Publications by authors named "Peter Tsvetkov"

30 Publications

  • Page 1 of 1

NQO1 Binds and Supports SIRT1 Function.

Front Pharmacol 2021 21;12:671929. Epub 2021 Jun 21.

Department of Molecular Genetics Weizmann Institute of Science, Rehovot, Israel.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD dependent deacetylase may act as a rheostat in sensing mitochondrial stress.
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http://dx.doi.org/10.3389/fphar.2021.671929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8255383PMC
June 2021

Degradation of Intrinsically Disordered Proteins by the NADH 26S Proteasome.

Biomolecules 2020 12 7;10(12). Epub 2020 Dec 7.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity.
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http://dx.doi.org/10.3390/biom10121642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762313PMC
December 2020

Persistent Activation of mRNA Translation by Transient Hsp90 Inhibition.

Cell Rep 2020 08;32(6):108001

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.

The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a role promoting the evolution of new heritable traits. To better understand how Hsp90 can affect mRNA translation, we screen more than 1,600 factors involved in mRNA regulation for physical interactions with Hsp90 in human cells. The mRNA binding protein CPEB2 strongly binds Hsp90 via its prion domain. In a yeast model, transient inhibition of Hsp90 results in persistent activation of a CPEB translation reporter even in the absence of exogenous CPEB that persists for 30 generations after the inhibitor is removed. Ribosomal profiling reveals that some endogenous yeast mRNAs, including HAC1, show a persistent change in translation efficiency following transient Hsp90 inhibition. Thus, transient loss of Hsp90 function can promote a nongenetic inheritance of a translational state affecting specific mRNAs, introducing a mechanism by which Hsp90 can promote phenotypic variation.
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http://dx.doi.org/10.1016/j.celrep.2020.108001DOI Listing
August 2020

Author Correction: In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2020 07 8;11(1):3498. Epub 2020 Jul 8.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17282-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343817PMC
July 2020

In-cell identification and measurement of RNA-protein interactions.

Nat Commun 2019 11 22;10(1):5317. Epub 2019 Nov 22.

Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, 75005, France.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.
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http://dx.doi.org/10.1038/s41467-019-13235-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876571PMC
November 2019

Mitochondrial metabolism promotes adaptation to proteotoxic stress.

Nat Chem Biol 2019 07 27;15(7):681-689. Epub 2019 May 27.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.
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http://dx.doi.org/10.1038/s41589-019-0291-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183600PMC
July 2019

Genetic and transcriptional evolution alters cancer cell line drug response.

Nature 2018 08 8;560(7718):325-330. Epub 2018 Aug 8.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.
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http://dx.doi.org/10.1038/s41586-018-0409-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522222PMC
August 2018

Oncogenic addiction to high 26S proteasome level.

Cell Death Dis 2018 07 10;9(7):773. Epub 2018 Jul 10.

Department of Molecular Genetics, Weizmann Institute of Science, 76100, Rehovot, Israel.

Proteasomes are large intracellular complexes responsible for the degradation of cellular proteins. The altered protein homeostasis of cancer cells results in increased dependency on proteasome function. The cellular proteasome composition comprises the 20S catalytic complex that is frequently capped with the 19S regulatory particle in forming the 26S proteasome. Proteasome inhibitors target the catalytic barrel (20S) and thus this inhibition does not allow the deconvolution of the distinct roles of 20S versus 26S proteasomes in cancer progression. We examined the degree of dependency of cancer cells specifically to the level of the 26S proteasome complex. Oncogenic transformation of human and mouse immortalized cells with mutant Ras induced a strong posttranscriptional increase of the 26S proteasome subunits, giving rise to high 26S complex levels. Depletion of a single subunit of the 19S RP was sufficient to reduce the 26S proteasome level and lower the cellular 26S/20S ratio. Under this condition the viability of the Ras-transformed MCF10A cells was severely compromised. This observation led us to hypothesize that cancer cell survival is dependent on maximal utilization of its 26S proteasomes. We validated this possibility in a large number of cancer cell lines and found that partial reduction of the 26S proteasome level impairs viability in all cancer cells examined and was not correlated with cell doubling time or reduction efficiency. Interstingly, normal human fibroblasts are refractory to the same type of 26S proteasome reduction. The suppression of 26S proteasomes in cancer cells activated the UPR and caspase-3 and cells stained positive with Annexin V. In addition, suppression of the 26S proteasome resulted in cellular proteasome redistribution, cytoplasm shrinkage, and nuclear deformation, the hallmarks of apoptosis. The observed tumor cell-specific addiction to the 26S proteasome levels sets the stage for future strategies in exploiting this dependency in cancer therapy.
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http://dx.doi.org/10.1038/s41419-018-0806-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039477PMC
July 2018

A Genetic Tool to Track Protein Aggregates and Control Prion Inheritance.

Cell 2017 Nov 19;171(4):966-979.e18. Epub 2017 Oct 19.

Biological Design Center, Boston University, Boston, MA 02215, USA; Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. Electronic address:

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.
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http://dx.doi.org/10.1016/j.cell.2017.09.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675731PMC
November 2017

Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

Proc Natl Acad Sci U S A 2017 01 27;114(2):382-387. Epub 2016 Dec 27.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142.

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.
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http://dx.doi.org/10.1073/pnas.1619067114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240730PMC
January 2017

Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome.

Elife 2015 Sep 1;4. Epub 2015 Sep 1.

Whitehead Institute for Biomedical Research, Cambridge, United States.

Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.
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http://dx.doi.org/10.7554/eLife.08467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551903PMC
September 2015

NADH binds and stabilizes the 26S proteasomes independent of ATP.

J Biol Chem 2014 Apr 4;289(16):11272-11281. Epub 2014 Mar 4.

Departments of Molecular Genetics Rehovot 76100, Israel. Electronic address:

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.
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http://dx.doi.org/10.1074/jbc.M113.537175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036265PMC
April 2014

The protein level of PGC-1α, a key metabolic regulator, is controlled by NADH-NQO1.

Mol Cell Biol 2013 Jul 6;33(13):2603-13. Epub 2013 May 6.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

PGC-1α is a key transcription coactivator regulating energy metabolism in a tissue-specific manner. PGC-1α expression is tightly regulated, it is a highly labile protein, and it interacts with various proteins--the known attributes of intrinsically disordered proteins (IDPs). In this study, we characterize PGC-1α as an IDP and demonstrate that it is susceptible to 20S proteasomal degradation by default. We further demonstrate that PGC-1α degradation is inhibited by NQO1, a 20S gatekeeper protein. NQO1 binds and protects PGC-1α from degradation in an NADH-dependent manner. Using different cellular physiological settings, we also demonstrate that NQO1-mediated PGC-1α protection plays an important role in controlling both basal and physiologically induced PGC-1α protein level and activity. Our findings link NQO1, a cellular redox sensor, to the metabolite-sensing network that tunes PGC-1α expression and activity in regulating energy metabolism.
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http://dx.doi.org/10.1128/MCB.01672-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3700121PMC
July 2013

The isolation and characterization of renal cancer initiating cells from human Wilms' tumour xenografts unveils new therapeutic targets.

EMBO Mol Med 2013 01 13;5(1):18-37. Epub 2012 Dec 13.

Pediatric Stem Cell Research Institute, Edmond and LiliSafra Children's Hospital, Sheba Medical Center, Ramat-Gan, Israel.

There are considerable differences in tumour biology between adult and paediatric cancers. The existence of cancer initiating cells/cancer stem cells (CIC/CSC) in paediatric solid tumours is currently unclear. Here, we show the successful propagation of primary human Wilms' tumour (WT), a common paediatric renal malignancy, in immunodeficient mice, demonstrating the presence of a population of highly proliferative CIC/CSCs capable of serial xenograft initiation. Cell sorting and limiting dilution transplantation analysis of xenograft cells identified WT CSCs that harbour a primitive undifferentiated-NCAM1 expressing-"blastema" phenotype, including a capacity to expand and differentiate into the mature renal-like cell types observed in the primary tumour. WT CSCs, which can be further enriched by aldehyde dehydrogenase activity, overexpressed renal stemness and genes linked to poor patient prognosis, showed preferential protein expression of phosphorylated PKB/Akt and strong reduction of the miR-200 family. Complete eradication of WT in multiple xenograft models was achieved with a human NCAM antibody drug conjugate. The existence of CIC/CSCs in WT provides new therapeutic targets.
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http://dx.doi.org/10.1002/emmm.201201516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569651PMC
January 2013

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1.

Mol Cell 2012 Jul;47(1):76-86

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.
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http://dx.doi.org/10.1016/j.molcel.2012.05.049DOI Listing
July 2012

Determination of IUP based on susceptibility for degradation by default.

Methods Mol Biol 2012 ;895:3-18

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Intrinsically unstructured proteins (IUPs) like the structured proteins are subjected to proteasomal degradation. However, unlike the structured ones, there is no crucial need of protein unfolding step to access the IUPs to the 20S catalytic subunit of the proteasome. This distinctive behavior set the stage for operational definition of the IUPs based on their susceptibility to the 20S degradation in a cell free system. Numerous studies revealed that this is the case in the cells as well, although no comprehensive analysis was performed to date. IUPs are degraded by the 20S proteasome subunit by default, without being polyubiquitinated or undergoing any other modifications. IUPs escape the process of degradation by default by a number of mechanisms, of which a more general one is interaction with a partner named nanny. Based on these attributes one can define IUP by conducting a set of cell free and cell culture experiments as outlined in this chapter.
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http://dx.doi.org/10.1007/978-1-61779-927-3_1DOI Listing
October 2012

Thermo-resistant intrinsically disordered proteins are efficient 20S proteasome substrates.

Mol Biosyst 2012 Jan 25;8(1):368-73. Epub 2011 Oct 25.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

Based on software prediction, intrinsically disordered proteins (IDPs) are widely represented in animal cells where they play important instructive roles. Despite the predictive power of the available software programs we nevertheless need simple experimental tools to validate the predictions. IDPs were reported to be preferentially thermo-resistant and also are susceptible to degradation by the 20S proteasome. Analysis of a set of proteins revealed that thermo-resistant proteins are preferred 20S proteasome substrates. Positive correlations are evident between the percent of protein disorder and the level of thermal stability and 20S proteasomal susceptibility. The data obtained from these two assays do not fully overlap but in combination provide a more reliable experimental IDP definition. The correlation was more significant when the IUPred was used as the IDPs predicting software. We demonstrate in this work a simple experimental strategy to improve IDPs identification.
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http://dx.doi.org/10.1039/c1mb05283gDOI Listing
January 2012

BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

J Cell Sci 2011 Mar;124(Pt 6):969-77

Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.
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http://dx.doi.org/10.1242/jcs.058438DOI Listing
March 2011

E3 ligase STUB1/CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1) accumulation in aged brain, a process impaired in certain Alzheimer disease patients.

J Biol Chem 2011 Mar 10;286(11):8839-45. Epub 2011 Jan 10.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that is important in maintaining the cellular redox state and regulating protein degradation. The NQO1 polymorphism C609T has been associated with increased susceptibility to various age-related pathologies. We show here that NQO1 protein level is regulated by the E3 ligase STUB1/CHIP (C terminus of Hsc70-interacting protein). NQO1 binds STUB1 via the Hsc70-interacting domain (tetratricopeptide repeat domain) and undergoes ubiquitination and degradation. We demonstrate here that the product of the C609T polymorphism (P187S) is a stronger STUB1 interactor with increased susceptibility to ubiquitination by the E3 ligase STUB1. Furthermore, age-dependent decrease of STUB1 correlates with increased NQO1 accumulation. Remarkably, examination of hippocampi from Alzheimer disease patients revealed that in half of the cases examined the NQO1 protein level was undetectable due to C609T polymorphism, suggesting that the age-dependent accumulation of NQO1 is impaired in certain Alzheimer disease patients.
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http://dx.doi.org/10.1074/jbc.M110.193276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058985PMC
March 2011

The nanny model for IDPs.

Nat Chem Biol 2009 Nov;5(11):778-81

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

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http://dx.doi.org/10.1038/nchembio.233DOI Listing
November 2009

Susceptibility of p53 unstructured N terminus to 20 S proteasomal degradation programs the stress response.

J Biol Chem 2009 Sep 17;284(39):26234-42. Epub 2009 Jul 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

The N-terminal transcription activation domain of p53 is intrinsically unstructured. We show in vitro and in vivo that this domain initiates p53 degradation by the 20 S proteasome in a ubiquitin-independent fashion. The decay of metabolically labeled p53 follows biphasic kinetics with an immediate fast phase that is ubiquitin-independent and a second slower phase that is ubiquitin-dependent. The 20 S proteasome executes the first phase by default, whereas the second phase requires the 26 S proteasome. p53 N-terminal binding proteins, such as Hdmx, can selectively block the first phase of degradation. Remarkably, gamma-irradiation inhibits both p53 decay phases, whereas UV selectively negates the second phase, giving rise to discrete levels of p53 accumulation. Our data of a single protein experiencing double mode degradation mechanisms each with unique kinetics provide the mechanistic basis for programmable protein homeostasis (proteostasis).
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http://dx.doi.org/10.1074/jbc.M109.040493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785311PMC
September 2009

BAG-1 associates with Hsc70.Tau complex and regulates the proteasomal degradation of Tau protein.

J Biol Chem 2007 Dec 22;282(51):37276-84. Epub 2007 Oct 22.

Department of Neurobiology, Weizmann Insitute of Science, Rehovot, Israel 76100.

Intraneuronal accumulation of phosphorylated Tau protein is a molecular pathology found in many forms of dementia, including Alzheimer disease. Research into possible mechanisms leading to the accumulation of modified Tau protein and the possibility of removing Tau protein from the system have revealed that the chaperone protein system can interact with Tau and mediate its degradation. Hsp70/Hsc70, a member of the chaperone protein family, interacts with Tau protein and mediates proper folding of Tau and can promote degradation of Tau protein under certain circumstances. However, because Hsp70/Hsc70 has many binding partners that can mediate its activity, there is still much to discover about how Hsp70 acts in vivo to regulate Tau protein. BAG-1, an Hsp70/Hsc70 binding partner, has been implicated as a mediator of neuronal function. In this work we show that BAG-1 associates with Tau protein in an Hsc70-dependent manner. Overexpression of BAG-1 induced an increase in Tau levels, which is shown to be due to an inhibition of protein degradation. We further show that BAG-1 can inhibit the degradation of Tau protein by the 20 S proteasome but does not affect the ubiquitination of Tau protein. RNA-mediated interference depletion of BAG-1 leads to a decrease in total Tau protein levels as well as promoting hyperphosphorylation of the remaining protein. Induction of Hsp70 by heat shock enhanced the increase of Tau levels in cells overexpressing BAG-1 but induced a decrease of Tau levels in cells that were depleted of BAG-1. Finally, BAG-1 is highly expressed in neurons bearing Tau tangles in a mouse model of Alzheimer disease. This data suggests a molecular mechanism through which Tau protein levels are regulated in the cell and possible consequences for the pathology and treatment of Alzheimer disease.
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http://dx.doi.org/10.1074/jbc.M706379200DOI Listing
December 2007

Operational definition of intrinsically unstructured protein sequences based on susceptibility to the 20S proteasome.

Proteins 2008 Mar;70(4):1357-66

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Intrinsically unstructured proteins (IUPs), also known as natively unfolded proteins, lack well-defined secondary and tertiary structure under physiological conditions. In recent years, growing experimental and theoretical evidence has accumulated, indicating that many entire proteins and protein sequences are unstructured under physiological conditions, and that they play significant roles in diverse cellular processes. Bioinformatic algorithms have been developed to identify such sequences in proteins for which structural data are lacking, but still generate substantial numbers of false positives and negatives. We describe here a simple and reliable in vitro assay for identifying IUP sequences based on their susceptibility to 20S proteasomal degradation. We show that 20S proteasomes digest IUP sequences, under conditions in which native, and even molten globule states, are resistant. Furthermore, we show that protein-protein interactions can protect IUPs against 20S proteasomal action. Taken together, our results thus suggest that the 20S proteasome degradation assay provides a powerful system for operational definition of IUPs.
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http://dx.doi.org/10.1002/prot.21614DOI Listing
March 2008

The crystal structure of NAD(P)H quinone oxidoreductase 1 in complex with its potent inhibitor dicoumarol.

Biochemistry 2006 May;45(20):6372-8

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.
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http://dx.doi.org/10.1021/bi0600087DOI Listing
May 2006

Inhibition of NAD(P)H:quinone oxidoreductase 1 activity and induction of p53 degradation by the natural phenolic compound curcumin.

Proc Natl Acad Sci U S A 2005 Apr 4;102(15):5535-40. Epub 2005 Apr 4.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.
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http://dx.doi.org/10.1073/pnas.0501828102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC556252PMC
April 2005

20S proteasomal degradation of ornithine decarboxylase is regulated by NQO1.

Mol Cell 2005 Mar;17(5):645-55

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a very labile protein. ODC is a homodimeric enzyme that undergoes ubiquitin-independent proteasomal degradation via direct interaction with antizyme, a polyamine-induced protein. Binding of antizyme promotes the dissociation of ODC homodimers and marks ODC for degradation by the 26S proteasomes. We describe here an alternative pathway for ODC degradation that is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). We show that NQO1 binds and stabilizes ODC. Dicoumarol, an inhibitor of NQO1, dissociates ODC-NQO1 interaction and enhances ubiquitin-independent ODC proteasomal degradation. We further show that dicoumarol sensitizes ODC monomers to proteasomal degradation in an antizyme-independent manner. This process of NQO1-regulated ODC degradation was recapitulated in vitro by using purified 20S proteasomes. Finally, we show that the regulation of ODC stability by NQO1 is especially prominent under oxidative stress. Our findings assign to NQO1 a role in regulating ubiquitin-independent degradation of ODC by the 20S proteasomes.
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http://dx.doi.org/10.1016/j.molcel.2005.01.020DOI Listing
March 2005

A mechanism of ubiquitin-independent proteasomal degradation of the tumor suppressors p53 and p73.

Genes Dev 2005 Feb;19(3):316-21

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level.
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http://dx.doi.org/10.1101/gad.319905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC546509PMC
February 2005

P53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1.

Proc Natl Acad Sci U S A 2003 Dec 21;100(25):15065-70. Epub 2003 Nov 21.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent "hot-spot" p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.
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http://dx.doi.org/10.1073/pnas.2436329100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC299908PMC
December 2003
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