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Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat.

ABSTRACT: BACKGROUND: During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. RESULTS: To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20 degrees C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37 degrees C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. CONCLUSION: Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

SUBMITTER: Egele C

PROVIDER: S-EPMC2483996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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NMDA receptor activation by HIV-Tat protein is clade dependent.

Project description:In countries infected with HIV clade B, some patients develop a rapidly progressive dementia that if untreated results in death. In regions of the world infected with HIV clade C, only milder forms of cognitive impairment have been recognized. HIV-infected macrophages are the principal mediators of dementia. HIV clade C, however, efficiently infects macrophages and HIV-infected macrophages are found in the brains of clade C-infected patients. HIV-infected macrophages release Tat protein, which may act directly on neurons to cause toxicity. We found that Tat released from Tat-expressing cells was at least 1000-fold more toxic than recombinant Tat protein. We determined whether Tat could interact with NMDA receptors and whether these interactions are clade dependent. It is demonstrated that Tat binds directly to the NMDA receptor leading to excitotoxicity. The Cys 30-Cys 31 motif in Tat is critical for exciting the NMDA receptor and the Cys31Ser mutation found in clade C Tat has a significantly attenuated neurotoxic response. Through molecular modeling and site-directed mutagenesis, we predict that Cys 31 disrupts the disulfide bond between Cys 744 and Cys 798 on the NR1 subunit of the NMDA receptor by directly interacting with Cys 744 leading to a free thiol group on Cys 798 and subsequent persistent activation of the NMDA receptor.

| S-EPMC6671692 | biostudies-literature

Tat-dependent translocation of an F420-binding protein of Mycobacterium tuberculosis.

Project description:F(420) is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F(420) has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N-terminal signal sequence and is annotated as an F(420)-dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co-purify with F(420) during expression experiments in M. smegmatis. We also show that the Rv0132c-F(420) complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F(420)-binding protein being a substrate for the Tat pathway and of the presence of F(420) outside of the cytosol in any F(420)-producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F(420) biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.

| S-EPMC3478262 | biostudies-literature

Genome-wide binding map of the HIV Tat protein to the human genome

Project description:This SuperSeries is composed of the SubSeries listed below.

2011-11-06 | GSE30739 | GEO

Genome-wide binding map of the HIV Tat protein to the human genome

Project description:This SuperSeries is composed of the following subset Series: GSE30734: Genome-wide binding map of the HIV Tat protein to the human genome (gene expression) GSE30736: Genome-wide binding map of the HIV Tat protein to the human genome (ChIP-chip) GSE30738: Genome-wide binding map of the HIV Tat protein to the human genome (ChIP-Seq) Refer to individual Series

2011-11-06 | E-GEOD-30739 | biostudies-arrayexpress

Microtubule-dependent modulation of adhesion complex composition.

Project description:The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5β1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.

| S-EPMC4272306 | biostudies-literature

Effects of HIV protein Tat on macrophage transcriptome

Project description:Purpose: To investigate the impact of HIV protein Tat on macrophage transcriptome related to atherosclerosis development Methods: Peritoneal macrophages were isolated from myeloid specific IKKβ deficient LDLR-/- (IKKβΔMyeLDLR-/-) mice and their littermates (IKKβF/FLDLR-/-). Then the macrophages were treated with HIV protein Tat or control for 12 hr. Total RNA was extracted for RNAseq Results: HIV protein Tat treatment induces 2640 differentially expressed genes (DEGs) with false discovery rate (FDR) < 1 % and fold change > 3 in control macrophages. Those DEGs enriched in several biological processes that may contribute to atherogenesis. FAIME analysis demonstrated higher geneset scores of those GO terms in the Tat-treated macrophages from IKKβF/FLDLR-/- mice compared with the control ones. IKKβ deletion resulted in loss of geneset score in the macrophages of IKKβΔMyeLDLR-/- treated with HIV Tat. Conclusions: These results indicate that HIV protein Tat may affect many genes in macrophages related atherosclerosis development through IKKβ signaling.

2021-07-21 | GSE157833 | GEO

Genome-wide binding map of the HIV-1 Tat protein to the human genome.

Project description:The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. While Tat's control of viral transcription is well-studied, much less is known about the interaction of Tat with the human genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells using chromatin immunoprecipitation combined with next-generation sequencing. Surprisingly, we found that ~53% of the Tat target regions are within DNA repeat elements, greater than half of which are Alu sequences. The remaining target regions are located in introns and distal intergenic regions; only ~7% of Tat-bound regions are near transcription start sites (TSS) at gene promoters. Interestingly, Tat binds to promoters of genes that, in Jurkat cells, are bound by the ETS1 transcription factor, the CBP histone acetyltransferase and/or are enriched for histone H3 lysine 4 tri-methylation (H3K4me3) and H3K27me3. Tat binding is associated with genes enriched with functions in T cell biology and immune response. Our data reveal that Tat's interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.

| S-EPMC3208564 | biostudies-literature

HIV-1 Tat interacts with LIS1 protein.

Project description:BackgroundHIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization.ResultsWe identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system.ConclusionOur results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.

| S-EPMC549217 | biostudies-literature

Genome-wide binding map of the HIV Tat protein to the human genome (ChIP-chip)

Project description:The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells (Jurkat-Tat cells) using chromatin immunoprecipitation combined with next-generation sequencing. cDNA microarray was used to monitor gene expression changes between Jurkat and Jurkat-Tat cells. Additionally, we compared distribution of H3K9ac near gene promoters between Jurkat and Jurkat-Tat cells using ChIP-chip method and hybridized onto Agilent promoter array. Our data reveal that Tat’s interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.

2011-11-06 | GSE30736 | GEO

Genome-wide binding map of the HIV Tat protein to the human genome (gene expression)

Project description:The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome. Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells (Jurkat-Tat cells) using chromatin immunoprecipitation combined with next-generation sequencing. cDNA microarray was used to monitor gene expression changes between Jurkat and Jurkat-Tat cells. Additionally, we compared distribution of H3K9ac near gene promoters between Jurkat and Jurkat-Tat cells using ChIP-chip method and hybridized onto Agilent promoter array. Our data reveal that Tat’s interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle. Agilent gene expression microarray was used to compare gene expression changes between Jurkat T cells and Jurkat T cells expressing HIV-Tat protein (Jurkat-Tat T cells)

2011-11-06 | GSE30734 | GEO

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