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Journal of Visualized Experiments : JoVE Dec 2009The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see (1,2)]. For this one can make use of...
The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see (1,2)]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration(2). In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre. Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells(3,4). The delivery of active Cre recombinase to cells, however, represents a limitation. We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.
Topics: Animals; Cell Membrane Permeability; Embryonic Stem Cells; Gene Products, tat; Genetic Vectors; Histidine; Integrases; Mice; Protein Engineering; Recombinant Fusion Proteins; Recombinant Proteins
PubMed: 20038937
DOI: 10.3791/1627 -
Cartilage Dec 2021Intra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide...
OBJECTIVE
Intra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide transduction domain (TAT-PTD) as a potential intra-articular drug delivery technology for synovial joints.
DESIGN
Experiments examined the ability of TAT conjugates to associate with primary chondrocytes and alter cellular function both and . Further experiments examined the ability of the TAT-PTD to bind to human osteoarthritic cartilage.
RESULTS
The results show that the TAT-PTD associates with chondrocytes, is capable of delivering siRNA for chondrocyte gene knockdown, and that the recombinant enzyme TAT-Cre is capable of inducing genetic recombination within the knee joint in a reporter mouse model. Last, binding studies show that osteoarthritic cartilage preferentially uptakes the TAT-PTD from solution.
CONCLUSIONS
The results suggest that the TAT-PTD is a promising delivery strategy for intra-articular therapeutics.
Topics: Animals; Cartilage; Chondrocytes; Gene Products, tat; Mice; Osteoarthritis; Technology
PubMed: 32954793
DOI: 10.1177/1947603520959392 -
BMC Infectious Diseases Mar 2023HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is... (Review)
Review
HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47-59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47-59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.
Topics: Humans; HIV-1; tat Gene Products, Human Immunodeficiency Virus; HIV Long Terminal Repeat; Amino Acids; RNA, Viral
PubMed: 36932337
DOI: 10.1186/s12879-023-08123-0 -
Journal of Virology Jul 2018Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially...
Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the -acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein. The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.
Topics: Gene Expression Regulation, Viral; Gene Products, tat; HEK293 Cells; HIV Infections; HIV-1; Humans; RNA Splicing; RNA, Viral; Virus Replication
PubMed: 29743356
DOI: 10.1128/JVI.01855-17 -
Scientific Reports Jun 2016HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by...
HIV-1 Tat is an intrinsically unfolded protein playing a pivotal role in viral replication by associating with TAR region of viral LTR. Unfolded proteins are degraded by 20S proteasome in an ubiquitin independent manner. Curcumin is known to activate 20S proteasome and promotes the degradation of intrinsically unfolded p53 tumor suppressor protein. Since HIV-1 Tat protein is largerly unfolded, we hypothesized that Tat may also be targeted through this pathway. Curcumin treated Tat transfected HEK-293T cells showed a dose and time dependent degradation of Tat protein. Contrary to this HIV-1 Gag which is a properly folded protein, remained unaffected with curcumin. Semi-quantitative RT-PCR analysis showed that curcumin treatment did not affect Tat gene transcription. Curcumin increased the rate of Tat protein degradation as shown by cycloheximide (CHX) chase assay. Degradation of the Tat protein is accomplished through proteasomal pathway as proteasomal inhibitor MG132 blocked Tat degradation. Curcumin also decreased Tat mediated LTR promoter transactivation and inhibited virus production from HIV-1 infected cells. Taken together our study reveals a novel observation that curcumin causes potent degradation of Tat which may be one of the major mechanisms behind its anti HIV activity.
Topics: Curcumin; Gene Expression Regulation, Viral; HIV Infections; HIV-1; Humans; Proteolysis; Transfection; Virus Replication; tat Gene Products, Human Immunodeficiency Virus
PubMed: 27283735
DOI: 10.1038/srep27539 -
FEBS Letters Feb 2002Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone... (Review)
Review
Bromodomains, an extensive family of evolutionarily conserved protein modules originally found in proteins associated with chromatin and in nearly all nuclear histone acetyltransferases, have been recently discovered to function as acetyl-lysine binding domains. More recent structural studies of bromodomain/peptide ligand complexes have enriched our understanding of differences in ligand selectivity of bromodomains. These new findings demonstrate that bromodomain/acetyl-lysine recognition can serve as a pivotal mechanism for regulating protein-protein interactions in numerous cellular processes including chromatin remodeling and transcriptional activation, and reinforce the concept that functional diversity of a conserved protein modular structure is achieved by evolutionary changes of amino acid sequences in the ligand binding site.
Topics: Acetylation; Acetyltransferases; Amino Acid Sequence; Animals; Binding Sites; Chromatin; Conserved Sequence; Evolution, Molecular; Gene Products, tat; HIV-1; Histone Acetyltransferases; Humans; Lysine; Molecular Sequence Data; Protein Structure, Secondary; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Transcription Factors; tat Gene Products, Human Immunodeficiency Virus
PubMed: 11911891
DOI: 10.1016/s0014-5793(01)03309-9 -
Current HIV Research 2019Methamphetamine abuse and human immunodeficiency virus (HIV) are common comorbidities. HIV-associated proteins, such as the regulatory protein TAT, may contribute to...
BACKGROUND
Methamphetamine abuse and human immunodeficiency virus (HIV) are common comorbidities. HIV-associated proteins, such as the regulatory protein TAT, may contribute to brain reward dysfunction, inducing an altered sensitivity to methamphetamine reward and/or withdrawal in this population.
OBJECTIVE
These studies examined the combined effects of TAT protein expression and, chronic and binge methamphetamine regimens on brain reward function.
METHODS
Transgenic mice with inducible brain expression of the TAT protein were exposed to either saline, a chronic, or a binge methamphetamine regimen. TAT expression was induced via doxycycline treatment during the last week of methamphetamine exposure. Brain reward function was assessed daily throughout the regimens, using the intracranial self-stimulation procedure, and after a subsequent acute methamphetamine challenge.
RESULTS
Both methamphetamine regimens induced withdrawal-related decreases in reward function. TAT expression substantially, but not significantly increased the withdrawal associated with exposure to the binge regimen compared to the chronic regimen, but did not alter the response to acute methamphetamine challenge. TAT expression also led to persistent changes in adenosine 2B receptor expression in the caudate putamen, regardless of methamphetamine exposure. These results suggest that TAT expression may differentially affect brain reward function, dependent on the pattern of methamphetamine exposure.
CONCLUSION
The subtle effects observed in these studies highlight that longer-term TAT expression, or its induction at earlier stages of methamphetamine exposure, may be more consequential at inducing behavioral and neurochemical effects.
Topics: Animals; Brain; Male; Methamphetamine; Mice; Mice, Transgenic; Receptors, Purinergic P1; Reward; Up-Regulation; tat Gene Products, Human Immunodeficiency Virus
PubMed: 31269883
DOI: 10.2174/1570162X17666190703165408 -
The Journal of Biological Chemistry Sep 2015HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms...
HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel β sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.
Topics: Cell Membrane; Electrophoresis, Polyacrylamide Gel; Fibroblast Growth Factor 2; Humans; Lipid Bilayers; Liposomes; Mutation; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Protein Binding; Protein Conformation; Protein Multimerization; Protein Structure, Secondary; Protein Transport; Spectroscopy, Fourier Transform Infrared; tat Gene Products, Human Immunodeficiency Virus
PubMed: 26183781
DOI: 10.1074/jbc.M115.667097 -
FEBS Letters Apr 1991A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was...
A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We then defined a rapid, three-step procedure for the purification of the Tat protein.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; DNA, Viral; Escherichia coli; Exons; Gene Products, tat; Genes, Synthetic; Genes, Viral; Genes, tat; HIV-1; Kinetics; Molecular Sequence Data; Polymerase Chain Reaction; tat Gene Products, Human Immunodeficiency Virus
PubMed: 2026253
DOI: 10.1016/0014-5793(91)80467-h -
Redox Biology Apr 2021The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but...
The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-β-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress.
Topics: Animals; HIV Infections; Mice; Microglia; Oxidative Stress; Rats; Sirtuin 3; tat Gene Products, Human Immunodeficiency Virus
PubMed: 33385630
DOI: 10.1016/j.redox.2020.101843