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Redox Biology Jun 2023This study was focused on exploring the role of the HIV-1 Tat protein in mediating microglial ferroptosis. Exposure of mouse primary microglial cells (mPMs) to HIV-1 Tat...
This study was focused on exploring the role of the HIV-1 Tat protein in mediating microglial ferroptosis. Exposure of mouse primary microglial cells (mPMs) to HIV-1 Tat protein resulted in induction of ferroptosis, which was characterized by increased expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4), in turn, leading to increased generation of oxidized phosphatidylethanolamine, elevated levels of lipid peroxidation, upregulated labile iron pool (LIP) and ferritin heavy chain-1 (FTH1), decreased glutathione peroxidase-4 and mitochondrial outer membrane rupture. Also, inhibition of ferroptosis by ferrostatin-1 (Fer-1) or deferoxamine (DFO) treatment suppressed ferroptosis-related changes in mPMs. Similarly, the knockdown of ACSL4 by gene silencing also inhibited ferroptosis induced by HIV-1 Tat. Furthermore, increased lipid peroxidation resulted in increased release of proinflammatory cytokines, such as TNFα, IL6, and IL1β and microglial activation. Pretreatment of mPMs with Fer-1 or DFO further blocked HIV-1 Tat-mediated microglial activation in vitro and reduced the expression and release of proinflammatory cytokines. We identified miR-204 as an upstream modulator of ACSL4, which was downregulated in mPMs exposed to HIV-1 Tat. Transient transfection of mPMs with miR-204 mimics reduced the expression of ACSL4 while inhibiting HIV-1 Tat-mediated ferroptosis and the release of proinflammatory cytokines. These in vitro findings were further validated in HIV-1 transgenic rats as well as HIV + ve human brain samples. Overall, this study underscores a novel mechanism(s) underlying HIV-1 Tat-mediated ferroptosis and microglial activation involving miR-204-ACSL4 signaling.
Topics: Animals; Humans; Mice; Rats; Coenzyme A Ligases; Cytokines; Ferroptosis; Gene Products, tat; HIV-1; Microglia; MicroRNAs; Rats, Transgenic
PubMed: 37023693
DOI: 10.1016/j.redox.2023.102689 -
Nature Communications Mar 2020Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is...
Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Topics: Base Sequence; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cell Survival; Dimerization; Exons; Extracellular Vesicles; Gene Editing; Genetic Vectors; HEK293 Cells; HIV Protease; Humans; Induced Pluripotent Stem Cells; Ligands; Luciferases; Nanoparticles; RNA Splicing; RNA, Catalytic; RNA, Guide, CRISPR-Cas Systems; Ribonucleoproteins; Tissue Donors; tat Gene Products, Human Immunodeficiency Virus
PubMed: 32170079
DOI: 10.1038/s41467-020-14957-y -
Aging May 2022
Topics: Aging; HIV Infections; Humans; tat Gene Products, Human Immunodeficiency Virus
PubMed: 35622387
DOI: 10.18632/aging.204105 -
Frontiers in Cellular and Infection... 2020HIV Tat protein is a critical protein that plays multiple roles in HIV pathogenesis. While its role as the transactivator of HIV transcription is well-established, other... (Review)
Review
HIV Tat protein is a critical protein that plays multiple roles in HIV pathogenesis. While its role as the transactivator of HIV transcription is well-established, other non-viral replication-associated functions have been described in several HIV-comorbidities even in the current antiretroviral therapy (ART) era. HIV Tat protein is produced and released into the extracellular space from cells with active HIV replication or from latently HIV-infected cells into neighboring uninfected cells even in the absence of active HIV replication and viral production due to effective ART. Neighboring uninfected and HIV-infected cells can take up the released Tat resulting in the upregulation of inflammatory genes and activation of pathways that leads to cytotoxicity observed in several comorbidities such as HIV associated neurocognitive disorder (HAND), HIV associated cardiovascular impairment, and accelerated aging. Thus, understanding how Tat modulates host and viral response is important in designing novel therapeutic approaches to target the chronic inflammatory effects of soluble viral proteins in HIV infection.
Topics: HIV Infections; HIV-1; Humans; tat Gene Products, Human Immunodeficiency Virus
PubMed: 32158701
DOI: 10.3389/fcimb.2020.00061 -
Nature May 2023Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes. These interactions are mediated by...
Cellular processes are the product of interactions between biomolecules, which associate to form biologically active complexes. These interactions are mediated by intermolecular contacts, which if disrupted, lead to alterations in cell physiology. Nevertheless, the formation of intermolecular contacts nearly universally requires changes in the conformations of the interacting biomolecules. As a result, binding affinity and cellular activity crucially depend both on the strength of the contacts and on the inherent propensities to form binding-competent conformational states. Thus, conformational penalties are ubiquitous in biology and must be known in order to quantitatively model binding energetics for protein and nucleic acid interactions. However, conceptual and technological limitations have hindered our ability to dissect and quantitatively measure how conformational propensities affect cellular activity. Here we systematically altered and determined the propensities for forming the protein-bound conformation of HIV-1 TAR RNA. These propensities quantitatively predicted the binding affinities of TAR to the RNA-binding region of the Tat protein and predicted the extent of HIV-1 Tat-dependent transactivation in cells. Our results establish the role of ensemble-based conformational propensities in cellular activity and reveal an example of a cellular process driven by an exceptionally rare and short-lived RNA conformational state.
Topics: HIV Long Terminal Repeat; Nucleic Acid Conformation; RNA, Viral; tat Gene Products, Human Immunodeficiency Virus; Transcriptional Activation; HIV-1
PubMed: 37198487
DOI: 10.1038/s41586-023-06080-x -
Theranostics 2023Recent studies indicate that microglial activation and the resulting inflammatory response could be potential targets of adjuvant therapy for ischemic stroke. Many...
Recent studies indicate that microglial activation and the resulting inflammatory response could be potential targets of adjuvant therapy for ischemic stroke. Many studies have emphasized a well-established function of Annexin-A1 (ANXA1) in the immune system, including the regulation of microglial activation. Nevertheless, few therapeutic interventions targeting ANXA1 in microglia for ischemic stroke have been conducted. In the present study, Tat-NTS, a small peptide developed to prevent ANXA1 from entering the nucleus, was utilized. We discovered the underlying mechanism that Tat-NTS peptide targets microglial ANXA1 to protect against ischemic brain injury. Preclinical studies of ischemic stroke were performed using an oxygen-glucose deprivation and reperfusion (OGD/R) cell model in vitro and the middle cerebral artery occlusion (MCAO) animal model of ischemic stroke in vivo. Confocal imaging and 3D reconstruction analyses for detecting the protein expression and subcellular localization of microglia in vivo. Co-immunoprecipitation (Co-IP), immunoblotting, ELISA, quantitative real-time PCR (qRT-PCR), Luciferase reporter assay for determining the precise molecular mechanism. Measurement on the cytotoxicity of Tat-NTS peptide for microglia was assessed by CCK-8 and LDH assay. TUNEL staining was used to detect the microglia conditioned medium-mediated neuronal apoptosis. Adeno-associated viruses (AAVs) were injected into the cerebral cortex, striatum and hippocampal CA1 region of adult male Cx3cr1-Cre mice, to further verify the neurofunctional outcome and mechanism of Tat-NTS peptide by TTC staining, the modified Neurological Severity Score (mNSS) test, the open field test (OFT), the novel object recognition task (NORT), the Morris water maze (MWM) test, the long-term potentiation (LTP) and the Transmission electron microscopy (TEM). It was observed that administration of Tat-NTS led to a shift of subcellular localization of ANXA1 in microglia from the nucleus to the cytoplasm in response to ischemic injury. Notably, this shift was accompanied by an increase in ANXA1 SUMOylation in microglia and a transformation of microglia towards an anti-inflammatory phenotype. We confirmed that Tat-NTS-induced ANXA1 SUMOylation in microglia mediated IKKα degradation via NBR1-dependent selective autophagy, then blocking the activation of the NF-κB pathway. As a result, the expression and release of the pro-inflammatory factors IL-1β and TNF-α were reduced in both in vitro and in vivo experiments. Furthermore, we found that Tat-NTS peptide's protective effect on microglia relieved ischemic neuron apoptosis. Finally, we demonstrated that Tat-NTS peptide administration, through induction of ANXA1 SUMOylation in microglia, reduced infarct volume, improved neurological function and facilitated behavioral recovery in MCAO mice. Our study provides evidence for a novel mechanism of Tat-NTS peptide in regulating microglial ANXA1 function and its substantial neuroprotective effect on neurons with ischemic injuries. These findings suggest that Tat-NTS peptides have a high potential for clinical application and may be a promising therapeutic candidate for treating cerebral ischemia.
Topics: Mice; Animals; Male; Microglia; Annexin A1; Sumoylation; Brain Ischemia; Infarction, Middle Cerebral Artery; Peptides; Ischemic Stroke; Reperfusion Injury; Neurons
PubMed: 37908731
DOI: 10.7150/thno.85390 -
Journal of Bacteriology Apr 2020The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the...
The transcription initiation signal elicited by the binding of ferric citrate to the outer membrane FecA protein is transmitted by the FecR protein across the cytoplasmic membrane to the FecI extracytoplasmic function (ECF) sigma factor. In this issue of , I. J. Passmore, J. M. Dow, F. Coll, J. Cuccui, et al. (J Bacteriol 202:e00541-19, 2020, https://doi.org/10.1128/JB.00541-19) report that the FecR sequence contains both the twin-arginine signal motif and the secretory (Sec) avoidance motif typical of proteins secreted by the twin-arginine translocation (TAT) system. The same study shows that FecR is indeed secreted by Tat and represents a new class of bitopic Tat-dependent membrane proteins.
Topics: Bacterial Secretion Systems; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Membrane Transport Proteins; Protein Transport; Receptors, Cell Surface; Sigma Factor
PubMed: 32015148
DOI: 10.1128/JB.00058-20 -
Journal of Neurovirology Jun 2019Human immunodeficiency virus-1 (HIV) infection of the central nervous system damages synapses and promotes axonal injury, ultimately resulting in HIV-associated... (Review)
Review
Human immunodeficiency virus-1 (HIV) infection of the central nervous system damages synapses and promotes axonal injury, ultimately resulting in HIV-associated neurocognitive disorders (HAND). The mechanisms through which HIV causes damage to neurons are still under investigation. The cytoskeleton and associated proteins are fundamental for axonal and dendritic integrity. In this article, we review evidence that HIV proteins, such as the envelope protein gp120 and transactivator of transcription (Tat), impair the structure and function of the neuronal cytoskeleton. Investigation into the effects of viral proteins on the neuronal cytoskeleton may provide a better understanding of HIV neurotoxicity and suggest new avenues for additional therapies.
Topics: AIDS Dementia Complex; Cytoskeleton; Human Immunodeficiency Virus Proteins; Humans; Neurons
PubMed: 30850975
DOI: 10.1007/s13365-019-00737-y -
Viruses Apr 2020The human immunodeficiency virus type 1 (HIV) establishes a chronic infection that can be well controlled, but not cured, by combined antiretroviral therapy (cART).... (Review)
Review
The human immunodeficiency virus type 1 (HIV) establishes a chronic infection that can be well controlled, but not cured, by combined antiretroviral therapy (cART). Interventions have been explored to accomplish a functional cure, meaning that a patient remains infected but HIV is undetectable in the blood, with the aim of allowing patients to live without cART. Tat, the viral transactivator of transcription protein, plays a critical role in controlling HIV transcription, latency, and viral rebound following the interruption of cART treatment. Therefore, a logical approach for controlling HIV would be to block Tat. Tackling Tat with inhibitors has been a difficult task, but some recent discoveries hold promise. Two anti-HIV proteins, Nullbasic (a mutant of Tat) and HT1 (a fusion of HEXIM1 and Tat functional domains) inhibit viral transcription by interfering with the interaction of Tat and cellular factors. Two small molecules, didehydro-cortistatin A (dCA) and triptolide, inhibit Tat by different mechanisms: dCA through direct binding and triptolide through enhanced proteasomal degradation. Finally, two Tat-based vaccines under development elicit Tat-neutralizing antibodies. These vaccines have increased the levels of CD4 cells and reduced viral loads in HIV-infected people, suggesting that the new vaccines are therapeutic. This review summarizes recent developments of anti-Tat agents and how they could contribute to a functional cure for HIV.
Topics: Animals; Anti-HIV Agents; Gene Expression Regulation, Viral; HIV Infections; HIV-1; Host-Pathogen Interactions; Humans; RNA-Binding Proteins; Recombinant Fusion Proteins; Transcription Factors; Transcription, Genetic; Virus Replication; tat Gene Products, Human Immunodeficiency Virus
PubMed: 32276443
DOI: 10.3390/v12040415 -
Journal of Neurovirology Feb 2021The transactivator of transcription (Tat) is a key HIV regulatory protein. We aimed to identify the frequency of key polymorphisms in HIV-1C compared with HIV-1B Tat... (Observational Study)
Observational Study
The transactivator of transcription (Tat) is a key HIV regulatory protein. We aimed to identify the frequency of key polymorphisms in HIV-1C compared with HIV-1B Tat protein, chiefly in the cysteine-, arginine-, and glutamine-rich domains and identify novel point mutations in HIV-1B and C sequences from Southern Brazil. This study was the first to investigate the genetic diversity and point mutations within HIV-1 Tat C in a Brazilian cohort. This was an observational, cross-sectional study, which included sequences of HIV-1B (n = 20) and HIV-1C (n = 21) from Southern Brazil. Additionally, 344 HIV-1C sequences were obtained from the Los Alamos database: 29 from Brazil and 315 from Africa, Asia, and Europe. The frequency of C31S substitution on HIV-1 Tat C in Brazil was 82% vs. 10% in the HIV-1B group (p < 0.0001). The frequency of the R57S substitution among the HIV-1C sequences from Brazil was 74% vs. 20% in HIV-1B (p = 0.004), and that of substitution Q63E in HIV-1C was 80% and 20% in HIV-1B (p < 0.0001). The mutation P60Q was more frequent in HIV-1B than in HIV-1C (55% and 6.12%, respectively, p < 0.0001)). Novel point mutations in the HIV-1C and B Tat functional domains were described. The frequency of C31S and other key point mutations in HIV-1 Tat C in Brazil were similar to those described in Africa, although lower than those in India. The Tat-B and C sequences found in Southern Brazil are consistent with biological differences and have potential implications for HIV-1 subtype pathogenesis.
Topics: Adult; Brazil; Cross-Sectional Studies; Female; HIV-1; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; tat Gene Products, Human Immunodeficiency Virus
PubMed: 33462791
DOI: 10.1007/s13365-020-00935-z