBMITTER: Arnst JL

PROVIDER: S-EPMC3784021 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity.

Arnst Jamie L JL Davies Christopher W CW Raja Srikumar M SM Das Chittaranjan C Natarajan Amarnath A

Analytical biochemistry 20130605 1

Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (associated molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent down-regulation of activated cell surface receptors via the endolysosomal degradative pathway. Therefore, small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently, there are no k ...[more]

PMID: 23747283

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Project description:AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to the lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view of better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, yields a substantial loss of catalytic activity without any significant change in the thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM; however, the precise mechanism and its significance are not known. On the basis of a number of biochemical and biophysical analyses, we are able to propose a model for activation according to which activation of AMSH is allowed by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence that supports a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate the transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.

Project description:Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5-5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry.

Dataset Information

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity.

Publications

High-throughput compatible fluorescence resonance energy transfer-based assay to identify small molecule inhibitors of AMSH deubiquitinase activity.

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