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GeroScience Feb 2021Hypogonadism is a common comorbidity associated with HIV-1 that is more prevalent among infected individuals over the age of 45. The underlying mechanisms are unknown,...
Hypogonadism is a common comorbidity associated with HIV-1 that is more prevalent among infected individuals over the age of 45. The underlying mechanisms are unknown, but both combined antiretroviral therapeutics and HIV-1 proteins, such as trans-activator of transcription protein (Tat), dysregulate steroid-synthetic mechanisms including lipid storage/synthesis and mitochondrial function. Thus, Tat expression may accelerate age-related comorbidities partly by impairing endocrine function. Few studies exist of Tat-mediated behavioral deficits in aged animals and effects of endocrine status have not been investigated. Accordingly, we tested whether conditional Tat expression in aged (~ 1.5 years old), female, Tat-transgenic [Tat(+)] mice increases anxiety-like behavior, impairs cognition, and augments mechanical allodynia, when compared to age-matched controls that do not express Tat protein [Tat(-)]. We further tested whether aged mice that maintained their endocrine status (pre-estropausal) were more resilient to Tat/age-related comorbidities than peri- or post-estropausal mice. Tat and endocrine aging status exerted separate and interacting effects that influenced anxiety-like and cognitive behaviors. Peri- and post-estropausal mice exhibited greater anxiety-like behavior in the elevated plus-maze and impaired learning in the radial arm water maze compared to pre-estropausal mice. Irrespective of estropause status, Tat(+) mice demonstrated impaired learning, reduced grip strength, and mechanical allodynia compared to Tat(-) mice. Tat exposure reduced circulating estradiol in post-estropausal mice and increased the estradiol-to-testosterone ratio in pre-estropausal mice. Changes in circulating estradiol, testosterone, and progesterone correlated with grip strength. Thus, endocrine status is an important factor in age-related anxiety, cognition, neuromuscular function, and allodynia that can be accelerated by HIV-1 Tat protein.
Topics: Aging; Analgesics; Animals; Anxiety; Cognition; Female; HIV-1; Mice; Mice, Transgenic; tat Gene Products, Human Immunodeficiency Virus
PubMed: 32940828
DOI: 10.1007/s11357-020-00268-z -
HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors.FEBS Letters Dec 2002Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal...
Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47-57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His(6)) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import.
Topics: Active Transport, Cell Nucleus; Animals; Apoptosis Regulatory Proteins; Base Sequence; Carrier Proteins; Cell Line; Cell Nucleus; Cytosol; Escherichia coli; Gene Products, tat; Genetic Vectors; Green Fluorescent Proteins; HIV-1; Humans; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Mitochondrial Proteins; Molecular Sequence Data; Nuclear Localization Signals; Peptides; Protein Structure, Tertiary; Protein Transport; Recombinant Fusion Proteins; Tumor Cells, Cultured; tat Gene Products, Human Immunodeficiency Virus
PubMed: 12459459
DOI: 10.1016/s0014-5793(02)03624-4 -
Brain, Behavior, and Immunity Oct 2017Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems...
Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV.
Topics: Animals; Dopamine; Dopamine Agents; Dopaminergic Neurons; Gene Products, tat; HIV-1; Humans; Locomotion; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nucleus Accumbens; Reward; Ventral Tegmental Area; tat Gene Products, Human Immunodeficiency Virus
PubMed: 28495611
DOI: 10.1016/j.bbi.2017.05.004 -
The EMBO Journal Jul 1991The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937...
The human immunodeficiency virus-1 (HIV-1) Tat protein has previously been shown to transactivate the HIV-1-LTR when added exogenously to HeLa, H9 lymphocytic and U937 promonocytic cells growing in culture. Here we show that Tat enters these cells by adsorptive endocytosis. Tat appears to bind non-specifically to the cell surface, with greater than 10(7) sites per cell. A specific receptor was not detected by protein crosslinking experiments, and uptake was not affected by treating cells with trypsin, heparinase or neuraminidase. Uptake and transactivation could be inhibited by incubation with heparin, dextran sulfate, an anti-Tat monoclonal antibody, or by incubation at 4 degrees C. In contrast, transactivation by Tat was markedly stimulated by the addition of basic peptides, such as Tat 38-58 or protamine. Fluorescence experiments with rhodamine-conjugated Tat show punctate staining on the cell surface and then localization to the cytoplasm and nucleus. The lack of a specific receptor makes it unclear whether Tat uptake is biologically important in HIV infection, however, the efficiency of uptake raises the possibility that Tat may be useful for delivery of protein molecules into cells.
Topics: Amino Acid Sequence; Antibodies, Monoclonal; Binding Sites; Biological Transport; Cell Nucleus; Cytoplasm; Dextran Sulfate; Endocytosis; Gene Products, tat; HIV-1; HeLa Cells; Heparin; Heparin Lyase; Humans; Kinetics; Molecular Sequence Data; Neuraminidase; Polysaccharide-Lyases; Temperature; Transcriptional Activation; Trypsin; Tumor Cells, Cultured; tat Gene Products, Human Immunodeficiency Virus
PubMed: 2050110
DOI: 10.1002/j.1460-2075.1991.tb07697.x -
Molecular Cell Nov 2000The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level,...
The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level, arginine-rich domains from different proteins adopt different conformations depending on the RNA site recognized, and in some cases fold only in the context of RNA. Here we show that the RNA binding domain of the Jembrana disease virus (JDV) Tat protein is able to recognize two different TAR RNA sites, from human and bovine immunodeficiency viruses (HIV and BIV, respectively), adopting different conformations in the two RNA contexts and using different amino acids for recognition. In addition to the conformational differences, the JDV domain requires the cyclin T1 protein for high-affinity binding to HIV TAR, but not to BIV TAR. The "chameleon-like" behavior of the JDV Tat RNA binding domain reinforces the concept that RNA molecules can provide structural scaffolds for protein folding, and suggests mechanisms for evolving distinct RNA binding specificities from a single multifunctional domain.
Topics: Amino Acid Motifs; Base Sequence; Binding Sites; Electrophoretic Mobility Shift Assay; Evolution, Molecular; Gene Expression Regulation, Viral; Gene Products, tat; HIV Long Terminal Repeat; Immunodeficiency Virus, Bovine; Lentivirus; Magnetic Resonance Spectroscopy; Models, Molecular; Mutation; Protein Binding; Protein Structure, Tertiary; RNA; RNA, Viral; RNA-Binding Proteins; Sequence Alignment; Substrate Specificity; Thermodynamics; Transcriptional Activation
PubMed: 11106746
DOI: 10.1016/s1097-2765(00)00105-2 -
Virology Dec 1998Fusions of the human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat to the green fluorescent protein (GFP) were used to study the intracellular...
Fusions of the human immunodeficiency virus type 1 (HIV-1) transactivator protein Tat to the green fluorescent protein (GFP) were used to study the intracellular localization, trafficking, and interactions of Tat in human cells. Tagging Tat with GFP did not change its nuclear localization or ability to act as a transactivator. Tat-GFP expressed at low levels was found in the nucleus, whereas overexpression resulted in nucleolar accumulation. A Tat-GFP hybrid protein containing in addition the HIV-1 Rev nuclear export signal (NES) localized predominantly to the cytoplasm. This shuttle protein, Tat-GFP-NES, transactivated the HIV-1 long terminal repeat. Thus a Tat molecule being only transiently present in the nucleus is active and nucleolar accumulation of Tat is not prerequisite for function. A coexpression assay previously used to define protein interaction domains in the HIV-1 Rev protein [R. H. Stauber, E. Afonina, S. Gulnik, J. Erickson, and G. N. Pavlakis (1998a). Virology 251, 38-48.] indicated that Tat exists predominantly as a monomer and does not form stable multimers with B23 in living cells. Using a heterokaryon fusion assay, we found that Tat-GFP was able to shuttle between the nucleus and the cytoplasm. Tat therefore has the potential to perform functions in the nucleus as well as in the cytoplasm.
Topics: Biological Transport; Cell Nucleolus; Cell Nucleus; Cytoplasm; Fluorescent Antibody Technique, Indirect; Gene Products, rev; Gene Products, tat; Green Fluorescent Proteins; HIV-1; HeLa Cells; Humans; Luminescent Proteins; Nuclear Proteins; Nucleophosmin; Protein Binding; RNA, Viral; Recombinant Fusion Proteins; Transcriptional Activation; Transfection; rev Gene Products, Human Immunodeficiency Virus; tat Gene Products, Human Immunodeficiency Virus
PubMed: 9875323
DOI: 10.1006/viro.1998.9400 -
Journal of Controlled Release :... Feb 2007Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several... (Review)
Review
Protein transduction with cell penetrating peptides over the past several years has been shown to be an effective way of delivering proteins in vitro and now several reports have also shown valuable in vivo applications in correcting disease states. An impressive bioinspired phenomenon of crossing biological barriers came from HIV transactivator Tat protein. Specifically, the protein transduction domain of HIV Tat has been shown to be a potent pleiotropic peptide in protein delivery. Various approaches such as molecular modeling, arginine guanidinium head group structural strategy, multimerization of PTD sequence and phage display system have been applied for taming of the PTD. This has resulted in identification of PTD variants which are efficient in cell membrane penetration and cytoplasmic delivery. In spite of these state of the art technologies, the dilemma of low protein transduction efficiency and target specific delivery of PTD fusion proteins remains unsolved. Moreover, some misconceptions about PTD of Tat in the literature require considerations. We have assembled critical information on secretory, plasma membrane penetration and transcellular properties of Tat and PTD using molecular analysis and available experimental evidences.
Topics: Cell Line; Drug Delivery Systems; Endocytosis; Gene Products, tat; Green Fluorescent Proteins; Humans; Models, Biological; Oligopeptides; Protein Transport; Recombinant Fusion Proteins
PubMed: 17196289
DOI: 10.1016/j.jconrel.2006.10.031 -
TheScientificWorldJournal Sep 2005Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current... (Review)
Review
Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cells in vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.
Topics: Animals; Autoantibodies; Biological Transport, Active; Cells, Cultured; Drug Delivery Systems; Endocytosis; Gene Products, tat; HIV-1; Humans; Immunoglobulin Fragments; Lymphokines; Mice; Peptide Fragments; Protein Transport; Proteins; Sialoglycoproteins; Signal Transduction; tat Gene Products, Human Immunodeficiency Virus
PubMed: 16170440
DOI: 10.1100/tsw.2005.98 -
Microbes and Infection Oct 2005The Tat protein is a viral transactivator that activates HIV transcription through complex interactions with RNA and host cell factors. Tat undergoes multiple... (Review)
Review
The Tat protein is a viral transactivator that activates HIV transcription through complex interactions with RNA and host cell factors. Tat undergoes multiple posttranslational modifications that regulate the dynamics and complexity of these interactions. The biology of these modifications and their role in Tat function are reviewed.
Topics: Arginine; Gene Products, tat; HIV-1; Humans; Protein Processing, Post-Translational; tat Gene Products, Human Immunodeficiency Virus
PubMed: 16046164
DOI: 10.1016/j.micinf.2005.06.003 -
Human Vaccines & Immunotherapeutics 2015The use of the Tat protein of HIV in vaccines against AIDS showed promising results in primate and human studies. To characterize the impact of the administration route...
The use of the Tat protein of HIV in vaccines against AIDS showed promising results in primate and human studies. To characterize the impact of the administration route on the induction of humoral responses at systemic and mucosal levels, we compared intradermal, intramuscular and mucosal immunizations with Tat and a Tat-derived peptide. Mice were immunized with the Tat protein by different routes and the titer and isotype of anti-Tat antibodies were assessed in serum and mucosal lavages. Intramuscular and intradermal administrations showed comparable immunogenicity, while the mucosal administration was unable to induce IgM in serum and IgG at mucosal sites but showed superior immunogenicity in terms of IgA induction. Anti-Tat antibodies were also obtained upon vaccination with the immunodominant Tat 1-20 peptide which was, however, less immunogenic than the whole Tat protein.
Topics: AIDS Vaccines; Animals; Blood; HIV Antibodies; Immunity, Humoral; Immunity, Mucosal; Mice; tat Gene Products, Human Immunodeficiency Virus
PubMed: 25875962
DOI: 10.1080/21645515.2015.1016676