@article {3150282, title = { yGPS-P: A yeast-based peptidome screen for studying quality control-associated proteolysis }, journal = {Biomolecules}, volume = {13}, year = {2023}, pages = {987}, abstract = {

Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. Here, damaged proteins that do not fold properly, are recognized by the ubiquitin-conjugating system that triggers their elimination by the 26S proteasome. How these proteins are selected for elimination is largely unknown. However, it has been suggested that cis-acting protein sequences, termed degrons, that are normally buried within the protein core are being exposed, resulting in the recruitment of the ubiquitin-conjugation machinery. To identify these cis-acting elements and learn about their unique features, we have developed an unbiased screening method that facilitates the determination of thousands of proteome-derived degrons. The application of a machine-learning algorithm further established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons. Here we provide details of the use of this new approach, including the setup, data acquisition, and analysis, and discuss its potential use for QCAP study in yeast and beyond.

}, url = {https://www.mdpi.com/2218-273X/13/6/987}, author = {Bayan Mashahreh and Shir Armony and Tommer Ravid} } @article {3150160, title = {The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation}, year = {2023}, month = {2023/06/12/}, abstract = {SummaryCdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in~vivo implications. }, keywords = {CDC48, CL-MS, crosslinking coupled with MS (XL-MS), integrative modeling, NPL4, protein quality control, structural mass spectrometry, UFD1}, isbn = {0969-2126}, url = {https://www.sciencedirect.com/science/article/pii/S0969212623001697}, author = {Oppenheim, Tal and Radzinski, Meytal and Braitbard, Merav and Brielle, Esther S. and Yogev, Ohad and Goldberger, Eliya and Yesharim, Yarden and Ravid, Tommer and Schneidman-Duhovny, Dina and Reichmann, Dana} } @article {3141213, title = {HSP70-binding motifs function as protein quality control degrons}, volume = {80}, year = {2023}, month = {2023}, pages = {32}, abstract = {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 that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.}, isbn = {1420-9071}, url = {https://doi.org/10.1007/s00018-022-04679-3}, author = {Abildgaard, Amanda B. and Voutsinos, Vasileios and Petersen, S{\o}ren D. and Larsen, Fia B. and Caroline Kampmeyer and Kristoffer E. Johansson and Stein, Amelie and Ravid, Tommer and Andr{\'e}asson, Claes and Jensen, Michael K. and Lindorff-Larsen, Kresten and Hartmann-Petersen, Rasmus} } @article {3125212, title = {Prediction of quality-control degradation signals in yeast proteins}, journal = {Journal of Molecular Biology}, year = {2023}, abstract = {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{\textemdash}degrons{\textemdash}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 entire 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.}, url = {https://www.biorxiv.org/content/10.1101/2022.04.06.487301v2.abstract}, author = {Kristoffer E. Johansson and Bayan Mashahreh and Hartmann-Petersen, Rasmus and Ravid, Tommer and Lindorff-Larsen, Kresten} } @article {Kampmeyer2022, title = {Disease-linked mutations cause exposure of a protein quality control degron}, journal = {Structure}, year = {2022}, month = {6}, publisher = {Cell Press}, issn = {0969-2126}, doi = {10.1016/J.STR.2022.05.016}, url = {https://linkinghub.elsevier.com/retrieve/pii/S0969212622001903}, author = {Caroline Kampmeyer and Sven Larsen-Ledet and Morten Rose Wagnkilde and Mathias Michelsen and Henriette K.M. Iversen and Nielsen, Sofie V. and S{\o}ren Lindemose and Alberto Caregnato and Ravid, Tommer and Stein, Amelie and Kaare Teilum and Lindorff-Larsen, Kresten and Hartmann-Petersen, Rasmus} } @article {David2022, title = {Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis}, journal = {Cells}, volume = {11}, year = {2022}, abstract = {Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.}, issn = {2073-4409}, doi = {10.3390/cells11091426}, url = {https://www.mdpi.com/2073-4409/11/9/1426}, author = {Yotam David and In{\^e}s Gomes Castro and Eden Yifrach and Chen Bibi and Enas Katawi and Dekel Yahav Har-Shai and Sagie Brodsky and Barkai, Naama and Ravid, Tommer and Miriam Eisenstein and Pietrokovski, Shmuel and Maya Schuldiner and Einat Zalckvar} } @article {3125210, title = {Conserved degronome features governing quality control-associated proteolysis}, journal = {Nature Communications (in press)}, year = {2022}, month = {2022}, abstract = {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{\textquoteright} half-life.}, url = {https://www.biorxiv.org/content/10.1101/2022.04.06.487275v2.abstract}, author = {Bayan Mashahreh and Shir Armony and Kristoffer En{\o}e Johansson and Alon Chappleboim and Friedman, Nir and G. Gardner, Richard and Hartmann-Petersen, Rasmus and Lindorff-Larsen, Kresten and Ravid, Tommer} } @article {3095148, title = {Folliculin variants linked to Birt-Hogg-Dub{\'e} syndrome are targeted for proteasomal degradation}, journal = {PLOS GeneticsPLOS Genetics}, volume = {16}, year = {2020}, month = {2020/11/02}, pages = {e1009187 - }, publisher = {Public Library of Science}, abstract = {Author summary Birt-Hogg-Dub{\'e} (BHD) syndrome is a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. The disease is linked to germline variants in the folliculin (FLCN) tumor suppressor gene. Here, we present a combined computational and experimental study, focusing on rare BHD-linked missense and single amino acid deletion variants. Our data show that many disease-causing FLCN variants lead to structural destabilization and rapid proteasomal degradation of the FLCN protein. The reduced level of FLCN, in turn, leads to degradation of the FLCN binding partners FNIP1 and FNIP2. Additional results show that the turnover of FLCN is regulated by the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones. We propose that for some missense variants, stabilization and resulting restoration of function may hold therapeutic potential, and that our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.}, url = {https://doi.org/10.1371/journal.pgen.1009187}, author = {Clausen, Lene and Stein, Amelie and Gr{\o}nb{\ae}k-Thygesen, Martin and Nygaard, Lasse and S{\o}ltoft, Cecilie L. and Nielsen, Sofie V. and Lisby, Michael and Ravid, Tommer and Lindorff-Larsen, Kresten and Hartmann-Petersen, Rasmus} } @article {3095147, title = {Releasing the Lockdown: An Emerging Role for the Ubiquitin-Proteasome System in the Breakdown of Transient Protein Inclusions}, journal = {Biomolecules}, volume = {10}, year = {2020}, month = {2020/08/10}, pages = {1168}, publisher = {MDPI}, abstract = {Intracellular protein inclusions are diverse cellular entities with distinct biological properties. They vary in their protein content, sequestration sites, physiological function, conditions for their generation, and turnover rates. Major distinctions have been recognized between stationary amyloids and dynamic, misfolded protein deposits. The former being a dead end for irreversibly misfolded proteins, hence, cleared predominantly by autophagy, while the latter consists of a protein-quality control mechanism, important for cell endurance, where proteins are sequestered during proteotoxic stress and resolved upon its relief. Accordingly, the disaggregation of transient inclusions is a regulated process consisting of protein solubilization, followed by a triage step to either refolding or to ubiquitin-mediated degradation. Recent studies have demonstrated an indispensable role in disaggregation for components of the chaperone and the ubiquitin-proteasome systems. These include heat-shock chaperones of the 40/70/100 kDa families, the proteasome, proteasome substrate shuttling factors, and deubiquitylating enzymes. Thus, a functional link has been established between the chaperone machinery that extracts proteins from transient deposits and 26S proteasome-dependent disaggregation, indicative of a coordinated process. In this review, we discuss data emanating from these important studies and subsequently consolidate the information in the form of a working model for the disaggregation mechanism.}, keywords = {*chaperones, *degradation, *disaggregation, *misfolding, *protein quality control, *refolding, *the ubiquitin-proteasome system, Animals, Cytoplasm/*metabolism, Humans, Inclusion Bodies/*metabolism, Molecular Chaperones/*metabolism, Proteasome Endopeptidase Complex/*metabolism, Saccharomyces cerevisiae/metabolism, Ubiquitin/*metabolism}, isbn = {2218-273X}, url = {https://pubmed.ncbi.nlm.nih.gov/32784966}, author = {Reiss, Yuval and Gur, Elisheva and Ravid, Tommer} } @article {3095151, title = {Assays for dissecting the in vitro enzymatic activity of yeast Ubc7}, journal = {Methods in Enzymology}, year = {2019}, abstract = {

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or ERAD. Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10 and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 co-factor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub-Ub linkages. Each isopeptide bond is formed by transfer of a Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub-Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its co-factor Cue1, and the RING domain of either Doa10 or Hrd1.\  Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub-Ub bond, specifically, the formation of a Ubc7~Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub-Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems.\ 

}, author = {Mashahreh, B. and Reiss, Y. and Wiener, R. and Ravid, T.} } @article {3095149, title = {The extent of Ssa1/Ssa2 Hsp70 chaperone involvement in nuclear protein quality control degradation varies with the substrate}, journal = {Molecular Biology of the CellMolecular Biology of the Cell}, volume = {31}, year = {2019}, note = {doi: 10.1091/mbc.E18-02-0121}, month = {2019}, pages = {221 - 233}, publisher = {American Society for Cell Biology (mboc)}, abstract = {Protein misfolding is a recurring phenomenon that cells must manage; otherwise misfolded proteins can aggregate and become toxic should they persist. To counter this burden, cells have evolved protein quality control (PQC) mechanisms that manage misfolded proteins. Two classes of systems that function in PQC are chaperones that aid in protein folding and ubiquitin?protein ligases that ubiquitinate misfolded proteins for proteasomal degradation. How folding and degradative PQC systems interact and coordinate their respective functions is not yet fully understood. Previous studies of PQC degradation pathways in the endoplasmic reticulum and cytosol have led to the prevailing idea that these pathways require the activity of Hsp70 chaperones. Here, we find that involvement of the budding yeast Hsp70 chaperones Ssa1 and Ssa2 in nuclear PQC degradation varies with the substrate. In particular, nuclear PQC degradation mediated by the yeast ubiquitin?protein ligase San1 often involves Ssa1/Ssa2, but San1 substrate recognition and ubiquitination can proceed without these Hsp70 chaperone functions in vivo and in vitro. Our studies provide new insights into the variability of Hsp70 chaperone involvement with a nuclear PQC degradation pathway.Protein misfolding is a recurring phenomenon that cells must manage; otherwise misfolded proteins can aggregate and become toxic should they persist. To counter this burden, cells have evolved protein quality control (PQC) mechanisms that manage misfolded proteins. Two classes of systems that function in PQC are chaperones that aid in protein folding and ubiquitin?protein ligases that ubiquitinate misfolded proteins for proteasomal degradation. How folding and degradative PQC systems interact and coordinate their respective functions is not yet fully understood. Previous studies of PQC degradation pathways in the endoplasmic reticulum and cytosol have led to the prevailing idea that these pathways require the activity of Hsp70 chaperones. Here, we find that involvement of the budding yeast Hsp70 chaperones Ssa1 and Ssa2 in nuclear PQC degradation varies with the substrate. In particular, nuclear PQC degradation mediated by the yeast ubiquitin?protein ligase San1 often involves Ssa1/Ssa2, but San1 substrate recognition and ubiquitination can proceed without these Hsp70 chaperone functions in vivo and in vitro. Our studies provide new insights into the variability of Hsp70 chaperone involvement with a nuclear PQC degradation pathway.}, isbn = {1059-1524}, url = {https://doi.org/10.1091/mbc.E18-02-0121}, author = {Jones, Ramon D. and Enam, Charisma and Ibarra, Rebeca and Borror, Heather R. and Mostoller, Kaitlyn E. and Fredrickson, Eric K. and Lin, JiaBei and Chuang, Edward and March, Zachary and Shorter, James and Ravid, Tommer and Kleiger, Gary and G. Gardner, Richard} } @article {3095150, title = {The Hunt for Degrons of the 26S Proteasome}, journal = {Biomolecules}, year = {2019}, abstract = {

Since the discovery of ubiquitin conjugation as a cellular mechanism that triggers proteasomal degradation, the mode of substrate recognition by the ubiquitin-ligation system has been the holy grail of research in the field. This entails the discovery of recognition determinants within protein substrates, which are part of a degron, and explicit E3 ubiquitin (Ub)-protein ligases that trigger their degradation. Indeed, many protein substrates and their cognate E3's have been discovered in the past 40 years. In the course of these studies, various degrons have been randomly identified, most of which are acquired through post-translational modification, typically, but not exclusively, protein phosphorylation. Nevertheless, acquired degrons cannot account for the vast diversity in cellular protein half-life times. Obviously, regulation of the proteome is largely determined by inherent degrons, that is, determinants integral to the protein structure. Inherent degrons are difficult to predict since they consist of diverse sequence and secondary structure features. Therefore, unbiased methods have been employed for their discovery. This review describes the history of degron discovery methods, including the development of high throughput screening methods, state of the art data acquisition and data analysis. Additionally, it summarizes major discoveries that led to the identification of cognate E3 ligases and hitherto unrecognized complexities of degron function. Finally, we discuss future perspectives and what still needs to be accomplished towards achieving the goal of understanding how the eukaryotic proteome is regulated via coordinated action of components of the ubiquitin-proteasome system.

}, url = {https://doi.org/10.3390/biom9060230}, author = {Hadar Ella and Reiss, Yuval and Ravid, Tommer} } @article {3095154, title = {From Precise Slicing to General SHREDding: The Ubiquitin Ligase Ubr1 Roqs as a Multipurpose Protein Terminator}, journal = {Molecular Cell}, volume = {70}, year = {2018}, pages = {989-990}, abstract = {

In the current issue of Molecular Cell, Szoradi et al. (2018) present compelling data demonstrating how the newly identified SHRED pathway in yeast selectively shifts the E3 ligase Ubr1 specificity from N-end rule substrates to misfolded proteins in cells under proteostatic stress.

}, url = {https://www.cell.com/molecular-cell/fulltext/S1097-2765(18)30448-9}, author = {Gardner, Richard~G. and Ravid, T} } @article {3095152, title = {The insulin/IGF signaling cascade modulates SUMOylation to regulate aging and proteostasis in Caenorhabditis elegans.}, journal = {eLife}, year = {2018}, abstract = {

Although aging-regulating pathways were discovered a few decades ago, it is not entirely clear how their activities are orchestrated, to govern lifespan and proteostasis at the organismal level. Here, we utilized the nematode\ Caenorhabditis elegans\ to examine whether the alteration of aging, by reducing the activity of the Insulin/IGF signaling (IIS) cascade, affects protein SUMOylation. We found that IIS activity promotes the SUMOylation of the germline protein, CAR-1, thereby shortening lifespan and impairing proteostasis. In contrast, the expression of mutated CAR-1, that cannot be SUMOylated at residue 185, extends lifespan and enhances proteostasis. A mechanistic analysis indicated that CAR-1 mediates its aging-altering functions, at least partially, through the notch-like receptor\ glp-1. Our findings unveil a novel regulatory axis in which SUMOylation is utilized to integrate the aging-controlling functions of the IIS and of the germline and provide new insights into the roles of SUMOylation in the regulation of organismal aging.

}, author = {Lorna Moll and Noa Roitenberg and Adi Biram and Ravid, Tommer and Ehud Cohen and Tzur YB} } @inbook {3095153, title = {Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells}, booktitle = {The Ubiquitin Proteasome System: Methods and Protocols}, volume = {1844}, year = {2018}, pages = {121-136}, publisher = {Springer }, organization = {Springer }, address = {New York}, abstract = {

Since its discovery nearly 40 years ago, many components of the ubiquitin-proteasome system (UPS) have been identified and characterized in detail. However, a key aspect of the UPS that remains largely obscure is the signals that initiate the interaction of a substrate with enzymes of the UPS machinery. Understanding these signals is of particular interest for studies that examine the mechanism of substrate recognition for proteins that have adopted a non-native structure, as part of the cellular protein quality control (PQC) defense mechanism. Such studies are quite salient as the entire proteome makes up the potential battery of PQC substrates, and yet only a limited number of ubiquitination pathways are known to handle misfolded proteins. Our current research aims at understanding how a small number of PQC ubiquitin-protein ligases specifically recognize and ubiquitinate the overwhelming assortment of misfolded proteins. Here, we present a new proteogenomic approach for identifying and characterizing recognition motifs within degradation elements (degrons) in a high-throughput manner. The method utilizes yeast growth under restrictive conditions for selecting protein fragments that confer instability. The corresponding cDNA fragments are analyzed by next-generation sequencing (NGS) that provides information about each fragment{\textquoteright}s identity, reading frame, and abundance over time. This method was used by us to identify PQC-specific and compartment-specific degrons. It can readily be modified to study protein degradation signals and pathways in other organisms and in various settings, such as different strain backgrounds and under various cell conditions, all of which can be sequenced and analyzed simultaneously.

}, author = {Geffen, Yifat and Appleboim, Alon and G. Gardner, Richard and Ravid, Tommer} } @article {3095155, title = {Protein Quality Control Degradation in the Nucleus}, journal = { Annual Review of Biochemistry}, volume = {87}, year = {2018}, pages = {725-749}, abstract = {

Nuclear proteins participate in diverse cellular processes, many of which are essential for cell survival and viability. To maintain optimal nuclear physiology, the cell employs the ubiquitin-proteasome system to eliminate damaged and misfolded proteins in the nucleus that could otherwise harm the cell. In this review, we highlight the current knowledge about the major ubiquitin-protein ligases involved in protein quality control degradation (PQCD) in the nucleus and how they orchestrate their functions to eliminate misfolded proteins in different nuclear subcompartments. Many human disorders are causally linked to protein misfolding in the nucleus, hence we discuss major concepts that still need to be clarified to better understand the basis of the nuclear misfolded proteins{\textquoteright} toxic effects. Additionally, we touch upon potential strategies for manipulating nuclear PQCD pathways to ameliorate diseases associated with protein misfolding and aggregation in the nucleus.

}, url = {https://www.annualreviews.org/doi/full/10.1146/annurev-biochem-062917-012730?url_ver=Z39.88-2003\&rfr_id=ori\%3Arid\%3Acrossref.org\&rfr_dat=cr_pub\%3Dpubmed}, author = {Enam, Charisma and Geffen, Yifat and Ravid, Tommer and G. Gardner, Richard} } @article {3095156, title = {Temporal profiling of redox-dependent heterogeneity in single cells}, journal = {eLife}, year = {2018}, abstract = {

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 Grx1-roGFP2. 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 a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. 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.

}, url = {https://elifesciences.org/articles/37623}, author = {Meytal Radzinski and Fassler, Rosi and Yogev, Ohad and Breuer, William and Shai, Nadav and Gutin, Jenia and Ilyas, Sidra and Geffen, Yifat and Sabina Tsytkin-Kirschenzweig and Yaakov Nahmias and Ravid, Tommer and Friedman, Nir and Shuldiner, Maya and Reichmann, Dana} } @article {3095157, title = {Mapping the Landscape of a Eukaryotic Degronome}, journal = {Molecular Cell}, volume = {63}, year = {2016}, month = {2016/9/15/}, pages = {1055 - 1065}, abstract = {

SummaryThe ubiquitin-proteasome system (UPS) for protein degradation has been under intensive study, and yet, we have only partial understanding of mechanisms by which proteins are selected to be targeted for proteolysis. One of the obstacles in studying these recognition pathways is the limited repertoire of known degradation signals (degrons). To better understand what determines the susceptibility of intracellular proteins to degradation by the UPS, we\ developed an unbiased method for large-scale identification of eukaryotic degrons. Using a reporter-based high-throughput competition assay, followed by deep sequencing, we measured a degradation potency index for thousands of native polypeptides in a single experiment. We further used this method to identify protein quality control (PQC)-specific and compartment-specific degrons. Our method provides an unprecedented insight into the yeast degronome, and it can readily be modified to study protein degradation signals and pathways in other organisms and in various settings.

}, isbn = {1097-2765}, url = {http://www.sciencedirect.com/science/article/pii/S1097276516304191}, author = {Geffen, Yifat and Appleboim, Alon and Gardner, Richard~G. and Friedman, Nir and Sadeh, Ronen and Ravid, Tommer} } @article {3095158, title = {Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase}, journal = {Molecular Cell}, year = {2016}, abstract = {

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of\ poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

}, url = {http://www.cell.com/molecular-cell/fulltext/S1097-2765(16)30373-2}, author = {Annika Weber and Cohen, Itamar and Oliver Popp and Gunnar Dittmar and Reiss, Yuval and Thomas Sommer and Ravid, Tommer and Ernst Jarosch} } @article {3095159, title = {Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.}, journal = {Nat Commun}, volume = {6}, year = {2015}, month = {2015}, pages = {7075}, abstract = {Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner{\textemdash}both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.}, author = {Sajman, Julia and Zenvirth, Drora and Mor Nitzan and Margalit, Hanah and Simpson-Lavy, Kobi J and Reiss, Yuval and Cohen, Itamar and Ravid, Tommer and Brandeis, Michael} } @article {3095160, title = {Distinct activation of an E2 ubiquitin-conjugating enzyme by its cognate E3 ligases.}, journal = {Proc Natl Acad Sci U S A}, volume = {112}, year = {2015}, month = {2015 Feb 17}, pages = {E625-32}, abstract = {A significant portion of ubiquitin (Ub)-dependent cellular protein quality control takes place at the endoplasmic reticulum (ER) in a process termed "ER-associated degradation" (ERAD). Yeast ERAD employs two integral ER membrane E3 Ub ligases: Hrd1 (also termed "Der3") and Doa10, which recognize a distinct set of substrates. However, both E3s bind to and activate a common E2-conjugating enzyme, Ubc7. Here we describe a novel feature of the ERAD system that entails differential activation of Ubc7 by its cognate E3s. We found that residues within helix α2 of Ubc7 that interact with donor Ub were essential for polyUb conjugation. Mutagenesis of these residues inhibited the in vitro activity of Ubc7 by preventing the conjugation of donor Ub to the acceptor. Unexpectedly, Ub chain formation by mutant Ubc7 was restored selectively by the Hrd1 RING domain but not by the Doa10 RING domain. In agreement with the in vitro data, Ubc7 α2 helix mutations selectively impaired the in vivo degradation of Doa10 substrates but had no apparent effect on the degradation of Hrd1 substrates. To our knowledge, this is the first example of distinct activation requirements of a single E2 by two E3s. We propose a model in which the RING domain activates Ub transfer by stabilizing a transition state determined by noncovalent interactions between the α2 helix of Ubc7 and Ub and that this transition state may be stabilized further by some E3 ligases, such as Hrd1, through additional interactions outside the RING domain.}, keywords = {Biocatalysis, Enzyme Activation, Lysine, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases}, author = {Cohen, Itamar and Wiener, Reuven and Reiss, Yuval and Ravid, Tommer} } @article {3095161, title = {Chaperoning proteins for destruction: diverse roles of Hsp70 chaperones and their co-chaperones in targeting misfolded proteins to the proteasome.}, journal = {Biomolecules}, volume = {4}, year = {2014}, month = {2014}, pages = {704-24}, abstract = {Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases.}, keywords = {Animals, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Humans, Proteasome Endopeptidase Complex, Proteostasis Deficiencies, ubiquitin}, author = {Shiber, Ayala and Ravid, Tommer} } @article {3095162, title = {The DNA2 nuclease/helicase is an estrogen-dependent gene mutated in breast and ovarian cancers.}, journal = {Oncotarget}, volume = {5}, year = {2014}, month = {2014 Oct 15}, pages = {9396-409}, abstract = {Genomic instability, a hallmark of cancer, is commonly caused by failures in the DNA damage response. Here we conducted a bioinformatical screen to reveal DNA damage response genes that are upregulated by estrogen and highly mutated in breast and ovarian cancers. This screen identified 53 estrogen-dependent cancer genes, some of which are novel. Notably, the screen retrieved 9 DNA helicases as well as 5 nucleases. DNA2, which functions as both a helicase and a nuclease and plays a role in DNA repair and replication, was retrieved in the screen. Mutations in DNA2, found in estrogen-dependent cancers, are clustered in the helicase and nuclease domains, suggesting activity impairment. Indeed, we show that mutations found in ovarian cancers impair DNA2 activity. Depletion of DNA2 in cells reduces their tumorogenicity in mice. In human, high expression of DNA2 correlates with poor survival of estrogen receptor-positive patients but not of estrogen receptor-negative patients. We also demonstrate that depletion of DNA2 in cells reduces proliferation, while addition of estrogen restores proliferation. These findings suggest that cells responding to estrogen will proliferate despite impaired in DNA2 activity, potentially promoting genomic instability and triggering cancer development.}, keywords = {Animals, Breast Neoplasms, Cell Line, Tumor, Cell Proliferation, Cell Transformation, Neoplastic, DNA Damage, DNA Helicases, DNA Repair, Estrogens, Female, Genomic Instability, Humans, MCF-7 Cells, Mice, Mice, Inbred NOD, Mice, SCID, Mutation, Neoplasm Transplantation, Ovarian Neoplasms, Receptors, Estrogen, RNA Interference, RNA, Small Interfering, Transplantation, Heterologous}, author = {Strauss, Carmit and Kornowski, Maya and Benvenisty, Avraham and Shahar, Amit and Masury, Hadas and Ittai Ben-Porath and Ravid, Tommer and Arbel-Eden, Ayelet and Goldberg, Michal} } @article {3095163, title = {Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.}, journal = {Prion}, volume = {8}, year = {2014}, month = {2014}, pages = {276-84}, abstract = {The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.}, keywords = {Flow Cytometry, Fungal Proteins, Green Fluorescent Proteins, Heat-Shock Proteins, Protein Aggregates, Saccharomyces cerevisiae}, author = {Shiber, Ayala and Breuer, William and Ravid, Tommer} } @article {3095164, title = {Reporter-based growth assay for systematic analysis of protein degradation.}, journal = {J Vis Exp}, year = {2014}, month = {2014}, pages = {e52021}, abstract = {Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels. Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation. This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.}, keywords = {Kinetics, Proteasome Endopeptidase Complex, Proteolysis, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, ubiquitin}, author = {Cohen, Itamar and Geffen, Yifat and Ravid, Guy and Ravid, Tommer} } @article {3095165, title = {Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.}, journal = {J Biol Chem}, volume = {288}, year = {2013}, month = {2013 May 3}, pages = {12645-53}, abstract = {Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.}, keywords = {Amino Acid Motifs, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Kinetochores, Mutation, Proteasome Endopeptidase Complex, Proteolysis, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Ubiquitination}, author = {Alfassy, Omri S and Cohen, Itamar and Reiss, Yuval and Boaz Tirosh and Ravid, Tommer} } @article {3095166, title = {Ubiquitin conjugation triggers misfolded protein sequestration into quality control foci when Hsp70 chaperone levels are limiting.}, journal = {Mol Biol Cell}, volume = {24}, year = {2013}, month = {2013 Jul}, pages = {2076-87}, abstract = {Ubiquitin accumulation in amyloid plaques is a pathological marker observed in the vast majority of neurodegenerative diseases, yet ubiquitin function in these inclusions is controversial. It has been suggested that ubiquitylated proteins are directed to inclusion bodies under stress conditions, when both chaperone-mediated refolding and proteasomal degradation are compromised or overwhelmed. Alternatively, ubiquitin and chaperones may be recruited to preformed inclusions to promote their elimination. We address this issue using a yeast model system, based on expression of several mildly misfolded degradation substrates in cells with altered chaperone content. We find that the heat shock protein 70 (Hsp70) chaperone pair Ssa1/Ssa2 and the Hsp40 cochaperone Sis1 are essential for degradation. Substrate ubiquitylation is strictly dependent on Sis1, whereas Ssa1 and Ssa2 are dispensable. Remarkably, in Ssa1/Ssa2-depleted cells, ubiquitylated substrates are sequestered into detergent-insoluble, Hsp42-positive inclusion bodies. Unexpectedly, sequestration is abolished by preventing substrate ubiquitylation. We conclude that Hsp40 is required for the targeting of misfolded proteins to the ubiquitylation machinery, whereas the decision to degrade or sequester ubiquitylated proteins is mediated by the Hsp70s. Accordingly, diminished Hsp70 levels, as observed in aging or certain pathological conditions, might be sufficient to trigger ubiquitin-dependent sequestration of partially misfolded proteins into inclusion bodies.}, keywords = {Adenosine Triphosphatases, Cytoplasm, Gene Expression Regulation, Fungal, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Inclusion Bodies, Proteasome Endopeptidase Complex, Protein Binding, Protein Folding, Protein Stability, Proteolysis, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, ubiquitin}, author = {Shiber, Ayala and Breuer, William and Brandeis, Michael and Ravid, Tommer} } @article {3095167, title = {Endoplasmic reticulum stress induces a caspase-dependent N-terminal cleavage of RBX1 protein in B cells.}, journal = {J Biol Chem}, volume = {287}, year = {2012}, month = {2012 Sep 7}, pages = {31223-32}, abstract = {Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.}, keywords = {Animals, Carrier Proteins, Caspases, Cell Differentiation, Cells, Cultured, Endoplasmic Reticulum Stress, Humans, Mice, Mutation, Plasma Cells, Proteolysis, Saccharomyces cerevisiae, Ubiquitination}, author = {Shteingart, Shimon and Rivka Hadar and Cohen, Itamar and Ravid, Tommer and Boaz Tirosh} } @article {3095168, title = {EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization.}, journal = {PLoS One}, volume = {6}, year = {2011}, month = {2011}, pages = {e23240}, abstract = {Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.}, keywords = {Animals, Cell Membrane, Ceramides, Cholesterol, Enzyme Activation, Humans, Hydrogen Peroxide, Ligands, Mice, Models, Biological, NIH 3T3 Cells, Oxidative Stress, Phosphorylation, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Protein Transport, Pyrazoles, Pyrimidines, Quinazolines, Receptor, Epidermal Growth Factor, src-Family Kinases, TEMPERATURE, Tyrphostins}, author = {Filosto, Simone and Khan, Elaine M and Tognon, Emiliana and Becker, Cathleen and Ashfaq, Majid and Ravid, Tommer and Goldkorn, Tzipora} } @article {3095169, title = {Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.}, journal = {Mol Biol Cell}, volume = {22}, year = {2011}, month = {2011 Dec}, pages = {4726-39}, abstract = {Proper functioning of the protein-folding quality control network depends on the network{\textquoteright}s ability to discern diverse structural perturbations to the native states of its protein substrates. Despite the centrality of the detection of misfolded states to cell home-ostasis, very little is known about the exact sequence and structural features that mark a protein as being misfolded. To investigate these features, we studied the requirements for the degradation of the yeast kinetochore protein Ndc10p. Mutant Ndc10p is a substrate of a protein-folding quality control pathway mediated by the E3 ubiquitin (Ub) ligase Doa10p at the endoplasmic reticulum (ER)/nuclear envelope membrane. Analysis of Ndc10p mutant derivatives, employing a reverse genetics approach, identified an autonomous quality control-associated degradation motif near the C-terminus of the protein. This motif is composed of two indispensable hydrophobic elements: a hydrophobic surface of an amphipathic helix and a loosely structured hydrophobic C-terminal tail. Site-specific point mutations expose these elements, triggering ubiquitin-mediated and HSP70 chaperone-dependent degradation of Ndc10p. These findings substantiate the ability of the ER quality control system to recognize subtle perturbation(s) in the native structure of a nuclear protein.}, keywords = {Amino Acid Motifs, DNA-Binding Proteins, Endoplasmic Reticulum, HSP70 Heat-Shock Proteins, Hydrophobic and Hydrophilic Interactions, Kinetochores, Mutation, Nuclear Envelope, Protein Folding, Protein Structure, Tertiary, Proteolysis, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Ubiquitin-Protein Ligases}, author = {Furth, Noa and Gertman, Or and Shiber, Ayala and Alfassy, Omri S and Cohen, Itamar and Rosenberg, Masha M and Doron, Nurit Kleinberger and Assaf Friedler and Ravid, Tommer} } @article {3095170, title = {Diversity of degradation signals in the ubiquitin-proteasome system.}, journal = {Nat Rev Mol Cell Biol}, volume = {9}, year = {2008}, month = {2008 Sep}, pages = {679-90}, abstract = {The ubiquitin-proteasome system degrades an enormous variety of proteins that contain specific degradation signals, or {\textquoteright}degrons{\textquoteright}. Besides the degradation of regulatory proteins, almost every protein suffers from sporadic biosynthetic errors or misfolding. Such aberrant proteins can be recognized and rapidly degraded by cells. Structural and functional data on a handful of degrons allow several generalizations regarding their mechanism of action. We focus on different strategies of degron recognition by the ubiquitin system, and contrast regulatory degrons that are subject to signalling-dependent modification with those that are controlled by protein folding or assembly, as frequently occurs during protein quality control.}, keywords = {Animals, Endoplasmic Reticulum, Humans, Proteasome Endopeptidase Complex, Protein Processing, Post-Translational, Signal Transduction, ubiquitin, Ubiquitin-Protein Ligases}, author = {Ravid, Tommer and Hochstrasser, Mark} } @article {3095171, title = {Molecular genetics of the ubiquitin-proteasome system: lessons from yeast.}, journal = {Ernst Schering Found Symp Proc}, year = {2008}, month = {2008}, pages = {41-66}, abstract = {Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome system. The initial work focused on the degradation of a transcription factor, the MATalpha2 repressor, but the pathways uncovered have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to alpha2 turnover. One of them depends on a large integral membrane ubiquitin ligase (E3) and a pair of ubiquitin-conjugating enzymes (E2s). The transmembrane E3 and E2 proteins must travel from their site of synthesis in the ER to the inner nuclear membrane in order to reach nuclear substrates such as alpha2. The 26S proteasome is responsible for alpha2 degradation, and several important features of proteasome assembly and active site formation were uncovered. Most recently, we have delineated major steps in 20S proteasome assembly and have also identified several novel 20S proteasome assembly factors. Surprisingly, alterations in 20S proteasome assembly lead to defects in the assembly of the proteasome regulatory particle (RP). The RP associates with the 20S proteasome to form the 26S proteasome. Our data suggest that the 20S proteasome can function as an assembly factor for the RP, which would make it the first such factor for RP assembly identified to date.}, keywords = {Animals, Humans, Molecular Biology, Proteasome Endopeptidase Complex, Protein Binding, Saccharomyces cerevisiae, Substrate Specificity, ubiquitin}, author = {Hochstrasser, M and Deng, M and Kusmierczyk, A R and Li, X. and Kreft, S G and Ravid, T and Funakoshi, M and Kunjappu, M and Xie, Y} } @article {3095172, title = {Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue.}, journal = {Nat Cell Biol}, volume = {9}, year = {2007}, month = {2007 Apr}, pages = {422-7}, abstract = {Cells have quality-control mechanisms to recognize non-native protein structures and either help the proteins fold or promote their degradation. Ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) work together to assemble polyubiquitin chains on misfolded or misassembled proteins, which are then degraded by the proteasome. Here, we find that Ubc7, a yeast E2, can itself undergo degradation when its levels exceed that of its binding partner Cue1, a transmembrane protein that tethers Ubc7 to the endoplasmic reticulum. Unassembled, and thus mislocalized, Ubc7 is targeted to the proteasome by Ufd4, a homologous to E6-AP C-terminus (HECT)-class E3. Ubc7 is autoubiquitinated by a novel mechanism wherein the catalytic cysteine, instead of a lysine residue, provides the polyubiquitin chain acceptor site, and this cysteine-linked chain functions as a degradation signal. The polyubiquitin chain can also be transferred to a lysine side chain, suggesting a mechanism for polyubiquitin chain assembly that precedes substrate modification.}, keywords = {Amino Acids, Catalytic Domain, Cysteine, Fungal Proteins, Gene Expression Regulation, Fungal, Green Fluorescent Proteins, Microscopy, Fluorescence, Plasmids, Polyubiquitin, Proteasome Endopeptidase Complex, Protein Binding, ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Yeasts}, author = {Ravid, Tommer and Hochstrasser, Mark} } @article {3095173, title = {An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery.}, journal = {Proc Natl Acad Sci U S A}, volume = {103}, year = {2006}, month = {2006 Jul 25}, pages = {11178-83}, abstract = {Serum- and glucocorticoid-induced kinase 1 (Sgk1) regulates many ion channels and transporters in epithelial cells and promotes cell survival under stress conditions. In this study we demonstrate that Sgk1 is a short-lived protein regulated by the endoplasmic reticulum (ER)-associated degradation system and subcellular localization to the ER. We identified a hydrophobic motif (residues 18-30) as the signal for ER localization and rapid degradation by the ubiquitin (Ub)/proteasome pathway in both yeast and mammalian cells. Deletion or reduction of hydrophobicity of the motif redistributes Sgk1 to the cytosol and nucleus and markedly increases its half-life. We determined that the Ub-conjugating UBC6 and UBC7 and the Ub ligase HRD1 are the ER-associated Ub enzymes that mediate degradation of Sgk1; thus, Sgk1 has been identified as a cytosolic substrate for mammalian HRD1. Compartmentalization of Sgk1 controls the functional and spatial specificities of Sgk1-mediated signaling pathways, whereas rapid protein turnover provides a means to rapidly adjust Sgk1 abundance in response to different hormonal and external stimuli that increase Sgk1 gene transcription.}, keywords = {Animals, Cell Nucleus, CHO Cells, Cricetinae, Cytosol, Endoplasmic Reticulum, Fungal Proteins, Humans, Immediate-Early Proteins, Mice, Models, Biological, Proteasome Endopeptidase Complex, Protein-Serine-Threonine Kinases, Signal Transduction, Transcription, Genetic, ubiquitin}, author = {Arteaga, Maria Francisca and Wang, Lin and Ravid, Tommer and Hochstrasser, Mark and Canessa, Cecilia M} } @article {3095174, title = {Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking.}, journal = {J Biol Chem}, volume = {281}, year = {2006}, month = {2006 May 19}, pages = {14486-93}, abstract = {The epidermal growth factor (EGF) receptor (EGFR) has been found to be overexpressed in several types of cancer cells, and the regulation of its oncogenic potential has been widely studied. The paradigm for EGFR down-regulation involves the trafficking of activated receptor molecules from the plasma membrane, through clathrin-coated pits, and into the cell for lysosomal degradation. We have previously shown that oxidative stress generated by H2O2 results in aberrant phosphorylation of the EGFR. This leads to the loss of c-Cbl-mediated ubiquitination of the EGFR and, consequently, prevents its degradation. However, we have found that c-Cbl-mediated ubiquitination is required solely for degradation but not for internalization of the EGFR under oxidative stress. To further examine the fate of the EGFR under oxidative stress, we used confocal analysis to show that the receptor not only remains co-localized with caveolin-1 at the plasma membrane, but at longer time points, is also sorted to a perinuclear compartment via a clathrin-independent, caveolae-mediated pathway. Our findings indicate that although the EGFR associates with caveolin-1 constitutively, caveolin-1 is hyperphosphorylated only under oxidative stress, which is essential in transporting the EGFR to a perinuclear location, where it is not degraded and remains active. Thus, oxidative stress may have a role in tumorigenesis by not only activating the EGFR but also by promoting prolonged activation of the receptor both at the plasma membrane and within the cell.}, keywords = {Active Transport, Cell Nucleus, Caveolin 1, Cell Line, Tumor, Cell Membrane, Cell Nucleus, Clathrin, Down-Regulation, Humans, Hydrogen Peroxide, Lysosomes, Models, Biological, Oxidative Stress, Receptor, Epidermal Growth Factor, Signal Transduction, src-Family Kinases}, author = {Khan, Elaine M and Heidinger, Jill M and Levy, Michal and Lisanti, Michael P and Ravid, Tommer and Goldkorn, Tzipora} } @article {3095175, title = {Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways.}, journal = {EMBO J}, volume = {25}, year = {2006}, month = {2006 Feb 8}, pages = {533-43}, abstract = {The yeast Doa10 ubiquitin (Ub) ligase resides in the endoplasmic reticulum (ER)/nuclear envelope (NE), where it functions in ER-associated degradation (ERAD). Doa10 substrates include non-ER proteins such as the transcription factor Mat alpha2. Here, we expand the range of Doa10 substrates to include a defective kinetochore component, a mutant NE membrane protein, and a substrate-regulated human ER enzyme. For all these substrates, Doa10 requires two Ub-conjugating enzymes, Ubc6 and Ubc7, as well as the Ubc7 cofactor Cue1. Based on a novel genomic screen of a comprehensive gene deletion library and other data, these four proteins appear to be the only nonessential and nonredundant factors generally required for Doa10-mediated ubiquitination. Notably, the Cdc48 ATPase facilitates degradation of membrane-embedded Doa10 substrates, but is not required for any tested soluble Doa10 substrates. This distinction is maintained even when comparing membrane and soluble proteins bearing the same degradation signal. Thus, while Doa10 ubiquitinates both membrane and soluble proteins, the mechanisms of subsequent proteasome targeting differ.}, keywords = {Cell Membrane, Endoplasmic Reticulum, Kinetochores, Membrane Proteins, Mutation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Signal Transduction, Solubility, Ubiquitin-Protein Ligases}, author = {Ravid, Tommer and Kreft, Stefan G and Hochstrasser, Mark} } @article {3095176, title = {Life and death decisions: ceramide generation and EGF receptor trafficking are modulated by oxidative stress.}, journal = {Antioxid Redox Signal}, volume = {7}, year = {2005}, month = {2005 Jan-Feb}, pages = {119-28}, abstract = {Reactive oxidants are associated with the pathogenesis of pulmonary diseases and affect various cell functions, from proliferation to apoptosis. We have shown that oxidants exert growth control on airway epithelial cells by modulating upstream receptor function. Additionally, hydrogen peroxide-mediated oxidative stress modulates ceramide levels to induce apoptosis in lung epithelium. Depletion of glutathione in lung epithelial cells results in ceramide accumulation, suggesting that ceramide elevation, coupled to oxidative stress, initiates apoptosis. While it is desirable to prevent cell death and tissue injury induced by oxidants in diseases such as asthma or acute respiratory distress syndrome, the opposite is sought in cancer. But oxidants may also activate growth factor receptors, enhancing cell proliferation and facilitating tumor promotion. Under oxidative stress, phosphorylation of the epidermal growth factor receptor (EGFR) is abrogated at tyrosine 1,045, the docking site for the ubiquitin ligase c-Cbl, rendering EGFR unable to recruit c-Cbl and be ubiquitylated and degraded. We thus proposed that this deficiency, which confers prolonged receptor signaling at the plasma membrane, links oxidative stress, EGFR, and tumorigenesis. Decoding the molecular interactions between oxidative stress and ceramide pathways and characterizing ubiquitylation control of receptor desensitization should provide new strategies for intervention in diverse pulmonary diseases and in diagnosing and eradicating cancer.}, keywords = {Animals, antioxidants, Apoptosis, Binding Sites, Caspase 3, Caspases, Cell Line, Tumor, Cell Membrane, Cell Proliferation, Cell Survival, Ceramides, Enzyme Activation, Epithelial Cells, Humans, Hydrogen Peroxide, Ligands, Models, Biological, Neoplasms, Oxidants, Oxidative Stress, Phosphorylation, Proteasome Endopeptidase Complex, Protein Binding, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Proto-Oncogene Proteins c-cbl, reactive oxygen species, Receptor, Epidermal Growth Factor, Tyrosine, ubiquitin, Ubiquitin-Protein Ligases}, author = {Goldkorn, Tzipora and Ravid, Tommer and Khan, Elaine M} } @article {3095177, title = {Tumor necrosis factor-{alpha} decreases Akt protein levels in 3T3-L1 adipocytes via the caspase-dependent ubiquitination of Akt.}, journal = {Endocrinology}, volume = {146}, year = {2005}, month = {2005 Jun}, pages = {2726-35}, abstract = {TNF-alpha is a mediator of insulin resistance in sepsis, obesity, and type 2 diabetes and is known to impair insulin signaling in adipocytes. Akt (protein kinase B) is a crucial signaling mediator for insulin. In the present study we examined the posttranslational mechanisms by which short-term (<6-h) exposure of 3T3-L1 adipocytes to TNF-alpha decreases Akt levels. TNF-alpha treatment both increased the ubiquitination of Akt and decreased its protein level. The decrease in protein was associated with the presence of an (immunoreactive) Akt fragment after TNF-alpha treatment, indicative of Akt cleavage. The broad-spectrum caspase inhibitor t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone markedly suppressed these effects of TNF-alpha. The caspase-6 inhibitor Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH(2)F potently suppressed Akt ubiquitination, degradation, and fragment formation, whereas the proteasome inhibitor Z-Leu-Leu-Leu-CHO modestly attenuated the decline in Akt levels. Exposure to TNF-alpha also enhanced the association of Akt with an E3 ligase activity. Adipocytes preexposed to TNF-alpha for 5 h and then stimulated with insulin for 30 min exhibited decreased levels of Akt, phosphorylated Akt, as well as phosphorylated Mdm2, which is a known direct substrate of Akt, and glucose uptake. Caspase inhibition attenuated these inhibitory effects of TNF-alpha. Collectively, our results suggest that TNF-alpha induces the caspase-dependent degradation of Akt via the cleavage and ubiquitination of Akt, which results in its degradation through the 26S proteasome. Furthermore, the caspase- and proteasome-mediated degradation of Akt due to TNF-alpha exposure leads to impaired Akt-dependent insulin signaling in adipocytes. These findings expand the mechanism by which TNF-alpha impairs insulin signaling.}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Caspase Inhibitors, Caspases, Cysteine Proteinase Inhibitors, Gene Expression, Insulin, Mice, Oligopeptides, Oncogene Proteins, Proteasome Endopeptidase Complex, Proteasome Inhibitors, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Signal Transduction, Tumor Necrosis Factor-alpha, ubiquitin}, author = {Medina, Edward A and Afsari, Robert R and Ravid, Tommer and Castillo, S Sianna and Erickson, Kent L and Goldkorn, Tzipora} } @article {3095178, title = {c-Cbl-mediated ubiquitinylation is required for epidermal growth factor receptor exit from the early endosomes.}, journal = {J Biol Chem}, volume = {279}, year = {2004}, month = {2004 Aug 27}, pages = {37153-62}, abstract = {Epidermal growth factor receptor (EGFR) controls cell growth and has a key role in tumorigenic processes. The extent of EGFR signaling is tightly regulated by post-transcriptional modifications leading to down-regulation of the levels of the receptor. Previous studies from our laboratory demonstrated that the reactive oxidant hydrogen peroxide activates the EGFR, yet, without down-regulation of the receptor levels, which results in prolonged receptor signaling. In the present study we examined the role of the E3 ligase c-Cbl, as a possible link between oxidative stress, EGFR signaling, and tumorigenic responses. First, we ectopically expressed a mutant EGFR (Tyr-1045 --> Phe) in cells lacking endogenous receptor, to determine whether the lack of phosphorylation at this site is the cause for EGFR retention at the membrane under oxidative stress, as we have previously suggested. Our findings suggest that abrogation of tyrosine 1045 phosphorylation alone is not enough to retain the EGFR at the plasma membrane under oxidative stress. Second, through the use of the Src inhibitor PP1, our findings establish EGFR movement out of the early endosomes as the exact location where c-Cbl-mediated ubiquitinylation is essential for EGFR trafficking. Finally, our studies substantiate the findings that c-Cbl-mediated ubiquitinylation is needed for degradation, but not for internalization of the EGFR in both transfection-dependent Chinese hamster ovary cells and transfection-independent A549 lung epithelial cells. These findings only begin to explain the features seen under oxidative stress, but they yield a greater understanding of the role of c-Cbl in EGFR trafficking.}, keywords = {Animals, Blotting, Western, Cell Line, Tumor, CHO Cells, Cricetinae, Down-Regulation, Endosomes, Humans, Hydrogen Peroxide, Microscopy, Fluorescence, Models, Biological, Mutation, Oxidative Stress, Phenylalanine, Phosphoprotein Phosphatases, Precipitin Tests, Protein Transport, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Receptor, Epidermal Growth Factor, RNA Processing, Post-Transcriptional, Signal Transduction, Transfection, Tyrosine, ubiquitin, Ubiquitin-Protein Ligases}, author = {Ravid, Tommer and Heidinger, Jill M and Gee, Peter and Khan, Elaine M and Goldkorn, Tzipora} } @article {3095179, title = {NF-kappaB signaling: flipping the switch with polyubiquitin chains.}, journal = {Curr Biol}, volume = {14}, year = {2004}, month = {2004 Oct 26}, pages = {R898-900}, abstract = {Protein modification by ubiquitin has emerged as an important cellular regulatory mechanism. Recent studies illustrate the surprising ways in which polyubiquitin chains are manipulated in the regulation of NF-kappaB signaling.}, keywords = {Gene Expression Regulation, Models, Biological, NF-kappa B, Phosphorylation, Polyubiquitin, Protein Processing, Post-Translational, Proteins, Signal Transduction, Tumor Necrosis Factor-alpha}, author = {Ravid, Tommer and Hochstrasser, Mark} } @article {3095180, title = {Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells.}, journal = {Am J Physiol Lung Cell Mol Physiol}, volume = {284}, year = {2003}, month = {2003 Jun}, pages = {L1082-92}, abstract = {Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.}, keywords = {Adenocarcinoma, Apoptosis, Caspase 3, Caspases, Ceramides, Enzyme Activation, Enzyme Inhibitors, Gene Expression, Humans, Hydrogen Peroxide, Lung Neoplasms, Morpholines, Oxidants, Proto-Oncogene Proteins c-bcl-2, Tumor Cells, Cultured}, author = {Ravid, Tommer and Tsaba, Adili and Gee, Peter and Rasooly, Reuven and Medina, Edward A and Goldkorn, Tzipora} } @article {3095181, title = {Epidermal growth factor receptor activation under oxidative stress fails to promote c-Cbl mediated down-regulation.}, journal = {J Biol Chem}, volume = {277}, year = {2002}, month = {2002 Aug 23}, pages = {31214-9}, abstract = {Activation of the epidermal growth factor (EGF) receptor by its ligand, EGF, rapidly enhances receptor internalization and degradation, which desensitizes receptor signaling. In contrast, we have shown previously that exposure to oxidative stress in the form of hydrogen peroxide (H(2)O(2)) activated the EGF receptor but that the levels of activated receptors did not decline, which resulted in prolonged receptor signaling. This study provides mechanistic insights into these different modes of EGF receptor activation. Here we demonstrate that the pattern of receptor tyrosine phosphorylation induced by H(2)O(2) differs from that induced by its ligand, EGF. Importantly, H(2)O(2) generates a receptor with negligible phosphorylation at tyrosine 1045, the major docking site for the ubiquitin ligase c-Cbl. As a result, H(2)O(2)-activated receptors fail to recruit c-Cbl and do not undergo ubiquitination and endocytosis. In summary, H(2)O(2) stimulation results in an activated receptor uncoupled from normal down-regulation, a process that may contribute to oxidant-mediated tumorigenesis.}, keywords = {Down-Regulation, Epidermal Growth Factor, Glucose Oxidase, Humans, Hydrogen Peroxide, Oxidative Stress, Phosphorylation, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Receptor, Epidermal Growth Factor, Tyrosine, ubiquitin, Ubiquitin-Protein Ligases}, author = {Ravid, Tommer and Sweeney, Colleen and Gee, Peter and Carraway, Kermit L and Goldkorn, Tzipora} } @article {3095182, title = {Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.}, journal = {Am J Respir Cell Mol Biol}, volume = {25}, year = {2001}, month = {2001 Dec}, pages = {676-84}, abstract = {Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.}, keywords = {Acetylcysteine, Amitrole, Annexin A5, antioxidants, Apoptosis, Bronchi, Buthionine Sulfoximine, Catalase, Cells, Cultured, Ceramides, Diacylglycerol Kinase, Dithiothreitol, DNA Fragmentation, Dose-Response Relationship, Drug, Enzyme Inhibitors, Epithelial Cells, Flow Cytometry, glutathione, Humans, Hydrogen Peroxide, In Situ Nick-End Labeling, Mercaptoethanol, Microscopy, Fluorescence, Oxidation-Reduction, Oxidative Stress, Pulmonary Alveoli, reactive oxygen species, Trachea}, author = {Lavrentiadou, S N and Chan, C and Kawcak, T and Ravid, T and Tsaba, A and van der Vliet, A and Rasooly, R and Goldkorn, T} } @article {3095183, title = {The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.}, journal = {J Biol Chem}, volume = {275}, year = {2000}, month = {2000 Nov 17}, pages = {35840-7}, abstract = {3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.}, keywords = {Animals, beta-Galactosidase, Bicyclo Compounds, Heterocyclic, Cell Line, Cholesterol, Cricetinae, Cycloheximide, Cysteine Proteinase Inhibitors, Farnesyl-Diphosphate Farnesyltransferase, Humans, Hydroxycholesterols, Hydroxymethylglutaryl CoA Reductases, Leupeptins, Lovastatin, Oligopeptides, Peptide Hydrolases, Precipitin Tests, Proteasome Endopeptidase Complex, Recombinant Fusion Proteins, Tricarboxylic Acids, Ubiquitins}, author = {Ravid, T and Doolman, R and Avner, R and Harats, D and Roitelman, J} } @article {3095184, title = {Impaired regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation in lovastatin-resistant cells.}, journal = {J Biol Chem}, volume = {274}, year = {1999}, month = {1999 Oct 8}, pages = {29341-51}, abstract = {L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.}, keywords = {Animals, Cell Division, Cell Line, CHO Cells, Cricetinae, Drug resistance, Gene Expression Regulation, Enzymologic, Genes, Reporter, Hydroxycholesterols, Hydroxymethylglutaryl CoA Reductases, Lipids, Lipoproteins, Lipoproteins, LDL, Lovastatin, Mevalonic Acid, Microscopy, Electron, Promoter Regions, Genetic, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, RNA, Messenger, Transfection}, author = {Ravid, T and Avner, R and Polak-Charcon, S and Faust, J R and Roitelman, J} } @article {3095185, title = {Lipid accumulation in the ovaries of a marine shrimp penaeus semisulcatus (de haan)}, journal = {J Exp Biol}, volume = {202 (Pt 13)}, year = {1999}, month = {1999 Jul}, pages = {1819-29}, abstract = {By the end of oocyte development, the ovaries of Penaeus semisulcatus have accumulated almost equal amounts (approximately 16 mg lipid g-1 protein) of phospholipids and triacylglycerols. The phospholipids consist mainly of phosphatidylcholine (75-80 \%) and phosphatidylethanolamine (20-25 \%). Approximately 30 \% of the total fatty acid content of both phospholipids and triacylglycerols is made up of polyunsaturated fatty acids. In fractions obtained by centrifugation of ovarian homogenates, most of the increase in levels of ovarian lipids during ovarian maturation was associated with an increase in triacylglycerol levels in the floating fat fraction and of phospholipids in the infranatant fraction. The presence of polyunsaturated fatty acids in the ovaries indicates the occurrence of lipid transport to the ovary during oocyte maturation. The gradual decrease in the relative abundance of polyunsaturated fatty acids as the ovaries matured supports previously published results suggesting intra-ovarian synthesis of saturated and mono-unsaturated fatty acids. Most of the lipids found in the female haemolymph (64.8 \%) were recovered in the high-density lipoprotein fraction after density ultracentrifugation. The haemocyanin fraction recovered from this stage of fractionation contained substantial amounts of lipid (16.8 \%) that could be removed by further sequential centrifugation at a higher NaBr density, leaving less than 0.9 \% of the total haemolymph lipids associated with this fraction. While 16.2 \% of the lipids were recovered from the very high-density lipoprotein fractions, these lipoproteins carried only 64-89 microg lipid mg-1 protein compared with 538.9 microg lipid mg-1 protein in the high-density lipoprotein fraction, indicating that the high-density lipoproteins are more likely to be the main transporters of lipids to the ovary. However, the contribution of very high-density lipoproteins to lipid transport cannot be ruled out at this stage. In this study, we present two models for lipid transport to the ovary based on the abundance of phospholipids and triacylglycerols in the haemolymph and on the amounts of polyunsaturated fatty acids accumulated within the ovary during vitellogenesis.}, author = {Ravid, T and Tietz, A and Khayat, M and Boehm, E and Michelis, R and Lubzens, E} } @article {Ravid1997, title = {Defective regulation of HMG-CoA reductase degradation in the lovastatin-resistant L-90 cells}, journal = {FASEB Journal}, volume = {11}, year = {1997}, abstract = {HMG-CoA reductase (HMGR) is a 97kDa glycoprotein of the eddoplasmic reticulum (ER) which catalyzes the formation of mevalonate (MVA), the rate limiting step in the cholesterol biosynthetic pathway. The turnover of HMGR is regulated by the cellular levels of sterol and nonsterol MVA-derived isoprenoids; when cells are starved for sterols/MVA, the enzyme is degraded with a halflife (tl/2) of 7-15h. The rate of HMGR degradation is accelerated 3-5 fold (tl/2 2-3h) in sterols/MVA replete cells. This regulation is imparted to the enzyme by its elaborated membrane spanning domain. We have selected for a stable Chinese hamster ovary-derived cell-line, designated L-90, that grows in the presence of 90M lovastatin, a potent inhibitor of HMGR. Similar to other statin-resistant cell lines (e.g., UT-1), L-90 cells overexpress tiMGR as a result of gene amplification. The cells also have crystaUoid ER. Surprisingly, L90 cells do not accelerate the rate of HMGR degradation in response to sterols or MVA. This abnormal phenotype appears to b due to a defect in tran,s-acting factor(s) because L-90 cells also do not regdlate the turnover of transfected HMGai, a 150kDa fusion protein between the membrane domain of tIMGR and bacterial t3-galactosidase. Therefore, L-90 cells should prove useful in identifying cellular factor(s) that operate in the regulated degradation of HMGR. One such factor may be a 97kDa protein, which is not HMGR, that co-immunoprecipitates with HMGal in transfected L-90 cells.}, issn = {08926638}, author = {Ravid, T. and R. Avner and S. Potak-Charcon and J. Roitelman} } @article {3095186, title = {Isolation and characterization of the high-density lipoproteins from the hemolymph and ovary of the penaeid shrimp Penaeus semisulcatus (de Haan): apoproteins and lipids.}, journal = {J Exp Zool}, volume = {278}, year = {1997}, month = {1997 Aug 15}, pages = {339-48}, abstract = {The high-density lipoproteins (HDLs) found in the male and female hemolymph of Penaeus semisulcatus de Haan were isolated by NaBr (1.22 g/ml) followed by sucrose gradient (5-25\%) ultracentrifugation. The male HDL contained one protein, lipoprotein 1 (LP1), composed of one 110-kDa peptide subunit. The female HDL contained two proteins: 1) the LP1 that was immunoidentical to the male LP1 and was similarly composed of one 110-kDa peptide subunit and 2) vitellogenin (Vg), reacting positively with the rabbit antiserum generated against vitellin (Vt) that was isolated from vitellogenic ovaries. Both Vg and Vt consisted mainly of three polypeptide subunits (200, 120, and 80 kDa) as revealed by denatured PAGE and Western blot. The LP1 from males or females did not react with the Vt rabbit antiserum. Similarly, Vg and Vt did not react with the rabbit antiserum prepared against LP1. Phospholipids (PL) constituted 71-76\% of the total lipids in the hemolymph and HDLs of both male and female hemolymph. Cholesterol (Ch) amounted to 17-20\%, and small amounts (5\%) of diacylglycerols (DAG) were also carried by these HDLs. Both the PL and DAG contained highly unsaturated fatty acids (20:5 omega 3 and 22:6 omega 3) that are transported from the food or hepatopancreas to the tissues, including the vitellogenic ovaries in females. In the present study we show for the first time the separate lipid composition of female LP1 and Vg and compare them with the lipids attached to the Vt. Vg had a lower lipid content than LP1 (540 and 1089 mg/g protein, respectively). Differences were also found in the relative abundance of PL, Ch, and DAG classes in the LP1 in comparison with Vg. Furthermore, small amounts (approximately 3.8\%) of triacylglycerols (TAG) were found only in the hemolymph of vitellogenic females, and they were associated with the Vg. Although Vg and Vt were composed of similar polypeptides, their lipid composition was different Vt, in contrast to Vg, carried considerable amounts of TAG (approximately 22\%) and only trace amounts of DAG. The significance of the TAG in the hemolymph of vitellogenic females is not known, and the functional relationship between Vg and Vt requires future extensive studies. Lipids were not detected in hemocyanin that was purified from clotted hemolymph.}, keywords = {Animals, Blotting, Western, Centrifugation, Density Gradient, Egg Proteins, Electrophoresis, Polyacrylamide Gel, Fatty Acids, Female, Hemolymph, Lipids, Lipoproteins, HDL, Male, Ovary, Penaeidae, Sex Characteristics, Vitellogenins}, author = {Lubzens, E and Ravid, T and Khayat, M and Daube, N and Tietz, A} }