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Journal of Bacteriology Apr 2022Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine...
Flagellum-mediated bacterial motility is important for bacteria to take up nutrients, adapt to environmental changes, and establish infection. The twin-arginine translocation system (Tat) is an important protein export system, playing a critical role in bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, a comparative transcriptomics analysis was performed with extraintestinal pathogenic Escherichia coli (ExPEC), which identified a considerable number of genes differentially expressed when the Tat system was disrupted. Among them, a large proportion of flagellar biosynthesis genes showed downregulation, indicating that transcription regulation plays an important role in mediating the motility defects. We further identified three Tat substrate proteins, MdoD, AmiA, and AmiC, that were responsible for the nonmotile phenotype. The Rcs system was deleted in the Δ, the Δ, and the ΔΔ strains, which restored the motility of Δ and partially restored the motility of Δ and ΔΔ. The flagella were also observed in all of the ΔΔ, ΔΔ, and ΔΔΔ strains, but not in the Δ, Δ, and ΔΔ strains, by using transmission electron microscopy. Quantitative reverse transcription-PCR data revealed that the regulons of the Rcs system displayed differential expression in the mutant, indicating that the Rcs signaling was activated. Our results suggest that the Rcs system plays an important role in mediating the motility defects of the mutant of ExPEC. The Tat system is an important protein export system critical for bacterial physiology and pathogenesis. It has been observed for a long time that the Tat system is critical for bacterial motility. However, the underlying mechanism remains unrevealed. In this study, we combine transcriptomics analysis and bacterial genetics, which reveal that transcription regulation plays an important role in mediating the motility defects of the mutant of extraintestinal pathogenic Escherichia coli. The Tat substrate proteins responsible for the motility defects are identified. We further show that the Rcs system contributes to the motility suppression. We for the first time reveal the link between the Tat system and bacterial motility, which is important for understanding the physiological functions of the Tat system.
Topics: Arginine; Escherichia coli Proteins; Extraintestinal Pathogenic Escherichia coli; Flagella; Protein Transport; Twin-Arginine-Translocation System
PubMed: 35311558
DOI: 10.1128/jb.00612-21 -
Frontiers in Microbiology 2022HIV-1 is responsible for a spectrum of neurocognitive deficits defined as HIV-associated neurocognitive disorders (HAND). The HIV transactivator of transcription (Tat)...
HIV-1 is responsible for a spectrum of neurocognitive deficits defined as HIV-associated neurocognitive disorders (HAND). The HIV transactivator of transcription (Tat) protein plays a key role in the neuropathophysiology of HAND. The Tat protein functions by transactivation of viral genes through its interaction with the transactivation response (TAR) RNA element. Subtype-specific Tat protein signatures including C31S, R57S and Q63E present in Tat subtype C has previously been linked to a lowered neuropathophysiology compared to Tat subtype B. In this study, we attempted to understand the molecular mechanism by which Tat subtype-specific variation, particularly, C31S, R57S, and Q63E influence the Tat-TAR interaction. We performed molecular modeling to generate accurate three-dimensional protein structures of the HIV-1 Tat subtypes C and B using the Swiss model webserver. Thereafter, we performed a molecular docking of the TAR RNA element to each of the Tat subtypes B and C protein structures using the HDOCK webserver. Our findings indicate that Tat subtype B had a higher affinity for the TAR RNA element compared to Tat subtype C based on a higher docking score of -187.37, a higher binding free energy value of -9834.63 ± 216.17 kJ/mol, and a higher number of protein-nucleotide interactions of 26. Furthermore, Tat subtype B displayed more flexible regions when bound to the TAR element and this flexibility could account for the stronger affinity of Tat subtype B to TAR. From the Tat signatures linked to neuropathogenesis, only R57/R57S are involved in Tat-TAR interaction. Due to the lack of electrostatic interactions observed between Tat subtype C and TAR, weaker affinity is observed, and this may contribute to a lower level of neuropathophysiology observed in subtype C infection.
PubMed: 35464972
DOI: 10.3389/fmicb.2022.866611 -
Journal of Neuroinflammation Sep 2016Synaptodendritic damage is a pathological hallmark of HIV-associated neurocognitive disorders, and HIV-1 Tat protein is known to cause such injury in the central nervous...
BACKGROUND
Synaptodendritic damage is a pathological hallmark of HIV-associated neurocognitive disorders, and HIV-1 Tat protein is known to cause such injury in the central nervous system. In this study, we aimed to determine the molecular mechanisms of Tat-induced neurite shortening, specifically the roles of miR-132, an important regulator of neurite morphogenesis in this process.
METHODS
The relationship between Tat expression and miR-132 expression was first determined using reverse transcription quantitative PCR (qRT-PCR) in Tat-transfected astrocytes and neurons, astrocytes from Tat-transgenic mice, and HIV-infected astrocytes. qRT-PCR and Western blotting were performed to determine Tat effects on expression of miR-132 target genes methyl CpG-binding protein 2, Rho GTPase activator p250GAP, and brain-derived neurotrophic factor. Exosomes were isolated from Tat-expressing astrocytes, and exosomal microRNA (miRNA) uptake into neurons was studied using miRNA labeling and flow cytometry. The lactate dehydrogenase release was used to determine the cytotoxicity, while immunostaining was used to determine neurite lengths and synapse formation. Tat basic domain deletion mutant and miR-132 mimic and inhibitor were used to determine the specificity of the relationship between Tat and miR-132 and its effects on astrocytes and neurons and the underlying mechanisms of Tat-induced miR-132 expression.
RESULTS
Tat significantly induced miR-132 expression, ensuing down-regulation of miR-132 target genes in astrocytes and neurons. miR-132 induction was associated with phosphorylation of cAMP response element-binding protein and required the basic domain of Tat. miRNA-132 induction had no effects on astrocyte activation or survival but was involved in the direct neurotoxicity of Tat. miR-132 was present in astrocyte-derived exosomes and was taken up by neurons, causing neurite shortening.
CONCLUSIONS
Tat-induced miR-132 expression contributes to both direct and astrocyte-mediated Tat neurotoxicity and supports the important roles of miR-132 in controlling neurite outgrowth.
Topics: Animals; Anti-Bacterial Agents; Astrocytes; Astrocytoma; CREB-Binding Protein; Cell Line, Transformed; Cytokines; Doxycycline; Epithelial Cells; Gene Expression Regulation, Viral; Glial Fibrillary Acidic Protein; Humans; Methyl-CpG-Binding Protein 2; Mice; MicroRNAs; Neurites; Neuroblastoma; Neurons; RNA, Small Nuclear; tat Gene Products, Human Immunodeficiency Virus
PubMed: 27634380
DOI: 10.1186/s12974-016-0716-2 -
Cell Dec 2016Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical...
Overlapping genes pose an evolutionary dilemma as one DNA sequence evolves under the selection pressures of multiple proteins. Here, we perform systematic statistical and mutational analyses of the overlapping HIV-1 genes tat and rev and engineer exhaustive libraries of non-overlapped viruses to perform deep mutational scanning of each gene independently. We find a "segregated" organization in which overlapped sites encode functional residues of one gene or the other, but never both. Furthermore, this organization eliminates unfit genotypes, providing a fitness advantage to the population. Our comprehensive analysis reveals the extraordinary manner in which HIV minimizes the constraint of overlapping genes and repurposes that constraint to its own advantage. Thus, overlaps are not just consequences of evolutionary constraints, but rather can provide population fitness advantages.
Topics: Biological Evolution; Entropy; Genetic Fitness; HIV Infections; HIV-1; Humans; Mutation; Open Reading Frames; rev Gene Products, Human Immunodeficiency Virus; tat Gene Products, Human Immunodeficiency Virus
PubMed: 27984726
DOI: 10.1016/j.cell.2016.11.031 -
Frontiers in Neuroscience 2018Addictive stimulant drugs, such as methamphetamine (METH), increase the risk of exposure to the human immunodeficiency virus-1 (HIV-1) infection and thus predispose...
Addictive stimulant drugs, such as methamphetamine (METH), increase the risk of exposure to the human immunodeficiency virus-1 (HIV-1) infection and thus predispose individuals to the development of HIV-associated neurocognitive disorders (HANDs). Previous studies have indicated that HIV-Tat (the transactivator of transcription) and METH can synergistically induce autophagy in SH-SY5Y neuroblastoma cells and that autophagy plays a pivotal role in the neuronal dysfunction in HANDs. However, the underlying mechanism of METH-and HIV-Tat-induced neuronal autophagy remains unclear. We cultured primary midbrain neuronal cells of tree shrews and treated them with METH and HIV-Tat to study the role of METH and HIV-Tat in inducing autophagy. We evaluated the effects of the single or combined treatment of METH and HIV-Tat on the protein expressions of the autophagy-related genes, including Beclin-1 and LC3B, ATG5, and ATG7 in METH and HIV-Tat-induced autophagy. In addition, the presence of autophagosomes in the METH and/or HIV-Tat treatment was revealed using transmission electron microscopy. The results indicated that METH increased the protein levels of LC3B and Beclin-1, and these effects were significantly enhanced by HIV-Tat. Moreover, the results suggested that ATG5 and ATG7 were involved in the METH and HIV-Tat-induced autophagy. In addition, it was found that mTOR inhibition via pharmacological intervention could trigger autophagy and promote METH and HIV-Tat-induced autophagy. Overall, this study contributes to the knowledge of the molecular underpinnings of METH and HIV-Tat-induced autophagy in primary midbrain neuronal cells. Our findings may facilitate the development of therapeutic strategies for METH-and HIV-Tat-induced autophagy in HANDs.
PubMed: 30574066
DOI: 10.3389/fnins.2018.00921 -
Current HIV Research 2014Earlier studies have established that infection with HIV-1 subtypes (clades) might differentially influence the neuropathogenesis of HIV-1-associated neurocognitive...
Earlier studies have established that infection with HIV-1 subtypes (clades) might differentially influence the neuropathogenesis of HIV-1-associated neurocognitive dysfunction (HAND). HIV-1 Trans activator of transcription protein (Tat) is of considerable significance and plays a major role in the central nervous system (CNS) dysfunction. However, these HIV-1 clades exert diverse cellular effects that leads to neuropathogenic dysfunction has not been well established. We hypothesized that the HIV-1 clade B and clade C Tat proteins effect synaptic plasticity expression in neuroblastoma cells (SK-N-MC) by diverse methods, and accordingly modulates the development of HAND. In the present study, we have analyzed important and highly expressed 84 key human synaptic plasticity genes expression which differentially impact in clade B and clade C Tat treated SK-N-MC cells using RT(2) Profile PCR Array human Synaptic Plasticity kit. Observed results demonstrate that out of 84 key synaptic plasticity genes, 36 and 25 synaptic genes were substantially (≥3 fold) up-regulated and 5 and 5 genes considerably (≥3 fold) down-regulated in clade B and clade C Tat treated cells, respectively, compared to the control SK-N-MC. We have also estimated the levels of glutamine and glutamate in HIV-1 clade B and C Tat exposed SK-N-MC cells compared to untreated cells. Our results indicate that levels of glutamate, glutamine and expression of synaptic plasticity genes were highly dysregulated by HIV-1 clade B Tat compared to clade C Tat in SK-N-MC cells. In summary, this study suggests that clade B Tat substantially potentiates neuronal toxicity and further dysregulated synaptic plasticity genes in SK-N-MC may contribute to the severe neuropathogenesis linked with HAND.
Topics: Cell Line, Tumor; Gene Expression Profiling; HIV-1; Host-Pathogen Interactions; Humans; Neuronal Plasticity; Neurons; tat Gene Products, Human Immunodeficiency Virus
PubMed: 25613138
DOI: 10.2174/1570162x13666150121104720 -
Journal of Neurovirology Aug 2019Astrocytes are susceptible to HIV infection and potential latent HIV reservoirs. Tat is one of three abundantly expressed HIV early genes in HIV-infected astrocytes and...
Astrocytes are susceptible to HIV infection and potential latent HIV reservoirs. Tat is one of three abundantly expressed HIV early genes in HIV-infected astrocytes and has been shown to be a major pathogenic factor for HIV/neuroAIDS. In this study, we sought to determine if and how Tat expression would affect HIV infection and latency in astrocytes. Using the glycoprotein from vesicular stomatitis virus-pseudotyped red-green HIV (RGH) reporter viruses, we showed that HIV infection was capable of establishing HIV latency in astrocytes. We also found that Tat expression decreased the generation of latent HIV-infected cells. Activation of latent HIV-infected astrocytes showed that treatment of GSK126, a selective inhibitor of methyltransferase enhancer of zeste homolog 2 (Ezh2) that is specifically responsible for tri-methylation of histone 3 lysine 27 (H3K27me3), led to activation of significantly more latent HIV-infected Tat-expressing astrocytes. Molecular analysis showed that H3K27me3, Ezh2, MeCP2, and Tat all exhibited a similar bimodal expression kinetics in the course of HIV infection and latency in astrocytes, although H3K27me3, Ezh2, and MeCP2 were expressed higher in Tat-expressing astrocytes and their expression were peaked immediately preceding Tat expression. Subsequent studies showed that Tat expression alone was sufficient to induce H3K27me3 expression, likely through its regulation of Ezh2 and MeCP2 expression. Taken together, these results showed for the first time that Tat expression induced H3K27me3 expression and contributed to HIV latency in astrocytes and suggest a new role and novel mechanism for Tat in HIV latency.
Topics: Astrocytes; Cell Line, Tumor; Enhancer of Zeste Homolog 2 Protein; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; HIV-1; Histones; Host-Pathogen Interactions; Humans; Indoles; Jurkat Cells; Luminescent Proteins; Methyl-CpG-Binding Protein 2; Methylation; Pyridones; Signal Transduction; Vesiculovirus; Virus Latency; tat Gene Products, Human Immunodeficiency Virus; Red Fluorescent Protein
PubMed: 31020497
DOI: 10.1007/s13365-019-00751-0 -
Blood May 2013As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells...
As a result of its interaction with transcription factors, HIV type 1 (HIV-1) Tat can modulate the expression of both HIV and cellular genes. In antigen-presenting cells Tat induces the expression of a subset of interferon (IFN)-stimulated genes (ISGs) in the absence of IFNs. We investigated the genome-wide Tat association with promoters in immature dendritic cells and in monocyte-derived macrophages. Among others, Tat associated with the MAP2K6, MAP2K3, and IRF7 promoters that are functionally part of IL-1 and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The association correlated with their increased gene expression, increased activation of p38 MAPK and of phosphorylated signal transducer and activator of transcription 1 (STAT1), and consequent induction of ISGs. Probing these pathways with RNA interference, pharmacological p38 MAPK inhibition, and in cell lines lacking STAT1s or the type I IFN receptor chain confirmed the role of MAPKKs and IRF7 in Tat-mediated modulation of ISGs and excluded the involvement of IFNs in this modulation. Tat interaction with the 2 MAPKK and IRF7 promoters in HIV-1-infected cells and the resulting persistent activation of ISGs, which include inflammatory cytokines and chemokines, can contribute to the increased immune activation that characterizes HIV infection.
Topics: Antigen-Presenting Cells; Cells, Cultured; Chemokines; Gene Products, tat; HIV Infections; HIV-1; Humans; Interferon Regulatory Factor-7; Interferons; Lymphocyte Activation; MAP Kinase Kinase 3; MAP Kinase Kinase 6; Promoter Regions, Genetic; Protein Binding; Receptors, Chemokine; Signal Transduction; Up-Regulation; p38 Mitogen-Activated Protein Kinases
PubMed: 23535064
DOI: 10.1182/blood-2012-10-461566 -
BioRxiv : the Preprint Server For... Mar 2024The complex and heterogeneous genetic architecture of schizophrenia inspires us to look beyond individual risk genes for therapeutic strategies and target their...
The complex and heterogeneous genetic architecture of schizophrenia inspires us to look beyond individual risk genes for therapeutic strategies and target their interactive dynamics and convergence. Postsynaptic NMDA receptor (NMDAR) complexes are a site of such convergence. Src kinase is a molecular hub of NMDAR function, and its protein interaction subnetwork is enriched for risk-genes and altered protein associations in schizophrenia. Previously, Src activity was found to be decreased in post-mortem studies of schizophrenia, contributing to NMDAR hypofunction. PSD-95 suppresses Src via interacting with its SH2 domain. Here, we devised a strategy to suppress the inhibition of Src by PSD-95 via employing a cell penetrating and Src activating PSD-95 inhibitory peptide (TAT-SAPIP). TAT-SAPIP selectively increased post-synaptic Src activity in humans and mice, and enhanced synaptic NMDAR currents in mice. Chronic ICV injection of TAT-SAPIP rescued deficits in trace fear conditioning in Src hypomorphic mice. We propose blockade of the Src-PSD-95 interaction as a proof of concept for the use of interfering peptides as a therapeutic strategy to reverse NMDAR hypofunction in schizophrenia and other illnesses.
PubMed: 38496466
DOI: 10.1101/2024.03.08.584132 -
Journal of Virology Apr 2023HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless,...
HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.
Topics: Humans; Capsid Proteins; Cyclophilin A; HIV Infections; HIV-1; tat Gene Products, Human Immunodeficiency Virus; Multiprotein Complexes; Surface Plasmon Resonance; Cytosol; Cell Line
PubMed: 37129415
DOI: 10.1128/jvi.00278-23