Publications by authors named "Yosef Shaul"

67 Publications

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

CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing.

Int J Mol Sci 2021 Apr 3;22(7). Epub 2021 Apr 3.

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

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is "scarless" since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system's utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.
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http://dx.doi.org/10.3390/ijms22073741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038335PMC
April 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

Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing.

Biomolecules 2019 10 8;9(10). Epub 2019 Oct 8.

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

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.
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http://dx.doi.org/10.3390/biom9100584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6843829PMC
October 2019

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1.

Biochem Biophys Res Commun 2019 02 14;509(4):1015-1020. Epub 2019 Jan 14.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address:

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes. The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.011DOI Listing
February 2019

Hippo Pathway Regulation by Tyrosine Kinases.

Methods Mol Biol 2019 ;1893:215-236

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

The Hippo pathway utilizes a well-characterized Ser/Thr kinase cascade to control the downstream effectors, Yap and Taz. In addition, Yap/Taz and other Hippo pathway components are directly regulated by tyrosine kinases (TKs). The methodological strategies described here use the example of the c-Abl non-receptor TK and the Yap substrate to outline the steps used to identify and to validate tyrosine phosphorylation sites, including bioinformatic approaches, ectopic expression of proteins in transfected tissue culture cells, and mutagenesis of endogenous proteins by CRISPR-Cas9. These general strategies can be applied to investigate regulation of protein signaling moieties by tyrosine phosphorylation in the context of distinct TKs.
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http://dx.doi.org/10.1007/978-1-4939-8910-2_17DOI Listing
June 2019

The Disordered Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated Granules.

Proteomics 2018 11 8;18(21-22):e1800076. Epub 2018 Aug 8.

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

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.
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http://dx.doi.org/10.1002/pmic.201800076DOI Listing
November 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

The nonreceptor tyrosine kinase c-Src attenuates SCF(β-TrCP) E3-ligase activity abrogating Taz proteasomal degradation.

Proc Natl Acad Sci U S A 2017 02 1;114(7):1678-1683. Epub 2017 Feb 1.

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

The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(β-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(β-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of β-TrCP, was rescued by Src, suggesting a wider effect of Src on β-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(β-TrCP).
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http://dx.doi.org/10.1073/pnas.1610223114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320963PMC
February 2017

c-Abl forces YAP to switch sides.

Mol Cell Oncol 2015 Jul-Sep;2(3):e995006. Epub 2015 Jan 23.

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

Cancer research has been significantly accelerated by viewing cancer as a functional collision between 2 dichotomous sets of genes: oncogenes and tumor suppressors. Signaling pathways turn oncogenes and tumor suppressors on and off to dictate cell fate decisions. We contend that signaling also dictates opposing behaviors of a given effector.
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http://dx.doi.org/10.4161/23723556.2014.995006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905508PMC
June 2016

Hepatitis B virus induces RNR-R2 expression via DNA damage response activation.

J Hepatol 2015 Oct 27;63(4):789-96. Epub 2015 May 27.

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

Background & Aims: Hepatitis B virus (HBV) infects and replicates in quiescent hepatocytes, which are deficient in dNTPs, the critical precursors of HBV replication. Most tumor viruses promote dNTP production in host cells by inducing cell proliferation. Although HBV is known as a major cause of hepatocellular carcinoma, it does not lead to cellular proliferation. Instead, HBV acquires dNTPs by activating the expression of the R2 subunit of the Ribonucleotide Reductase (RNR) holoenzyme, the cell cycle gene that is rate-limiting for generation of dNTPs, without inducing the cell cycle. We wished to elucidate the molecular basis of HBV-dependent R2 expression in quiescent cells.

Methods: Quiescent HepG2 cells were transduced with an HBV-containing lentiviral vector, and primary human hepatocytes were infected with HBV. DNA damage response and RNR-R2 gene expression were monitored under this condition.

Results: We report here that HBV-induced R2 expression is mediated by the E2F1 transcription factor, and that HBV induces E2F1 accumulation, modification and binding to the R2 promoter. We found that Chk1, a known E2F1 kinase that functions in response to DNA damage, was activated by HBV. In cells where Chk1 was pharmacologically inhibited, or depleted by shRNA-mediated knockdown, HBV-mediated R2 expression was severely attenuated. Furthermore, we found that HBV attenuates DNA repair, thus reducing cellular dNTP consumption.

Conclusions: Our findings demonstrate that HBV exploits the Chk1-E2F1 axis of the DNA damage response pathway to induce R2 expression in a cell cycle-independent manner. This suggests that inhibition of this pathway may have a therapeutic value for HBV carriers.
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http://dx.doi.org/10.1016/j.jhep.2015.05.017DOI Listing
October 2015

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage.

J Biol Chem 2015 Jul 5;290(27):16478-88. Epub 2015 May 5.

From the Department of Molecular Genetics and

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.
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http://dx.doi.org/10.1074/jbc.M114.628982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505402PMC
July 2015

Tyrosine phosphorylation of WW proteins.

Exp Biol Med (Maywood) 2015 Mar 26;240(3):375-82. Epub 2015 Jan 26.

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

A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein-protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins.
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http://dx.doi.org/10.1177/1535370214565991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935225PMC
March 2015

c-Abl tyrosine kinase promotes adipocyte differentiation by targeting PPAR-gamma 2.

Proc Natl Acad Sci U S A 2014 Nov 3;111(46):16365-70. Epub 2014 Nov 3.

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

Adipocyte differentiation, or adipogenesis, is a complex and highly regulated process. A recent proteomic analysis has predicted that the nonreceptor tyrosine kinase Abelson murine leukemia viral oncogene (c-Abl) is a putative key regulator of adipogenesis, but the underlying mechanism remained obscure. We found that c-Abl was activated during the early phase of mouse 3T3-L1 preadipocyte differentiation. Moreover, c-Abl activity was essential and its inhibition blocked differentiation to mature adipocytes. c-Abl directly controlled the expression and activity of the master adipogenic regulator peroxisome proliferator-activator receptor gamma 2 (PPARγ2). PPARγ2 physically associated with c-Abl and underwent phosphorylation on two tyrosine residues within its regulatory activation function 1 (AF1) domain. We demonstrated that this process positively regulates PPARγ2 stability and adipogenesis. Remarkably, c-Abl binding to PPARγ2 required the Pro12 residue that has a phenotypically well-studied common human genetic proline 12 alanine substitution (Pro12Ala) polymorphism. Our findings establish a critical role for c-Abl in adipocyte differentiation and explain the behavior of the known Pro12Ala polymorphism.
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http://dx.doi.org/10.1073/pnas.1411086111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246298PMC
November 2014

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

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

Context-dependent resistance to proteolysis of intrinsically disordered proteins.

Protein Sci 2011 Aug 8;20(8):1285-97. Epub 2011 Jun 8.

The Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot, Israel.

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.
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http://dx.doi.org/10.1002/pro.657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189518PMC
August 2011

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

Hepatocyte metabolic signalling pathways and regulation of hepatitis B virus expression.

Liver Int 2011 Mar 11;31(3):282-90. Epub 2011 Jan 11.

The Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.

Hepatitis B virus (HBV) is a small DNA virus responsible for significant morbidity and mortality worldwide. The liver, which is the main target organ for HBV infection, provides the virus with the machinery necessary for persistent infection and propagation, a process that might ultimately lead to severe liver pathologies such as chronic hepatitis, cirrhosis and liver cancer. HBV gene expression is regulated mainly at the transcriptional level by recruitment of a whole set of cellular transcription factors (TFs) and co-activators to support transcription. Over the years, many of these TFs were identified and interestingly enough most are associated with the body's nutritional state. These include the hepatocyte nuclear factors, forkhead Box O1, Farnesoid X receptor, cyclic-AMP response element-binding (CREB), CCAAT/enhancer-binding protein (C/EBP) and glucocorticoid receptor TFs and the transcription coactivator PPARγ coactivator-1α. Consequently, HBV gene expression is linked to hepatic metabolic processes such as glucose and fat production and utilization as well as bile acids' production and secretion. Furthermore, recent evidence indicates that HBV actively interferes with some of these hepatic metabolic processes by manipulating key TFs, such as CREB and C/EBP, to meet its requirements. The discovery of the mechanisms by which HBV is controlled by the hepatic metabolic milieu may broaden our understanding of the unique regulation of HBV expression and may also explain the mechanisms by which HBV induces liver pathologies. The emerging principle of the intimate link between HBV and liver metabolism can be further exploited for host-targeted therapeutic strategies.
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http://dx.doi.org/10.1111/j.1478-3231.2010.02423.xDOI 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 role of p53 in nutrients levels.

Cell Cycle 2010 Sep 15;9(18):3646-7. Epub 2010 Sep 15.

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http://dx.doi.org/10.4161/cc.9.18.13222DOI Listing
September 2010

c-Fos proteasomal degradation is activated by a default mechanism, and its regulation by NAD(P)H:quinone oxidoreductase 1 determines c-Fos serum response kinetics.

Mol Cell Biol 2010 Aug 24;30(15):3767-78. Epub 2010 May 24.

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

The short-lived proto-oncoprotein c-Fos is a component of the activator protein 1 (AP-1) transcription factor. A large region of c-Fos is intrinsically unstructured and susceptible to a recently characterized proteasomal ubiquitin-independent degradation (UID) pathway. UID is active by a default mechanism that is inhibited by NAD(P)H:quinone oxidoreductase 1 (NQO1), a 20S proteasome gatekeeper. Here, we show that NQO1 binds and induces robust c-Fos accumulation by blocking the UID pathway. c-Jun, a partner of c-Fos, also protects c-Fos from proteasomal degradation by default. Our findings suggest that NQO1 protects monomeric c-Fos from proteasomal UID, a function that is fulfilled later by c-Jun. We show that this process regulates c-Fos homeostasis (proteostasis) in response to serum stimulation, phosphorylation, nuclear translocation, and transcription activity. In addition, we show that NQO1 is important to ensure immediate c-Fos accumulation in response to serum, since a delayed response was observed under low NQO1 expression. These data suggest that in vivo, protein unstructured regions determine the kinetics and the homeostasis of regulatory proteins. Our data provide evidence for another layer of regulation of key regulatory proteins that functions at the level of protein degradation and is designed to ensure optimal formation of functional complexes such as AP-1.
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http://dx.doi.org/10.1128/MCB.00899-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916405PMC
August 2010

Curcumin inhibits hepatitis B virus via down-regulation of the metabolic coactivator PGC-1alpha.

FEBS Lett 2010 Jun 29;584(11):2485-90. Epub 2010 Apr 29.

The Institute of Gastroenterology and Liver Disease, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.

Hepatitis B virus (HBV) infects the liver and uses its cell host for gene expression and propagation. Therefore, targeting host factors essential for HBV gene expression is a potential anti-viral strategy. Here we show that treating HBV expressing cells with the natural phenolic compound curcumin inhibits HBV gene expression and replication. This inhibition is mediated via down-regulation of PGC-1alpha, a starvation-induced protein that initiates the gluconeogenesis cascade and that has been shown to robustly coactivate HBV transcription. We suggest curcumin as a host targeted therapy for HBV infection that may complement current virus-specific therapies.
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http://dx.doi.org/10.1016/j.febslet.2010.04.067DOI Listing
June 2010

Hepatitis B virus activates deoxynucleotide synthesis in nondividing hepatocytes by targeting the R2 gene.

Hepatology 2010 May;51(5):1538-46

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

Unlabelled: Hepatitis B virus (HBV) causes liver diseases from acute hepatitis to cirrhosis and liver cancer. Currently, more than 350 million people are chronic HBV carriers, with devastating prognosis. HBV is a small enveloped noncytopathic virus, containing a circular partially double-stranded DNA genome, and exhibits strong tropism for human liver cells. Infected individuals (acute and chronic) secrete about 10(7) to 10(11) virions per day to the bloodstream, with each infected cell releasing 50-300 viruses per day. HBV infects nondividing hepatocytes and replicates by reverse-transcribing the pregenomic RNA to DNA in the host cells. The level of deoxyribonucleotide triphosphates (dNTPs) in nondividing cells is too low to support viral replication and enable the high yield of secreted virions. Here, we report production of dNTPs by viral-dependent transcription activation of R2, the key component of ribonucleotide reductase (RNR), and show that this process is critical for the HBV life-cycle. This was found in an established HBV-positive cell line and was reproduced by HBV DNA-transduced cells, in both culture and mice. Furthermore, the viral hepatitis B X protein is essential in activating R2 expression by blocking access of Regulatory factor x1, a repressor of the R2 gene.

Conclusion: Our findings demonstrate that the hepatitis B X protein is critical in infecting nonproliferating hepatocytes, which contain a low dNTP level. In addition, we provide molecular evidence for a new mechanism of HBV-host cell interaction where RNR-R2, a critical cell-cycle gene, is selectively activated in nonproliferating cells. This mechanism may set the stage for formulating a new category of anti-HBV drugs.
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http://dx.doi.org/10.1002/hep.23519DOI Listing
May 2010

The Hippo tumor suppressor pathway: a brainstorming workshop.

Sci Signal 2009 Nov 3;2(95):mr6. Epub 2009 Nov 3.

Department of Molecular Medicine, Regina Elena Cancer Institute, Rome, Italy.

Researchers from around the world met for two days in April this year in Rome, Italy, to discuss progress in the rapidly developing field of Hippo signaling, which is relevant to cancer and the control of organ size. Most of the participants presented data related to previously uncharacterized proteins that physically and functionally interact with known components of the Hippo pathway and regulate its biological output.
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http://dx.doi.org/10.1126/scisignal.295mr6DOI Listing
November 2009

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

The "Trojan horse" model-delivery of anti-HBV small interfering RNAs by a recombinant HBV vector.

Biochem Biophys Res Commun 2009 Dec 8;390(3):619-23. Epub 2009 Oct 8.

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

Hepatitis B virus (HBV) is a small virus that infects the liver. The major obstacle in applying the RNA interference method as an anti-HBV weapon is the challenge to deliver the small interfering RNA molecules to the liver efficiently and specifically. Here we show that HBV-specific short hairpin RNAs (shRNAs) are efficiently expressed from a recombinant HBV into which an shRNA-expressing cassette was inserted, resulting in a significant knock-down of HBV gene expression. Notably, this recombinant HBV still expresses the HBV Core protein, which is targeted by the shRNAs produced by the same vector. Our results set the stage for further use of this recombinant HBV virus with the potential to function as a "Trojan horse"; one that specifically targets the liver and uses the resident virus as an helper for its own propagation, and at the same time eliminate itself and the resident HBV by knocking-down their gene expression.
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http://dx.doi.org/10.1016/j.bbrc.2009.10.016DOI Listing
December 2009
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