Biology subjects

Dantuma, N. P.

Publications and source records attributed to Dantuma, N. P..

3 recordsLinked to original sources

bioRxiv2025

Mitochondria serve as a Holdout Compartment for Aggregation-Prone Proteins hindering Efficient Ubiquitin-Dependent Degradation

The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. In this study, we have conducted a genome-wide CRISPR-Cas9 screen to identify cellular factors that stimulate the degradation of an aggregation-prone reporter protein. Our findings revealed that genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, were highly enriched among suppressors of degradation of an aggregation-prone reporter. Conversely, endoplasmic reticulum (ER)-associated ubiquitin ligases facilitated degradation, indicating opposing roles for these cellular compartments in the clearance of aggregation-prone proteins. Genetic or chemical inhibition of eIF5A led to the dissociation of the aggregation-prone substrate from mitochondria, which was accompanied by enhanced degradation through ER-associated ubiquitination. The presence of an aggregation-prone, amphipathic helix that localized the reporter to mitochondria was crucial for the stimulatory effect of eIF5A inhibition. Additionally, the steady-state levels of -synuclein, a disease-associated protein containing an amphipathic helix that mislocalizes to mitochondria, were reduced upon eIF5A inhibition. We propose that mitochondria behave as a holdout compartment for aggregation-prone proteins, keeping them out of reach of ubiquitin ligases that target them for proteasomal degradation. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing their levels in neurodegenerative disorders.

cell biology↗

bioRxiv2024

Chemical inhibition of the integrated stress response impairs the ubiquitin-proteasome system

The Integrated Stress Response Inhibitor (ISRIB) is an experimental compound that has been used to explore the potential beneficial effects of reducing the activation of the integrated stress response (ISR). As the ISR is a protective response, there is, however, a risk that its inhibition may compromise the cells ability to restore protein homeostasis. Here, we show that ISRIB treatment impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Degradation of proteins intercepted by ribosome quality control (RQC) was particularly affected as accumulation of a UPS reporter substrate for ribosome quality control (RQC) was comparable to the level observed after proteasome inhibition. Consistent with impaired RQC, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. As depletion of the RQC ubiquitin ligase listerin partially restored ubiquitin-dependent proteasomal degradation, these data suggest that the persistent protein translation during proteotoxic stress in ISRIB-treated cells increases the pool of newly synthesized proteins targeted by RQC, which aggravates UPS dysfunction by overloading the cytosolic UPS.

cell biology↗

bioRxiv2017

The Deubiquitylating Enzyme Ubp12 Regulates Rad23-Dependent Substrate Delivery at the Proteasome

The consecutive actions of the ubiquitin-selective segregase Cdc48 and the ubiquitin shuttle factor Rad23 result in the delivery of ubiquitylated proteins at the proteasome. Here, we show that the deubiquitylating enzyme Ubp12 interacts with Cdc48 and regulates proteasomal degradation of Rad23-dependent substrates. Overexpression of Ubp12 results in stabilization of Rad23-dependent substrates. We show that Ubp12 removes short ubiquitin chains from the N-terminal ubiquitin-like domain (UbL) of Rad23. Preventing ubiquitylation of Rad23 by substitution of lysine residues within the UbL domain, Rad23UbLK0, does not affect the non-proteolytic role of Rad23 in DNA repair but causes an increase in ubiquitylated cargo bound to the UBA2 domains of Rad23 and recapitulates the stabilization of Rad23-dependent substrates observed upon overexpression of Ubp12. Expression of Rad23UbLK0 or overexpression of Ubp12 impairs the ability of yeast to cope with proteotoxic stress consistent with inefficient clearance of misfolded proteins by the ubiquitin/proteasome system. Our data suggest that ubiquitylation of Rad23 plays a stimulatory role in facilitating the transfer of ubiquitylated substrates to the proteasome.\n\nSummary statementUbiquitylation of a ubiquitin shuttle factor regulates the delivery of substrates at the proteasome, uncovering a novel regulatory link between ubiquitin and proteasomal degradation.

cell biology↗