Publications by authors named "Nina Reuven"

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

Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis.

Front Cell Dev Biol 2021 20;9:671210. Epub 2021 May 20.

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such "error of nature," namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.
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http://dx.doi.org/10.3389/fcell.2021.671210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173195PMC
May 2021

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

J Cell Sci 2021 05 11;134(9). Epub 2021 May 11.

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

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.
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http://dx.doi.org/10.1242/jcs.254979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182410PMC
May 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

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

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

Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response.

J Virol 2014 Sep 25;88(17):10146-56. Epub 2014 Jun 25.

Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, Connecticut, USA

Unlabelled: The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Consistent with previous reports, we estimate that there are approximately 15 gaps per genome, and we calculate the average gap length to be approximately 30 bases. Virion DNA was isolated and treated with DNA-modifying enzymes in order to fill in the gaps and modify the ends. Interestingly, filling in gaps, blunting the ends, or adding random sequences to the 3' ends of DNA, producing 3' flaps, did not impair the infectivity of treated DNA following transfection of Vero cells. On the other hand, the formation of 5' flaps in the DNA following treatment resulted in a dramatic reduction (95 to 100%) in infectivity. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5' flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. The infectivity of 5'-flapped DNA was restored in cells that do not express DNA-PKcs and in cells cotransfected with the immediate early protein ICP0, which degrades DNA-PKcs. These results are consistent with previous reports that DNA-dependent protein kinase (DNA-PK) and the nonhomologous end joining (NHEJ) repair pathway are intrinsically antiviral and that ICP0 can counteract this effect. We suggest that HSV-1 DNA with 5' flaps may induce an antiviral state due to the induction of a DNA damage response, primarily mediated by NHEJ, that renders the HSV-1 genome less efficient for lytic infection.

Importance: For productive lytic infection to occur, HSV-1 must counteract a variety of cellular intrinsic antiviral mechanisms, including the DNA damage response (DDR). DDR pathways have been associated with silencing of gene expression, cell cycle arrest, and induction of apoptosis. In addition, the fate of viral genomes is likely to play a role in whether viral genomes adopt a configuration suitable for lytic DNA replication. This study demonstrates that virion DNA activates the cellular DDR kinase, DNA-PK, and that this response is inhibitory to viral infection. Furthermore, we show that HSV-1 ubiquitin ligase, ICP0, plays an important role in counteracting the negative effects of DNA-PK activation. These findings support the notion that DNA-PK is antiviral and suggest that the fate of incoming viral DNA has important consequences for the progression of lytic infection. This study underscores the complex evolutionary relationships between HSV and its host.
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http://dx.doi.org/10.1128/JVI.01723-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136335PMC
September 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 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

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 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

A regulatory circuit controlling Itch-mediated p73 degradation by Runx.

J Biol Chem 2008 Oct 12;283(41):27462-27468. Epub 2008 Aug 12.

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

The members of the tumor suppressor p53 family are under tight regulation by distinct ubiquitin-protein isopeptide (E3) ligases. The level of p73 is regulated by the E3 ligase Itch. Itch levels are sharply reduced in response to DNA damage with concomitant p73 accumulation and activation. The mechanism of controlling Itch level is not known. We show that the Itch promoter is a target of the transcription activator Runx. Yes-associated protein (Yap1) is a shared transcription co-activator of Runx and p73. Under normal conditions, the Runx-Yap1 complex binds the Itch promoter and supports its transcription and p73 degradation. In response to DNA damage, Yap1 is phosphorylated by c-Abl at the position Tyr-357. The modified Yap1 does not co-activate Runx in supporting Itch transcription. The subsequent reduction in the Itch level gives rise to p73 accumulation. These results demonstrate how Yap1 supports degradation of p73 via Runx and how it plays an opposite role in response to DNA damage.
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http://dx.doi.org/10.1074/jbc.M803941200DOI Listing
October 2008

Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage.

Mol Cell 2008 Feb;29(3):350-61

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

Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.
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http://dx.doi.org/10.1016/j.molcel.2007.12.022DOI Listing
February 2008

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

20S proteasomes and protein degradation "by default".

Bioessays 2006 Aug;28(8):844-9

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

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin-dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. Alternatively, certain proteins are inherently unstable and undergo "default" degradation by the 20S proteasomes. Puzzlingly, proteins are by large subjected to both degradation pathways. Proteins with unstructured regions have been found to be substrates of the 20S proteasomes in vitro and, therefore, unstructured regions may serve as signals for protein degradation "by default" in the cell. The literature is loaded with examples where engagement of a protein into larger complexes increases protein stability, possibly by escaping degradation "by default". Our model suggests that formation of protein complexes masks the unstructured regions, making them inaccessible to the 20S proteasomes. This model not only provides molecular explanations for a recent theoretical "cooperative stability" principle, but also provokes new predictions and explanations in the field of protein regulation and functionality.
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http://dx.doi.org/10.1002/bies.20447DOI Listing
August 2006

Autorepression of rfx1 gene expression: functional conservation from yeast to humans in response to DNA replication arrest.

Mol Cell Biol 2005 Dec;25(23):10665-73

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

The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.
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http://dx.doi.org/10.1128/MCB.25.23.10665-10673.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1291218PMC
December 2005

Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity.

J Virol 2005 Jul;79(14):9356-8

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3205, USA.

UL12 is a 5'- to 3'-exonuclease encoded by herpes simplex virus type 1 (HSV-1) which degrades single- and double-stranded DNA. UL12 and the single-strand DNA binding protein ICP8 mediate a strand exchange reaction. We found that ICP8 inhibited UL12 digestion of single-stranded DNA but stimulated digestion of double-stranded DNA threefold. The stimulatory effect of ICP8 was independent of a strand exchange reaction; furthermore, the effect was specific to ICP8, as it could not be reproduced by Escherichia coli single-stranded DNA binding protein. The effect of ICP8 on the rate of UL12 double-stranded DNA digestion is attributable to an increase in processivity in the presence of ICP8.
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http://dx.doi.org/10.1128/JVI.79.14.9356-9358.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1168754PMC
July 2005

Catalysis of strand exchange by the HSV-1 UL12 and ICP8 proteins: potent ICP8 recombinase activity is revealed upon resection of dsDNA substrate by nuclease.

J Mol Biol 2004 Sep;342(1):57-71

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington 06030-3205, USA.

The replication of herpes simplex virus type 1 (HSV-1) is associated with a high degree of homologous recombination, which is likely to be mediated, in part, by HSV-1-encoded proteins. We have previously shown that the HSV-1 encoded ICP8 protein and alkaline nuclease UL12 are capable of catalyzing an in vitro strand-exchange reaction. Here, we show, by electron microscopy, that the products of the strand exchange reaction between linear double-stranded DNA and circular single-stranded DNA consist of the expected joint molecule forms: sigma, alpha, and gapped circles. Other exonucleases, such as lambda Red alpha, which, like UL12, digests 5'-3', as well as Escherichia coli exonuclease III (ExoIII), which digests 3'-5', could substitute for UL12 in the strand exchange reaction by providing a resected DNA end. ICP8 generated the same intermediates and strand exchange products when the double-stranded DNA substrate was preresected by any of the nucleases. Using substrates with large regions of non-homology we found that pairing by ICP8 could be initiated from the middle of a DNA molecule and did not require a homologous end. In this reaction, the resection of a DNA end by the nuclease is required to reveal homologous sequences capable of being paired by ICP8. This study further illustrates the complexity of the multi-functional ICP8 protein.
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http://dx.doi.org/10.1016/j.jmb.2004.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412345PMC
September 2004

The UL12.5 gene product of herpes simplex virus type 1 exhibits nuclease and strand exchange activities but does not localize to the nucleus.

J Virol 2004 May;78(9):4599-608

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3205, USA.

The herpes simplex virus type 1 (HSV-1) alkaline nuclease, encoded by the UL12 gene, plays an important role in HSV-1 replication, as a null mutant of UL12 displays a severe growth defect. Although the precise in vivo role of UL12 has not yet been determined, several in vitro activities have been identified for the protein, including endo- and exonuclease activities, interaction with the HSV-1 single-stranded DNA binding protein ICP8, and an ability to promote strand exchange in conjunction with ICP8. In this study, we examined a naturally occurring N-terminally truncated version of UL12 called UL12.5. Previous studies showing that UL12.5 exhibits nuclease activity but is unable to complement a UL12 null virus posed a dilemma and suggested that UL12.5 may lack a critical activity possessed by the full-length protein, UL12. We constructed a recombinant baculovirus capable of expressing UL12.5 and purified soluble UL12.5 from infected insect cells. The purified UL12.5 exhibited both endo- and exonuclease activities but was less active than UL12. Like UL12, UL12.5 could mediate strand exchange with ICP8 and could also be coimmunoprecipitated with ICP8. The primary difference between the two proteins was in their intracellular localization, with UL12 localizing to the nucleus and UL12.5 remaining in the cytoplasm. We mapped a nuclear localization signal to the N terminus of UL12, the domain absent from UL12.5. In addition, when UL12.5 was overexpressed so that some of the enzyme leaked into the nucleus, it was able to partially complement the UL12 null mutant.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC387724PMC
http://dx.doi.org/10.1128/jvi.78.9.4599-4608.2004DOI Listing
May 2004

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro.

J Virol 2003 Jul;77(13):7425-33

Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3205, USA.

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'-->3' exonuclease that shares homology with Redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164775PMC
http://dx.doi.org/10.1128/jvi.77.13.7425-7433.2003DOI Listing
July 2003
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