Biology subjects

Gallant, K.

Publications and source records attributed to Gallant, K..

3 recordsLinked to original sources

bioRxiv2024

Chimeric deubiquitinase engineering reveals structural basis for specific inhibition of USP30 and a framework for DUB ligandability

The mitochondrial deubiquitinase USP30 negatively regulates Pink1/Parkin-driven mitophagy. Whether enhanced mitochondrial quality control through inhibition of USP30 can protect dopaminergic neurons is currently explored in a clinical trial for Parkinsons disease. However, the molecular basis for specific inhibition of USP30 by small molecules has remained elusive. Here, we report the crystal structure of human USP30 in complex with a specific inhibitor, enabled by chimeric protein engineering. Our study uncovers how the inhibitor extends into a cryptic pocket facilitated by a compound-induced conformation of the USP30 switching loop. Our work underscores the potential of exploring induced pockets and conformational dynamics to obtain specific deubiquitinase inhibitors and identifies underlying USP30-specific residues. More broadly, we delineate a conceptual framework for specific USP deubiquitinase inhibition based on a common ligandability hotspot in the Leu73-Ubiquitin binding site and on diverse compound extensions. Collectively, our work establishes a generalizable chimeric protein engineering strategy to aid deubiquitinase crystallization and enables structure-based drug design with relevance to neurodegeneration.

biochemistry↗

bioRxiv2024

Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53

Ubiquitin-specific proteases (USPs) are the largest class of human deubiquitinases (DUBs) and comprise its phylogenetically most distant members USP53 and USP54, which are annotated as catalytically inactive pseudo-enzymes. Conspicuously, mutations in the USP domain of USP53 cause familial intrahepatic cholestasis. Here we report the discovery that USP53 and USP54 are in fact active DUBs with high specificity for K63-linked polyubiquitin. We demonstrate how USP53 patient mutations abrogate catalytic activity, implicating loss of DUB activity in USP53-mediated pathology. Depletion of USP53 increases K63-linked ubiquitination of tricellular junction components. Assays with substrate-bound polyubiquitin reveal that USP54 cleaves within K63-linked chains, whereas USP53 can deubiquitinate a substrate in a K63-linkage-dependent manner. Biochemical and structural analyses uncover underlying K63-specific S2-ubiquitin-binding sites within their catalytic domains. Collectively, our work revises the annotation of USP53 and USP54, provides chemical reagents and a mechanistic framework to broadly investigate K63-polyubiquitin chain length decoding, and establishes K63-linkage-directed deubiquitination as novel DUB activity.

biochemistry↗

bioRxiv2021

HiIDDD: A high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells

DNA damage is a prominent biomarker for numerous diseases, including cancer and aging. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, semiquantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and {gamma}-H2AX. We demonstrate sensitive detection of DNA damage in a wide set of freshly isolated and cryopreserved primary human immune cells, including CD4+ and CD8+ T cells, B cells and monocytes with low inter-assay variability. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

cell biology↗