Biology subjects

Ravid, T.

Publications and source records attributed to Ravid, T..

7 recordsLinked to original sources

bioRxiv2022

Prediction of quality-control degradation signals in yeast proteins

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs--degrons--that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

bioinformatics↗

bioRxiv2022

Conserved degronome features governing quality control-associated proteolysis

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identified a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm established constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons were identified in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of proteins half-life.

cell biology↗

bioRxiv2021

HSP70-binding motifs function as protein quality control degrons

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and PQC-linked degradation therefore overlaps in specificity with chaperone binding. This sheds new light on how the PQC system has evolved to exploit the intrinsic capacity of chaperones to recognize misfolded proteins, thereby placing them at the nexus of protein folding and degradation. Significance StatementIt is broadly accepted that misfolded proteins are often rapidly degraded by the ubiquitin-proteasome system, but how cells specifically recognize this immensely diverse group of proteins is largely unknown. Here we show that upon uncoupling of protein folding from protein degradation, a canonical chaperone-binding motif doubles as a degradation signal (degron), and that within the context of a Hsp70-binding region, many sequences are compatible with degron function. We find that degradation is correlated with the number of Hsp70-binding sites within a protein, and that the number of exposed Hsp70-binding sites in the yeast proteome correlates with more rapid degradation.

biochemistry↗

bioRxiv2021

Disease-linked mutations trigger exposure of a protein quality control degron in the DHFR protein

Degrons are short stretches of amino acids or structural motifs that are embedded in proteins. They mediate recognition by E3 ubiquitin-protein ligases and thus confer protein degradation via the ubiquitin-proteasome system. Well-described degrons include the N-degrons, destruction boxes, and the PIP degrons, which mediate the controlled degradation of various proteins including signaling components and cell cycle regulators. In comparison, the so-called protein quality control (PQC) degrons that mediate the degradation of structurally destabilized or misfolded proteins are not well described. Here, we show that disease-linked DHFR missense variants are structurally destabilized and chaperone-dependent proteasome targets. We systematically mapped regions within DHFR to assess those that act as cytosolic PQC degrons in yeast cells. Two regions, DHFR-Deg13-36 (here Deg1) and DHFR-Deg61-84 (here Deg2), act as degrons and conferred degradation to unrelated fusion partners. The proteasomal turnover of Deg2 was dependent on the molecular chaperone Hsp70. Structural analyses by NMR and hydrogen/deuterium exchange revealed that Deg2 is buried in wild-type DHFR, but becomes transiently exposed in the disease-linked missense variants.

biochemistry↗

bioRxiv2020

Folliculin variants linked to Birt-Hogg-Dube syndrome are targeted for proteasomal degradation

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dube (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present computational and experimental studies showing that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant ({Delta}E510) forms perinuclear protein aggregates. A yeast-based screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature to 29 {degrees}C led to a stabilization of two FLCN missense proteins, and for one variant (R362C), FLCN function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization of certain disease-linked variants may hold therapeutic potential.

molecular biology↗

bioRxiv2019

A molecular switch for Cdc48 activity and localization during oxidative stress and aging

Control over a healthy proteome begins with the birth of the polypeptide chain and ends with coordinated protein degradation. One of the major players in eukaryotic protein degradation is the essential and highly conserved ATPase, Cdc48 (p97/VCP in mammals). Cdc48 mediates clearance of misfolded proteins from the nucleus, cytosol, ER, mitochondria, and more. Here we dissect the crosstalk between cellular oxidation and Cdc48 activity by identification of a redox-sensitive site, Cys115. By integrating proteomics, biochemistry, microscopy, and bioinformatics, we show that removal of Cys115s redox-sensitive thiol group leads to accumulation of Cdc48 in the nucleus and consequently, results in severe defects in the oxidative stress response, mitochondrial fragmentation, and a decrease in ERAD and sterol biogenesis. We have thus identified a unique redox switch in Cdc48, which may provide a clearer picture of the importance of Cdc48s localization in maintaining a \"healthy\" proteome during oxidative stress and chronological aging in yeast.

molecular biology↗

bioRxiv2017

Temporal profiling of redox-dependent heterogeneity in single cells

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe roGFP. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach we defined redox-dependent heterogeneity of yeast cells, and characterized growth, as well as proteomic and transcriptomic profiles of subpopulations of cells that differ in their redox status, but are similar in age. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

biochemistry↗