scholarly journals Anti-Vpr Activity of a Yeast Chaperone Protein

2004 ◽  
Vol 78 (20) ◽  
pp. 11016-11029 ◽  
Author(s):  
Zsigmond Benko ◽  
Dong Liang ◽  
Emmanuel Agbottah ◽  
Jason Hou ◽  
Karen Chiu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Suppressive Effect ◽  
Dependent Manner ◽  
Protein Protein Interaction ◽  
Chaperone Protein ◽  
Genome Wide ◽  
Multicopy Suppressors ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) exerts multiple effects on viral and host cellular activities during viral infection, including nuclear transport of the proviral integration complex, induction of cell cycle G2 arrest, and cell death. In this report, we show that a fission yeast chaperone protein Hsp16 inhibits HIV-1 by suppressing these Vpr activities. This protein was identified through three independent genome-wide screens for multicopy suppressors of each of the three Vpr activities. Consistent with the properties of a heat shock protein, heat shock-induced elevation or overproduction of Hsp16 suppressed Vpr activities through direct protein-protein interaction. Even though Hsp16 shows a stronger suppressive effect on Vpr in fission yeast than in mammalian cells, similar effects were also observed in human cells when fission yeast hsp16 was expressed either in vpr-expressing cells or during HIV-1 infection, indicating a possible highly conserved Vpr suppressing activity. Furthermore, stable expression of hsp16 prior to HIV-1 infection inhibits viral replication in a Vpr-dependent manner. Together, these data suggest that Hsp16 inhibits HIV-1 by suppressing Vpr-specific activities. This finding could potentially provide a new approach to studying the contribution of Vpr to viral pathogenesis and to reducing Vpr-mediated detrimental effects in HIV-infected patients.

HSP70 Induces IL-6 in Stromal Cells and Stat-3 Activation in Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3353-3353
Author(s):  
Ramadevi Nimmanapalli ◽  
Elvira Gerbino ◽  
William S. Dalton ◽  
Melissa Alsina
Keyword(s):  
Bone Marrow ◽  
Heat Shock ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Mammalian Cells ◽  
Marrow Stromal Cells ◽  
Hsp70 Expression ◽  
High Dose ◽  
Dependent Manner ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells that accumulate preferentially in the bone marrow. In spite of high dose chemotherapy and novel targeted therapies this disease remains incurable with a median survival of 3–6 years mainly because of the emergence of drug resistance. Improved survival requires new strategies to prevent relapse. Heat shock proteins (HSPs) are a super family of highly conserved proteins, which are induced in plant, yeast, bacterial and mammalian cells in response to an array of physiological and environmental stress cues. Among heat shock protein families, HSP70 is one of the most highly conserved and is the only protein expressed in response to cellular stress. Exogenous HSP70 has been demonstrated to act as a cytokine to human monocytes by stimulating rapid calcium influx, activating nuclear factor (NF)-kB and up-regulating the expression of IL-1b, IL-6 and tumor necrosis factor alpha (TNF-a) (Asea A et al., 2000). Adhesion of myeloma cells to bone marrow stromal cells mediates IL-6 secretion and tumor cell proliferation in part mediated by STAT-3 activation (Cheung WC et al., 2001). We have shown that adhesion of myeloma cells to bone marrow stromal cells enhances IL-6 secretion by stromal cells and HSP70 secretion by myeloma cells. When we inhibited the HSP70 expression using either KNK437 (HSF-1 inhibitor) or RNAi to HSP70, IL-6 secretion by stromal cells as well as activation of STAT-3 in myeloma cells was inhibited in dose-dependent manner. These results suggest that HSP70 released from myeloma cells is enhancing IL-6 secretion from stromal cells. Incubation of stromal cells with recombinant HSP70 did not enhance IL-6 secretion in stromal cells suggesting that some other soluble factor released from myeloma cells cooperates with HSP70 to enhance IL-6 secretion by stromal cells, We examined whether HSP70 can modulate IL-6 mediated STAT-3 activation by stimulating 8226 cells with IL-6 in the presence or absence of KNK437 and RNAi to HSP70 and measuring phospho-STAT-3 by western analysis. HSP70 inhibition attenuated IL-6 induced STAT-3 activity, but not ERK1/2 activity, indicating that HSP70 mediated IL-6 signaling is very specific to STAT-3. The signal transduction cascade by which HSP70 induces IL-6 secretion and the mechanism by which HSP70 mediates IL-6 induced STAT-3 activity are currently under investigation.

USP7 deubiquitinase controls HIV-1 production by stabilizing Tat protein

2017 ◽  
Vol 474 (10) ◽  
pp. 1653-1668 ◽  
Author(s):  
Amjad Ali ◽  
Rameez Raja ◽  
Sabihur Rahman Farooqui ◽  
Shaista Ahmad ◽  
Akhil C. Banerjea
Keyword(s):  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Virus Production ◽  
Dependent Manner ◽  
Tat Protein ◽  
Viral Production ◽  
Cellular Processes ◽  
Latently Infected ◽  
Dub Inhibitor ◽  
Hiv 1

Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.

scholarly journals Interaction of Recombinant Norwalk Virus Particles with the 105-Kilodalton Cellular Binding Protein, a Candidate Receptor Molecule for Virus Attachment

2000 ◽  
Vol 74 (24) ◽  
pp. 11589-11597 ◽  
Author(s):  
Masaru Tamura ◽  
Katsuro Natori ◽  
Masahiko Kobayashi ◽  
Tatsuo Miyamura ◽  
Naokazu Takeda
Keyword(s):  
Mammalian Cells ◽  
Protein A ◽  
Binding Activity ◽  
Conformational Structure ◽  
Dependent Manner ◽  
Norwalk Virus ◽  
Capsid Protein Gene ◽  
Virus Attachment ◽  
Protein Protein Interaction ◽  
Mammalian Cell Lines

ABSTRACT Norwalk virus (NV), responsible for outbreaks of acute gastroenteritis, comprises the species of the genus Norwalk-like viruses in the family Caliciviridae. Although the study of the molecular biology of NV has been hampered by a lack of culture systems or small experimental animal models, virus-like particles (VLPs) generated with recombinant baculoviruses harboring the capsid protein gene of NV provide a useful tool for investigating NV-cell interactions. In this study, the attachment of the recombinant VLPs derived from the Ueno virus (UEV), a strain belonging to the genogroup II NVs, to mammalian and insect cells was examined. Kinetic analyses of the binding of the recombinant VLPs of the UEV (rUEVs) to Caco-2 cells demonstrated that the binding was specific and occurred in a dose-dependent manner. Approximately 7.5% of the prebound rUEVs were internalized into the Caco-2 cells. Enzymatic and chemical modification of Caco-2 cell surface molecules suggested that the binding was directly mediated by a protein-protein interaction. A virus overlay protein-binding assay (VOPBA) indicated that rUEVs appeared to bind to a 105-kDa molecule, designated as the NV attachment (NORVA) protein. Furthermore, the assay indicated that its native conformational structure was indispensable for the binding activity. In Caco-2 cells, the NORVA protein was detected when VOPBA was carried out with the VLPs from Seto and Funabashi viruses, which are serologically different NVs from UEV, used as probes. The binding of rUEVs to NORVA protein was also observed in six mammalian cell lines other than Caco-2. These data suggest that the attachment of NV to mammalian cells is mediated by NORVA protein, which is ubiquitously expressed in the mammalian cells. The present study is the first report on the role of the cellular molecule in the binding of recombinant VLPs of NV.

scholarly journals Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

1995 ◽  
Vol 15 (10) ◽  
pp. 5288-5293 ◽  
Author(s):  
M L Goodson ◽  
O K Park-Sarge ◽  
K D Sarge
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mammalian Cells ◽  
Protein Gene ◽  
Heat Shock Factor ◽  
Dependent Manner ◽  
Heat Shock Protein Gene ◽  
Mouse Tissues

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

scholarly journals Genome-wide Screening of Genes Associated with Momilactone B Sensitivity in the Fission Yeast

2021 ◽  
Author(s):  
Keisuke Tomita ◽  
Yoko Yashiroda ◽  
Yasuhiro Matsuo ◽  
Jeff S Piotrowski ◽  
Sheena C Li ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Mode Of Action ◽  
Mammalian Cells ◽  
Biological Activities ◽  
Genetic Determinants ◽  
Cellular Mechanisms ◽  
Plant Chemical Defense ◽  
Genome Wide ◽  
Plant Chemical ◽  
Yeast Mutants

Abstract Momilactone B is a natural product with dual biological activities, including antimicrobial and allelopathic properties, and play a major role in plant chemical defense against competitive plants and pathogens. The pharmacological effects of momilactone B on mammalian cells have also been reported. However, little is known about the molecular and cellular mechanisms underlying its broad bioactivity. In this study, the genetic determinants of momilactone B sensitivity in yeast were explored to gain insight into its mode of action. We screened fission yeast mutants resistant to momilactone B from a pooled culture containing genome-wide gene-overexpressing strains in a drug-hypersensitive genetic background. Overexpression of pmd1, bfr1, pap1, arp9, or SPAC9E9.06c conferred resistance to momilactone B. In addition, a drug-hypersensitive, barcoded deletion library was newly constructed and the genes that imparted altered sensitivity to momilactone B upon deletion were identified. Gene Ontology and fission yeast phenotype ontology enrichment analyses predicted the biological pathways related to the mode of action of momilactone B. The validation of predictions revealed that momilactone B induced abnormal phenotypes such as multiseptated cells and disrupted organization of the microtubule structure. This is the first investigation of the mechanism underlying the antifungal activity of momilactone B against yeast. The results and datasets obtained in this study narrow the possible targets of momilactone B and facilitate further studies regarding its mode of action.

scholarly journals Antagonistic interaction of HIV-1 Vpr with Hsf-mediated cellular heat shock response and Hsp16 in fission yeast (Schizosaccharomyces pombe)

Retrovirology ◽  
2007 ◽  
Vol 4 (1) ◽  
pp. 16 ◽  
Author(s):  
Zsigmond Benko ◽  
Dong Liang ◽  
Emmanuel Agbottah ◽  
Jason Hou ◽  
Lorena Taricani ◽  
...  
Keyword(s):  
Heat Shock ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Heat Shock Response ◽  
Antagonistic Interaction ◽  
Shock Response ◽  
Hiv 1

scholarly journals The Role of Heat Shock Transcription Factor 1 in the Genome-wide Regulation of the Mammalian Heat Shock Response

2004 ◽  
Vol 15 (3) ◽  
pp. 1254-1261 ◽  
Author(s):  
Nathan D. Trinklein ◽  
John I. Murray ◽  
Sara J. Hartman ◽  
David Botstein ◽  
Richard M. Myers
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Transcriptional Response ◽  
Heat Shock Transcription Factor ◽  
Genome Wide ◽  
Shock Response ◽  
Inducible Genes ◽  
Transcription Factor 1

Previous work has implicated heat shock transcription factor 1 (HSF1) as the primary transcription factor responsible for the transcriptional response to heat stress in mammalian cells. We characterized the heat shock response of mammalian cells by measuring changes in transcript levels and assaying binding of HSF1 to promoter regions for candidate heat shock genes chosen by a combination of genome-wide computational and experimental methods. We found that many heat-inducible genes have HSF1 binding sites (heat shock elements, HSEs) in their promoters that are bound by HSF1. Surprisingly, for 24 heat-inducible genes, we detected no HSEs and no HSF1 binding. Furthermore, of 182 promoters with likely HSE sequences, we detected HSF1 binding at only 94 of these promoters. Also unexpectedly, we found 48 genes with HSEs in their promoters that are bound by HSF1 but that nevertheless did not show induction after heat shock in the cell types we examined. We also studied the transcriptional response to heat shock in fibroblasts from mice lacking the HSF1 gene. We found 36 genes in these cells that are induced by heat as well as they are in wild-type cells. These results provide evidence that HSF1 does not regulate the induction of every transcript that accumulates after heat shock, and our results suggest that an independent posttranscriptional mechanism regulates the accumulation of a significant number of transcripts.

scholarly journals Evolutionarily conserved regulation of immunity by the splicing factor RNP-6/PUF60

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chun Kew ◽  
Wenming Huang ◽  
Julia Fischer ◽  
Raja Ganesan ◽  
Nirmal Robinson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Bacterial Pathogen ◽  
Active Role ◽  
Suppressive Effect ◽  
Mapk Signaling ◽  
Splicing Factor ◽  
Dependent Manner ◽  
Loss Of Function ◽  
C Elegans ◽  
Evolutionarily Conserved

Splicing is a vital cellular process that modulates important aspects of animal physiology, yet roles in regulating innate immunity are relatively unexplored. From genetic screens in C. elegans, we identified splicing factor RNP-6/PUF60 whose activity suppresses immunity, but promotes longevity, suggesting a tradeoff between these processes. Bacterial pathogen exposure affects gene expression and splicing in a rnp-6 dependent manner, and rnp-6 gain and loss-of-function activities reveal an active role in immune regulation. Another longevity promoting splicing factor, SFA-1, similarly exerts an immuno-suppressive effect, working downstream or parallel to RNP-6. RNP-6 acts through TIR-1/PMK-1/MAPK signaling to modulate immunity. The mammalian homolog, PUF60, also displays anti-inflammatory properties, and its levels swiftly decrease after bacterial infection in mammalian cells, implying a role in the host response. Altogether our findings demonstrate an evolutionarily conserved modulation of immunity by specific components of the splicing machinery.

scholarly journals Heat Shock Protein 70 Inhibits HIV-1 Vif-mediated Ubiquitination and Degradation of APOBEC3G

2011 ◽  
Vol 286 (12) ◽  
pp. 10051-10057 ◽  
Author(s):  
Ryuichi Sugiyama ◽  
Hironori Nishitsuji ◽  
Ayako Furukawa ◽  
Masato Katahira ◽  
Yuichiro Habu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Antiviral Activity ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cytidine Deaminase ◽  
Host Protein ◽  
Hsp70 Expression ◽  
Dependent Manner ◽  
Reverse Transcription Step ◽  
Hiv 1

The cytidine deaminase APOBEC3G, which is incorporated into nascent virus particles, possesses potent antiviral activity and restricts Vif-deficient HIV-1 replication at the reverse transcription step through deamination-dependent and -independent effects. HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inducing APOBEC3G polyubiquitination and its subsequent proteasomal degradation. In this study, we show that overexpression of heat shock protein 70 (HSP70) blocked the degradation of APOBEC3G in the ubiquitin-proteasome pathway by HIV-1 Vif, rendering the viral particles non-infectious. In addition, siRNA targeted knock-down of HSP70 expression enhanced the Vif-mediated degradation of APOBEC3G. A co-immunoprecipitation study revealed that overexpression of HSP70 inhibited APOBEC3G binding to HIV-1 Vif. Thus, we provide evidence for a host protein-mediated suppression of HIV-1 replication in an APOBEC3G-dependent manner.

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