Biology subjects

Strieter, E. R.

Publications and source records attributed to Strieter, E. R..

5 recordsLinked to original sources

bioRxiv2025

Site-Specific Nanobody Inhibitors of the Proteasomal Deubiquitinase UCH37

Dysregulation of the ubiquitin (Ub) proteasome system (UPS) is linked to numerous human diseases, making its components attractive therapeutic targets. Among these, the proteasomal deubiquitinase UCH37 (also known as UCHL5) has two distinct Ub-processing activities: C-terminal Ub hydrolysis and K48-linkage-specific chain debranching. Separating these activities has not been possible with existing small-molecule inhibitors, which target the catalytic site and broadly affect both functions along with many other DUBs. Here, we report the development of two site-specific nanobody inhibitors of UCH37 that together enable this separation. Using yeast surface display, we generated Nb-cS1, which binds the canonical Ub-binding (S1) site and blocks both activities, and Nb-ncS1, which binds the non-canonical K48-specific (ncS1) site and selectively impairs chain debranching without affecting C-terminal Ub hydrolysis. X-ray crystallography and hydrogen-deuterium exchange mass spectrometry define the distinct binding surfaces engaged by each nanobody. In cells, both nanobodies selectively engage UCH37, with Nb-ncS1 showing the cleanest target engagement. These tools provide the first means to independently interrogate UCH37s two enzymatic activities and offer new approaches for dissecting the distinct roles of UCH37 in proteasomal and chromatin-associated contexts.

biochemistry↗

bioRxiv2025

Computationally Driven Top-Down Mass Spectrometry of Ubiquitinated Proteins

Ubiquitination regulates numerous cellular processes through the attachment of polyubiquitin (Ub) chains that vary in linkage type, length, and branching topology. However, current mass spectrometry approaches cannot simultaneously define both the site of ubiquitination and the topology of the attached Ub chain on intact protein substrates. Here, we present the first integrated strategy that enables simultaneous determination of ubiquitin site and chain architecture using top-down mass spectrometry (TD-MS). Central to this approach is UbqTop, a custom computational platform that predicts Ub chain topology from tandem MS (MS{superscript 2}) fragmentation data by utilizing Bayesian-like scoring algorithm. To address the challenge of analyzing complex substrates, we combine this with selective Asp-N proteolysis, which digests the substrate while preserving intact Ub chains. This enables direct, site-resolved mapping of Ub chain topology on proteins. We demonstrate the broad utility of this method on both free Ub chains and multiply ubiquitinated protein substrates, including the resolution of isomeric chains and branched architectures. Together, this work establishes a powerful new framework for proteoform-level analysis of ubiquitin signaling with unprecedented structural resolution.

bioinformatics↗

bioRxiv2023

Uncovering DUB Selectivity Through Ion-Mobility-Based Assessment of Ubiquitin Chain Isomers

Ubiquitination is a reversible posttranslational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological function. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native, unlabeled ubiquitin conjugates. Here we report on an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-MS can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM coupled with mass spectrometry (IM-MS) is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single component mixtures.

biochemistry↗

bioRxiv2021

Ubiquitination drives COPI priming and Golgi SNARE localization

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime COPI assembly to ensure incorporation of these essential cargoes into vesicles but the regulation of these events is poorly understood. Here we report a roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs. The ability of COPI to bind ubiquitin through its N-terminal WD repeat domain of {beta}COP or through an unrelated ubiquitin-binding domain (UBD) is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3 and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is thought to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

cell biology↗

bioRxiv2020

Proteasome-Bound UCH37 Debranches Ubiquitin Chains to Promote Degradation

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, however the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, exclusively cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using proteasome complexes reconstituted with either active or inactive UCH37 together with protein substrates modified with branched chains, we find that chain debranching promotes degradation under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

biochemistry↗