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Christopher P Walczak, Dara E Leto, Lichao Zhang, Celeste Riepe, Ryan Y Muller, Paul A DaRosa, Nicholas T Ingolia, Joshua E Elias, Ron R Kopito
Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes...
January 9, 2019: Proceedings of the National Academy of Sciences of the United States of America
Dara E Leto, David W Morgens, Lichao Zhang, Christopher P Walczak, Joshua E Elias, Michael C Bassik, Ron R Kopito
The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD)...
December 6, 2018: Molecular Cell
Camille I Pataki, João Rodrigues, Lichao Zhang, Junyang Qian, Bradley Efron, Trevor Hastie, Joshua E Elias, Michael Levitt, Ron R Kopito
Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles...
August 28, 2018: Proceedings of the National Academy of Sciences of the United States of America
Annemieke T van der Goot, Margaret M P Pearce, Dara E Leto, Thomas A Shaler, Ron R Kopito
Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition...
May 3, 2018: Molecular Cell
Jiwon Hwang, Christopher P Walczak, Thomas A Shaler, James A Olzmann, Lichao Zhang, Joshua E Elias, Ron R Kopito
Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1...
June 2, 2017: Journal of Biological Chemistry
Patrick Sweeney, Hyunsun Park, Marc Baumann, John Dunlop, Judith Frydman, Ron Kopito, Alexander McCampbell, Gabrielle Leblanc, Anjli Venkateswaran, Antti Nurmi, Robert Hodgson
A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network...
2017: Translational Neurodegeneration
Kyung Suk Lee, Shachar Iwanir, Ronen B Kopito, Monika Scholz, John A Calarco, David Biron, Erel Levine
Animals integrate physiological and environmental signals to modulate their food uptake. The nematode C. elegans, whose food uptake consists of pumping bacteria from the environment into the gut, provides excellent opportunities for discovering principles of conserved regulatory mechanisms. Here we show that worms implement a graded feeding response to the concentration of environmental bacteria by modulating a commitment to bursts of fast pumping. Using long-term, high-resolution, longitudinal recordings of feeding dynamics under defined conditions, we find that the frequency and duration of pumping bursts increase and the duration of long pauses diminishes in environments richer in bacteria...
February 1, 2017: Nature Communications
Margaret M P Pearce, Ron R Kopito
Transmissible spongiform encephalopathies are infectious neurodegenerative diseases caused by the conversion of prion protein (PrP) into a self-replicating conformation that spreads via templated conversion of natively folded PrP molecules within or between cells. Recent studies provide compelling evidence that prion-like behavior is a general property of most protein aggregates associated with neurodegenerative diseases. Many of these disorders are associated with spontaneous protein aggregation, but genetic mutations can increase the aggregation propensity of specific proteins, including expansion of polyglutamine (polyQ) tracts, which is causative of nine inherited neurodegenerative diseases...
February 1, 2018: Cold Spring Harbor Perspectives in Medicine
Adi Ben Yehuda, Marwa Risheq, Ofra Novoplansky, Kirill Bersuker, Ron R Kopito, Michal Goldberg, Michael Brandeis
Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci...
2017: PloS One
Bianca Schrul, Ron R Kopito
Lipid droplets (LDs) are endoplasmic reticulum (ER)-derived lipid storage organelles uniquely encapsulated by phospholipid monolayers. LD membrane proteins are embedded into the monolayer in a monotopic hairpin topology and are therefore likely to have requirements for their biogenesis distinct from those inserting as bitopic and polytopic proteins into phospholipid bilayers. UBXD8 belongs to a subfamily of hairpin proteins that localize to both the ER and LDs, and are initially inserted into the cytoplasmic leaflet of the ER bilayer before partitioning to the LD monolayer...
July 2016: Nature Cell Biology
Ron R Kopito
No abstract text is available yet for this article.
August 2016: Trends in Cell Biology
Mohammed Inayathullah, Jayakumar Rajadas
Protein misfolding and aggregation are responsible for a large number of diseases called protein conformational diseases or disorders that include Alzheimer׳s disease, Huntington׳s diseases, Prion related encephalopathies and type-II diabetes ( (Kopito and Ron, 2000) [1]. A variety of studies have shown that some small organic molecules, known as osmolytes have the ability to stabilize native conformation of proteins and prevent misfolding and aggregation (
June 2016: Data in Brief
Kirill Bersuker, Michael Brandeis, Ron R Kopito
Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease...
April 25, 2016: Journal of Cell Biology
Margaret M P Pearce, Ellen J Spartz, Weizhe Hong, Liqun Luo, Ron R Kopito
The brain has a limited capacity to self-protect against protein aggregate-associated pathology, and mounting evidence supports a role for phagocytic glia in this process. We have established a Drosophila model to investigate the role of phagocytic glia in clearance of neuronal mutant huntingtin (Htt) aggregates associated with Huntington disease. We find that glia regulate steady-state numbers of Htt aggregates expressed in neurons through a clearance mechanism that requires the glial scavenger receptor Draper and downstream phagocytic engulfment machinery...
April 13, 2015: Nature Communications
Ronen B Kopito, Erel Levine
Animal response to changes in environmental cues is a complex dynamical process that occurs at diverse molecular and cellular levels. To gain a quantitative understanding of such processes, it is desirable to observe many individuals, subjected to repeatable and well defined environmental cues over long time periods. Here we present WormSpa, a microfluidic system where worms are individually confined in optimized chambers. We show that worms in WormSpa are neither stressed nor starved, and in particular exhibit pumping and egg-laying behaviors equivalent to those of freely behaving worms...
February 21, 2014: Lab on a Chip
Kirill Bersuker, Mark S Hipp, Barbara Calamini, Richard I Morimoto, Ron R Kopito
The cellular heat shock response (HSR) protects cells from toxicity associated with defective protein folding, and this pathway is widely viewed as a potential pharmacological target to treat neurodegenerative diseases linked to protein aggregation. Here we show that the HSR is not activated by mutant huntingtin (HTT) even in cells selected for the highest expression levels and for the presence of inclusion bodies containing aggregated protein. Surprisingly, HSR activation by HSF1 overexpression or by administration of a small molecule activator lowers the concentration threshold at which HTT forms inclusion bodies in cells expressing aggregation-prone, polyglutamine-expanded fragments of HTT...
August 16, 2013: Journal of Biological Chemistry
Aaron M Streets, Yannick Sourigues, Ron R Kopito, Ronald Melki, Stephen R Quake
An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils...
2013: PloS One
James A Olzmann, Caleb M Richter, Ron R Kopito
UBXD8 is a membrane-embedded recruitment factor for the p97/VCP segregase that has been previously linked to endoplasmic reticulum (ER)-associated degradation and to the control of triacylglycerol synthesis in the ER. UBXD8 also has been identified as a component of cytoplasmic lipid droplets (LDs), but neither the mechanisms that control its trafficking between the ER and LDs nor its functions in the latter organelle have been investigated previously. Here we report that association of UBXD8 with the ER-resident rhomboid pseudoprotease UBAC2 specifically restricts trafficking of UBXD8 to LDs, and that the steady-state partitioning of UBXD8 between the ER and LDs can be experimentally manipulated by controlling the relative expression of these two proteins...
January 22, 2013: Proceedings of the National Academy of Sciences of the United States of America
James A Olzmann, Ron R Kopito, John C Christianson
The endoplasmic reticulum (ER) is the site of synthesis for nearly one-third of the eukaryotic proteome and is accordingly endowed with specialized machinery to ensure that proteins deployed to the distal secretory pathway are correctly folded and assembled into native oligomeric complexes. Proteins failing to meet this conformational standard are degraded by ER-associated degradation (ERAD), a complex process through which folding-defective proteins are selected and ultimately degraded by the ubiquitin-proteasome system...
September 2013: Cold Spring Harbor Perspectives in Biology
Dara P Dowlatshahi, Virginie Sandrin, Sandro Vivona, Thomas A Shaler, Stephen E Kaiser, Francesco Melandri, Wesley I Sundquist, Ron R Kopito
The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub...
December 11, 2012: Developmental Cell
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