UMLS:C0019693 - FACTA Search

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Query: UMLS:C0019693 (HIV)
170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal titration calorimetry. The thermodynamic parameters are K0 = (6.0 +/- 0.8) x 10(5) M(-1) and kcal/mol for heparin and K0 = (2.5 +/- 0.5) x 10(5) M(-1) and kcal/mol for chondroitin sulfate B at 28 degrees C. The close thermodynamic similarity of the three binding molecules also implies a close structural relationship. The ubiquitous occurrence of glycosaminoglycans on the cell surface together with their tight and rapid interaction with the TAT protein transduction domain makes complex formation a strong candidate as the primary step of protein translocation.
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PMID:Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters. 1469 67

A technology has recently been developed that allows for the rapid transduction of full-length functionally active proteins into intact tissue through intravenous injection and into cultured cells. This technology involves the fusion of an 11 amino acid sequence of the HIV TAT protein to the protein of interest. In the current investigation, we determined whether functionally active TAT fusion proteins could be transduced into intact corneas by topical application. TAT-beta-galactosidase was purified from bacterial cells and applied in serial dilutions (12.5-250 nm) to cultured epithelial cells for 5 or 15 min. In addition, enucleated globes and excised corneas with or without a central 3-mm epithelial debridement were incubated with TAT-beta-galactosidase for 1 or 2 hr. Excised corneas were allowed to heal in organ culture. Transduction of active beta-galactosidase was detected by incubating the cells or corneas with X-gal. TAT-beta-galactosidase was transduced into nearly all cultured cells in a concentration-dependent manner. When TAT-beta-galactosidase was topically applied to intact corneas, only the most superficial layer of epithelium was highly transduced. When the superficial layer was removed with nitrocellulose, two to four layers of cells were transduced. In corneas with a central debridement, epithelial cells at the edge of the debridement were transduced as well as the stromal cells subjacent to the debridement. Active beta-galactosidase was maintained at least 1 day in organ culture. No X-gal reaction was seen in either cells or corneas not incubated with TAT-beta-galactosidase. Functionally active proteins can be efficiently transduced into corneal epithelial and stromal cells using TAT fusion protein technology. The intact epithelium provides a barrier to penetration of TAT proteins. This barrier can be overcome by disrupting the epithelium. TAT-mediated protein transduction may be extremely useful in studies of corneal wound healing and homeostasis.
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PMID:Transduction of functionally active TAT fusion proteins into cornea. 1505 80

Human granulocytes clearly play an important role in host defence against invading organisms, however they have also been implicated in the pathogenesis and progression of many chronic inflammatory diseases. In addition, these cells have been paramount in gaining a better understanding of many key-signalling pathways regulating fundamental biological processes. Since granulocytes are terminally differentiated and undergo relatively rapid constitutive apoptosis it has been difficult to manipulate intracellular events by transfection or transduction procedures. It has been shown in recent years that the HIV-TAT protein transduction system can be successfully used in granulocytes to manipulate key signalling mechanisms regulating functional responsiveness and survival. In this paper, we review recent literature highlighting important developments using this system in isolated human granulocytes and in inflammatory process in vivo where these cells play a prominent role.
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PMID:Getting to grips with the granulocyte: manipulation of granulocyte behaviour and apoptosis by protein transduction methods. 1515 58

To elucidate the role of enhanced phosphoinositide-3-kinase (PI3-kinase) activity in memory, a synthetic phosphopeptide (TAT-YPMDM) containing the p85 regulatory subunit receptor-binding motif (YXXM) coupled to the cell transduction domain of HIV-TAT protein was employed. This phosphopeptide bound the p85 subunit of PI3-kinase, and was internalized by both granule and pyramidal neurons when injected into the hippocampus. Increased lipid kinase activity and enhanced phosphorylation of the PI3-kinase substrates Akt (protein kinase B) and ribosomal S6 kinase were associated with TAT-YPMDM administration. Bilateral infusion of the phosphopeptide into the dorsal hippocampus after training improved performance in three hippocampus-dependent memory tasks: contextual fear conditioning, trace fear conditioning, and the Morris water maze. Both the biochemical and behavioral effects of the TAT-YPMDM phosphopeptide could be blocked by wortmannin. No effect was observed when a nonphosphorylated peptide (TAT-YMDM), or a second, unrelated phosphopeptide (TAT-YPLDL) was utilized. In addition, infusion of the TAT-YPMDM phosphopeptide did not interfere with memory acquisition or 4 hr memory. In addition, pretesting administration did not affect the ability to recall a previously established long-term memory. These findings suggest that stimulation of PI3-kinase activity by phosphorylated receptor fragments containing the YMDM motif augments long-term memory.
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PMID:Performance in long-term memory tasks is augmented by a phosphorylated growth factor receptor fragment. 1521 87

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

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

Clostridium botulinum exoenzyme C3 is responsible for the inactivation of members of the Rho GTPase family that are implicated in actin-cytoskeleton reorganization. This property has been extensively used in the field to investigate the functionality of the Rho GTPases. However, systematic analysis of Rho GTPase functions requires large amounts of such inhibitors and consequently an optimization of the production yield of these proteins. Bacterial production of soluble proteins often requires a refolding step that noticeably affects the production yields and necessitates additional experiments to verify functional activity. This is particularly true for TAT-C3, the production yields of which are generally low. In this report, we describe a rapid and efficient method for the production of soluble C3 exoenzyme developed by screening a collection of bacterial strains. The recombinant C3 protein was fused to the TAT protein-transduction domain from HIV, to allow protein delivery into cells, and to a hexahistidine tag, that permitted purification by Nickel affinity chromatography. We have demonstrated the production of large amounts of soluble and functional protein using the bacterial strain AD494 (DE3)pLysS. This rapid and efficient method for the production of soluble C3 exoenzyme could also be useful for the production of other proteins with solubility problems.
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PMID:Efficient production of Clostridium botulinum exotoxin C3 in bacteria: a screening method to optimize production yields. 1572 84

The protein transduction domain (PTD) from the HIV-1 TAT protein has been widely utilized to deliver biologically active macromolecules, including full-length proteins, into a variety of cell types in vitro and in vivo. Without additional targeting signals, the intracellular localization of the proteins delivered in this fashion appears to be cytoplasmic, nuclear or, as recently reported, endosomal. In this study, we show that the presence of the mitochondrial targeting signal (MTS) from hMnSOD on the N-terminus of TAT-fusion proteins directs them into mitochondria of breast cancer cells. We generated and purified fusion proteins containing GFP (MTS-GFP-TAT) or Exonuclease III (MTS-ExoIII-TAT) from Escherichia coli. The results of Western blots of subcellular fractions and fluorescent microscopic analyses revealed efficient protein transduction and mitochondrial localization of the fusion proteins. Specific exonuclease activity was found in the mitochondrial extracts isolated from MTS-ExoIII-TAT transduced cells. This increased exonuclease activity reduced the repair of mtDNA damage following oxidative stress. This diminished mtDNA repair led to a decrease in survival of breast cancer cells. Thus, the present study demonstrates the applicability of this new approach for intramitochondrial targeting of TAT-fusion proteins capable of modulating mitochondrial function and cell survival.
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PMID:TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. 1572 31

Stem cell technologies hold great potential for the treatment of type 1 diabetes, provided that functional transplantable beta-cells can be selectively generated in an efficient manner. Such a process should recapitulate, at least to a certain extent, the embryonic development of beta-cells in vitro. However, progress at identifying the transcription factors involved in beta-cell development has not been accompanied by a parallel success at unraveling the pattern of their instructive extracellular signals. Here we present proof of principle of a novel approach to circumvent this problem, based on the use of the HIV/TAT protein transduction domain. Neurogenin 3 (ngn3), a factor whose expression is essential for pancreatic endocrine differentiation, was fused to the TAT domain. Administration of TAT/ngn3 to cultured pancreatic explants results in efficient uptake, nuclear translocation, and stimulation of downstream reporter and endogenous genes. Consistent with the predicted activity of the protein, e9.5 and e13.5 mouse pancreatic explants cultured in the presence of TAT/ngn3 show an increased level of endocrine differentiation compared with control samples. Our results raise the possibility of sequentially specifying stem/progenitor cells toward the beta-cell lineage, by using the appropriate sequence and combination of TAT-fused transcription factors.
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PMID:TAT-mediated neurogenin 3 protein transduction stimulates pancreatic endocrine differentiation in vitro. 1573 48

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