Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.
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PMID:Characterization of TAT-mediated transport of detachable kinase substrates. 1522 64

S100 proteins belong to the EF-hand Ca2+-binding protein family and are involved in the regulation of a variety of cellular processes. Individual S100 proteins are expressed in cell- and tissue-specific manners, and functional deterioration of S100 proteins leads to a number of human diseases, including cancer. We previously demonstrated that S100C/A11 was translocated to nuclei and inhibited DNA synthesis in human keratinocytes when exposed to high Ca2+. In the present study we examined the effects of synthetic partial peptides of S100C/A11 on human carcinoma cell lines. Only an N-terminal peptide with 19 amino acid residues (MAK19) showed cytotoxicity to the cell lines in dose- and time-dependent manners when introduced into cells by flanking the HIV-TAT protein transduction domain (TAT-MAK19). Pulse field electrophoresis revealed that DNA of the treated cells was partially degradated. Annexin V, a marker of cellular apoptosis, was detected in the cells treated with TAT-MAK19 by immunostaining and flow cytometry. The induction of apoptotic cell death was apparently independent of p53, p21WAF1/CIP1, and caspase activity, but treatment with TAT-MAK19 resulted in partial translocation of apoptosis-inducing factor (AIF) from the cytoplasm to nuclei. These results indicate that MAK19 induces apoptosis in human cell lines and may therefore lead to the establishment of a new molecular target for the treatment of human cancer.
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PMID:Introduction of an N-terminal peptide of S100C/A11 into human cells induces apoptotic cell death. 1524

Tumor hypoxia in a solid tumor mass has long been recognized as a cause of resistance to current cancer therapies, and has also been suggested to be a potent driving force towards malignancy. Recent progress in the understanding of the molecular mechanism of the tumor response to hypoxia has increased attention on targeting hypoxia for cancer therapy. We have generated a hypoxia-targeting fusion protein, TOP3, which is composed of a protein transduction domain (PTD) of HIV TAT, an oxygen-dependent degradation domain (ODD) of HIF-1 alpha, and procaspase-3. Here, we examine the effects of TOP3 in a rat ascites model. First, we clarified that the fluid in ascites from MM1 cells, which are derivatives of AH130 rat ascites hepatoma cells, was highly hypoxic. In vitro, MM1 cells retained protein degradation machinery through the ODD domain, and TOP3 effectively impaired MM1 cell growth in culture under hypoxic conditions by inducing apoptosis. Intraperitoneal administration of TOP3 prolonged the life span of rats bearing a significant amount of malignant ascites, and 60% of the treated animals were cured without recurrence of ascites. Thus, TOP3 had a dramatic effect on malignant ascites and, hence, we propose that rodent malignant ascites is an appropriate platform for testing hypoxia-targeted drugs.
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PMID:Targeting hypoxic cancer cells with a protein prodrug is effective in experimental malignant ascites. 1528 74

Viability of isolated islets is one of the main obstacles limiting islet transplantation success. It has been reported that overexpression of Bcl-2/Bcl-XL proteins enhances islet viability. To avoid potential complications associated with long-term expression of anti-apoptotic proteins, we investigated the possibility of delivering Bcl-XL or its anti-apoptotic domain BH4 to islets by protein transduction. Bcl-XL and BH4 molecules were fused to TAT/PTD, the 11-aa cell penetrating peptide from HIV-1 transactivating protein, generating TAT-Bcl-XL and TAT-BH4, respectively. Transduction efficiency was assessed by laser scanning confocal microscopy of live islets. Biological activity was tested as the ability to protect NIT-1 insulinoma cell line from death induced by staurosporine or serum deprivation. Spontaneous caspase activation in human islets and cytotoxicity caused by IL-1beta were significantly reduced in the presence of TAT-Bcl-XL and TAT-BH4. We conclude that both TAT proteins are biologically active after transduction and could be an asset in the improvement of islet viability.
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PMID:Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets. 1536 75

For cytosolic delivery of liposomes containing macromolecular drugs, such as proteins or nucleic acids, it would be beneficial to bypass endocytosis to prevent degradation in the lysosomes. Recent reports pointed to the possibility that coupling of TAT-peptides to the outer surface of liposome particles would enable translocation over the cellular plasma membrane. Here, we demonstrate that cellular uptake of TAT-liposomes occurs via endocytosis rather than plasma membrane translocation. The coupling of HIV-1 derived TAT-peptide to liposomes enhances their binding to ovarian carcinoma cells. The binding was inhibited by the presence of heparin or dextran sulfate, indicating that cell surface proteoglycans are involved in the binding interaction. Furthermore, living confocal microscopy studies revealed that binding of the TAT-liposomes to the plasma membrane is followed by intracellular uptake in vesicular structures. Staining the endosomes and lysosomes demonstrated that fluorescent liposomal labels are present within the endosomal and lysosomal compartments. Furthermore, incubation at low temperature or addition of a metabolic or an endocytosis inhibitor blocked cellular uptake. In conclusion, coupling TAT-peptide to the outer surface of liposomes leads to enhanced endocytosis of the liposomes by ovarian carcinoma cells, rather than direct cytosolic delivery by plasma membrane translocation.
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PMID:OVCAR-3 cells internalize TAT-peptide modified liposomes by endocytosis. 1547 70

The resistance of melanoma to apoptosis, as well as its growth and metastasis capabilities, can be overcome by expression of a peptide derived from amino acid (aa) 51 to 100 of ATF2. Here we show that expression of ATF2((51-100)) in human melanoma cells reduced their growth in nude mice, which was additionally inhibited upon treatment with protein kinase inhibitors UCN-01 or SB203580. Injection of a fusion protein consisting of HIV-TAT and aa 51 to 100 of ATF2 into SW1 melanomas efficiently inhibits their growth and their metastasis up to complete regression. Additionally, expression of a 10aa peptide that corresponds to aa 51 to 60 of ATF2 sensitizes melanoma cells to spontaneous apoptosis, which coincides with activation of caspase 9 and poly(ADP-ribose) polymerase cleavage, and inhibit their growth in vivo. The 10aa peptide increases the association of c-Jun NH(2)-terminal kinase with c-Jun but not with ATF2, resulting in concomitant increase in TRE-mediated transcription. Our study points to mechanisms underlying the activities of the ATF2 peptide while highlighting its possible use in drug design.
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PMID:Inhibition of melanoma growth and metastasis by ATF2-derived peptides. 1554 88

Here we demonstrate that an inducible anti-HIV short hairpin RNA (shRNA) expressed from a Pol II promoter inhibits HIV-1 gene expression in mammalian cells. Our strategy is based on a promoter system in which the HIV-1 LTR is fused to the Drosophila hsp70 minimal heat shock promoter. This system is inducible by HIV-1 TAT, which functions in a negative feedback loop to activate transcription of an shRNA directed against HIV-1 rev. Upon induction the shRNA is processed to an siRNA that guides inhibition of HIV replication in cultured T-lymphocytes and hematopoietic stem cell-derived monocytes. The fusion promoter system may be safer than drug-inducible systems for shRNA-mediated gene therapy against HIV as the shRNAs are only expressed following HIV infection.
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PMID:Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. 1556 18

An 11 amino acid HIV-TAT peptide can deliver target proteins into a variety of cells in a receptor-independent manner. To generate a highly specific inhibitor of the transcription factor NF-kappa B, we have fused the TAT-peptide to a version of I kappa B alpha that is resistant to signal-induced degradation. TAT-I kappa B alpha(S32A, S36A) inhibited NF-kappa B-dependent transcription in HeLa and A549 cells by retaining NF-kappa B p65 in the cytoplasm. Introduction of TAT-I kappa B alpha(S32A, S36A) into human eosinophils inhibited the nuclear translocation of NF-kappa B and induced apoptosis. Thus, continuous NF-kappa B-dependent transcription is important for eosinophil survival. While eosinophils are normally refractive to standard methods of gene delivery, the ability of TAT fusion proteins to be taken up by these cells should enable a detailed molecular analysis of survival pathways in these cells.
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PMID:Inhibition of NF-kappa B by a cell permeable form of I kappa B alpha induces apoptosis in eosinophils. 1559 46

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.
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PMID:The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. 1562 54

For the aims of studying molecular mechanisms of functioning of adenylyl cyclase signaling systems (ACS), we investigated the influence of synthetic polycationic peptides of the star-like structure (dendrons), containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of ACS components in smooth muscles of the mollusc Anodonta cygnea and in rat skeletal muscles. It has been shown that the following peptides (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx(= epsilon-aminohexanoic acid)-Cys(Acm), referred to as peptide I, (Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys(Acm) (peptide II), [(Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide III), and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide IV) inhibit in a dose-dependent manner the adenylyl cyclase (AC) activity stimulated by both nonhormanal agents (GppNHp and forskolin) and hormones, such as serotonin (mollusc) and isoproterenol (rat). Peptides III and IV (tetrameric dendrons) were most effective in comparison with peptides I and II (dimeric dendrons). The AC activity stimulated by hormones and forskolin was most sensitive to the action of dendrons. All dendrons stimulated GTP-binding activity of G-proteins: dimeric dendrons were most effective at 10(-5) M concentration, whereas tetrameric dendrons at 10(-6) M. In the presence of dendrons, the affinity of beta-antagonist [3H]-dihydroalprenolol to P-adrenergic receptor in rat muscle mem- branes was unchanged. At the same time, the affinity of beta-agonist isoproterenol to the receptor decreased, and no shift to the right was observed on the curve of isoproterenol-induced [3H]-dihydroalprenolol displacement in the presence of GTP. The obtained data show the disturbance of the coupling between the receptor and G-protein, which is the main reason of dendron inhibitory action on AC stimulation by hormones. Besides, these data demonstrated that hormones could disturb the functional activity of AC, i.e. a catalytic component of ACS.
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PMID:[Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems]. 1570 84


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