Biology subjects

Rossio, V.

Publications and source records attributed to Rossio, V..

3 recordsLinked to original sources

bioRxiv2026

CDK/mTOR-dependent phosphorylation of UBE2H restrains its charging with ubiquitin and regulates CTLH-dependent degradation

The C-terminal to LisH (CTLH) complex is a modular multi-subunit E3 ligase with diverse biological functions, yet how its overall ubiquitylation activity is tuned remains unclear. Here, we identify CDK- and mTOR-dependent phosphorylation of the cognate E2 enzyme UBE2H as a key regulator of CTLH E3 catalytic capacity. Phosphorylation of two N-terminal serine residues (S3/S5) reduces UBE2H charging with ubiquitin, thereby limiting the pool of active E2 available to CTLH. Mitotic CDK activity inactivates UBE2H during mitosis, whereas mTOR restrains UBE2H charging in interphase to couple CTLH-dependent ubiquitylation to nutrient status. Preventing this phosphorylation maintains UBE2H charging, enhances CTLH-mediated substrate degradation, promotes CTLH subunit turnover, and causes proliferation and mitotic defects. Using hyperactive UBE2H, we identify two additional CTLH substrates, the mitotic kinase NEK9 and Angio-associated migratory cell protein (AAMP) and define a DR-like C-degron recognized by the CTLH subunit MKLN1. These findings reveal how regulation of an E2 enzyme by cell cycle and nutrient signaling pathways dynamically shape CTLH activity.

cell biology↗

bioRxiv2023

Substrate identification and specificity profiling of deubiquitylases against endogenously-generatedubiquitin-protein conjugates

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high impact DUBs (USP7, USP9X, USP36, USP15 and USP24) that each reduced ubiquitylation of over ten percent of the isolated proteins. Candidate substrates of high impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

biochemistry↗

bioRxiv2019

Unmasking functional redundancy of deubiquitylases enables specificity profiling and discovery of novel proteasome substrates

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs act on the same substrates, thwarting genetic approaches. Here, we circumvented redundancy by broadly inhibiting DUBs in Xenopus egg extract. DUB inhibition increases ubiquitylation of hundreds of proteins, depleting free ubiquitin without inducing widespread degradation. Restoring available ubiquitin led to proteasomal degradation of over thirty proteins, indicating that deubiquitylation is essential to maintain their stability. We confirmed their DUB-dependent stability with recombinant human proteins, demonstrating evolutionary conservation. We profiled the ability of DUBs to rescue protein stability, and found that USP7 has a unique ability to broadly antagonize proteasomal degradation. Together, we provide a comprehensive characterization of ubiquitin dynamics in the Xenopus system, identify new DUB substrates, and present a new approach to characterize physiological DUB specificity that overcomes challenges posed by DUB redundancy

biochemistry↗