scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2020 ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
maria Delucia ◽  
jinwoo ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether , our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria Delucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
Protein Interactions ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
Hiv 1

Abstract Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1.Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase,Conclusions: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals Targeting the p27 E3 ligase SCFSkp2 results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy

Blood ◽  
2008 ◽  
Vol 111 (9) ◽  
pp. 4690-4699 ◽  
Author(s):  
Qing Chen ◽  
Weilin Xie ◽  
Deborah J. Kuhn ◽  
Peter M. Voorhees ◽  
Antonia Lopez-Girona ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
High Throughput Screening ◽  
Antitumor Agents ◽  
Plasma Cells ◽  
E3 Ligase ◽  
Poor Prognostic Factor ◽  
Cycle Arrest ◽  
Ligase Function

Abstract Decreased p27Kip1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCFSkp2 E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27Kip1, agents inhibiting SCFSkp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCFSkp2 ligase function in vitro, and induced specific accumulation of p21 and other SCFSkp2 substrates in cells without activating a heat-shock protein response. CpdA prevented incorporation of Skp2 into the SCFSkp2 ligase, and induced G1/S cell-cycle arrest as well as SCFSkp2- and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCFSkp2 inhibitors as a novel class of antitumor agents.

The Novel Histone Deacetylase Inhibitor RAS2410 (Resminostat) Interferes with Important Signalling Pathways and Induces Apoptosis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4928-4928
Author(s):  
Philipp Baumann ◽  
Sonja Mandl-Weber ◽  
Felix Meinel ◽  
Ruediger Jankowsky ◽  
Fuat S. Oduncu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Synergistic Effects ◽  
In Vivo Studies ◽  
The Novel ◽  
Cycle Arrest

Abstract Abstract 4928 Inhibition of histone deacetylase (HDAC) is a promising target for novel, anti-myeloma agents. In this study we investigated the biologic effects of the novel HDAC inhibitor RAS2410 (also known as “4SC-201”, “resminostat”) on Multiple Myeloma (MM) cells in vitro. RAS2410 is a potent, direct inhibitor of HDACs 1, 3 and 6 (IC50 = 43-72nM) representing the HDAC classes I and II. Accordingly, RAS2410 induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of RAS2410 abrogate cell growth and strongly induce apoptosis (IC50 = 2.5-3μM in 3 out of 4 cell lines) in MM cell lines (NCI-H929, U-266, RPMI-8226, OPM-2) as well as in primary MM cells isolated from patients. At 1μM, RAS2410 induces G0/G1 cell cycle arrest in 3 out of 4 MM cell lines associated with decreased levels of cyclin D1, cdc25a, Cdk4, pRb and p53 as well as upregulation of p21. This cell cycle arrest is reflected by an inhibition of cell proliferation. RAS2410 decreases phosphorylation of 4EBP-1 and P70S6K indicating that RAS2410 induces apoptosis by interfering with Akt pathway signalling downstream of Akt. Treatment with RAS2410 results in increased protein levels of Bim and Bax and decreased levels of Bcl-xL. Caspases 3, 8 and 9 are activated by RAS2410. Furthermore, additive and synergistic effects in terms of apoptosis induction are observed for combinations of RAS2410 with melphalan, doxorubicin and the proteasome inhibitors bortezomib and S2209. In conclusion, we have identified potent anti-myeloma activity for the novel HDACi RAS2410. This study has yielded further insight into the biological sequelae of HDAC inhibition in MM and provides the rationale for in vivo studies and clinical trials using RAS2410 to improve patient outcome in MM. Disclosures Jankowsky: 4SC: Employment. Schmidmaier:4SC : Research Funding.

scholarly journals G2/M Cell Cycle Arrest Correlates with Primate Lentiviral Vpr Interaction with the SLX4 Complex

2014 ◽  
Vol 89 (1) ◽  
pp. 230-240 ◽  
Author(s):  
Gregory Berger ◽  
Madeleine Lawrence ◽  
Stephane Hué ◽  
Stuart J. D. Neil
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Fanconi Anemia ◽  
Human Cells ◽  
Repair Pathway ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTThe accessory genevpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vprin vivo.IMPORTANCEThe Vpr protein of HIV-1 and related viruses is essential for the virusin vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replicationin vivo.

Polyphyllin I Induces Cell Cycle Arrest and Cell Apoptosis in Human Retinoblastoma Y-79 Cells through Targeting p53

2018 ◽  
Vol 18 (6) ◽  
pp. 875-881 ◽  
Author(s):  
Xue Zhu ◽  
Ke Wang ◽  
Kai Zhang ◽  
Yi Pan ◽  
Fanfan Zhou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Apoptosis ◽  
Potential Effect ◽  
External Beam Radiation ◽  
Beam Radiation ◽  
P53 Expression ◽  
Cycle Arrest ◽  
Wide Range ◽  
Human Retinoblastoma

Background: Retinoblastoma is the most common intraocular malignant tumor in childhood. Although external beam radiation and enucleation are effective to control retinoblastoma, eye salvage and vision preservation are still significant challenges. Polyphyllin I (PPI), a natural compound extracted from Paris polyphylla rhizomes, has a wide range of activities against many types of cancers. However, the potential effect of this herbal compound on retinoblastoma has not yet been investigated. Method: In the present study, we evaluated the cytotoxic effect of PPI on human retinoblastoma Y-79 cells as well as its underlying molecular mechanism. Our results indicated that PPI treatment significantly inhibited cell proliferation, arrested the cell cycle at G2/M phase and induced cell apoptosis of Y79 cells through the mitochondrial- dependent intrinsic pathway. Moreover, p53 is involved in PPI-induced cytotoxicity in human retinoblastoma Y-79 cells. Exposure to 10 μM PPI for 48 h dramatically induced the expression levels of p53, phosphorylated- p53 and acetylated-p53. Furthermore, blockade of p53 expression effectively attenuated PPI-induced cell cycle arrest and cell apoptosis in Y-79 cells. Result: These results demonstrated that PPI exhibits anti-proliferation effect on human retinoblastoma Y-79 cells through modulating p53 expression, stabilization and activation. This information shed light on the potential application of PPI in retinoblastoma therapy.

scholarly journals Role of Mis Localization of DNA Repair Factors in Cell Cycle Arrest

2019 ◽  
Vol 116 (3) ◽  
pp. 76a
Author(s):  
Manasvita Vashisth ◽  
Sangkyun Cho ◽  
Dennis Discher
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Cycle Arrest
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