Biology subjects

Jones, H. B.

Publications and source records attributed to Jones, H. B..

4 recordsLinked to original sources

bioRxiv2026

STI1 domains coordinate partitioning of UBQLN2 into stress-induced condensates

UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation in vitro and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that in vitro phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions. HighlightsO_LIUBQLN2 is recruited to both stress granules and puncta formed by the autophagy receptor protein p62 in response to heat stress. C_LIO_LIBoth endogenous and overexpressed UBQLN2 tend to colocalize with p62 puncta in cells where p62 is abundant, even without acute stress treatment. C_LIO_LIUsing an extensive domain deletion library, ability of UBQLN2 constructs to form puncta correlates with their in vitro phase separation propensity in the absence of stress. C_LIO_LIEach of the two STI1 domains contribute non-redundantly to formation of UBQLN2 puncta in response to heat stress. C_LIO_LIThe STI1-II domain is independently required for UBQLN2 oligomerization, phase separation, and puncta formation. C_LI

cell biology↗

bioRxiv2024

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinsons Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

biochemistry↗

bioRxiv2022

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinsons Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, 39. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of 39 for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of 39 enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with 39, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that 39 binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

biochemistry↗

bioRxiv2021

Chemical mapping exposes the importance of active site interactions in governing the temperature dependence of enzyme turnover

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, {Delta}CP{ddagger}, can reveal links between the protein free energy landscape, global protein dynamics and enzyme turnover, suggesting that subtle changes molecular interactions at the active site can affect long range protein dynamics and link to enzyme temperature activity. Here we use a model promiscuous enzyme (Glucose dehydrogenase from Sulfolobus Solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, REES spectroscopy and computational simulation we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in {Delta}CP{ddagger} and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

biochemistry↗