Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

TAT protein is an essential regulatory protein of the human immunodeficiency virus type 1 (HIV-1). Inhibition of TAT activity blocks the virus cycle, and a drug that blocks TAT is one of the possibilities to cure AIDS. Circular dichroism (CD) was measured for TAT peptides covering the TAT sequence with overlaps. The CD spectrum of each peptide was measured in different solvents to evaluate the ability of each TAT region to form different secondary structures. The most variation or conformational heterogeneity is observed with the two regions adjacent to the TAT basic region. CD data show that the basic region can adopt an extended structure in a full TAT protein, which is not the case for the isolated peptide. TAT sequences from the different HIV-1 isolates were analyzed, and the results showed that the sequences could be gathered into six groups. Molecular modeling was done on the various isolates based on a TAT structure from two-dimensional NMR. After minimization and dynamic steps, the modeled three-dimensional structures were compared. The results showed structural variations of the TAT protein as a function of the HIV-1 isolates. These structural variations were mainly in the two regions adjacent to the basic region, confirming the conformational heterogeneity indicated by the CD measurements. Furthermore, Chou-Fasman analysis shows significant changes in propensities for each secondary structure only for regions III and V. This conformational heterogeneity should be essential for TAT activity and points out that regions III and V are a poor potential target to design a TAT ligand. We propose a target involving TAT structurally conserved regions, accessible whatever the size of the TAT C terminus.
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PMID:Conformational heterogeneity in two regions of TAT results in structural variations of this protein as a function of HIV-1 isolates. 879 35

Clinical and experimental observations suggest that human herpesvirus-6 (HHV-6), a T-lymphotropic herpesvirus, may act as a cofactor in the acquired immunodeficiency syndrome (AIDS). Moreover, a possible role of HHV-6 in the increased incidence and severity of cervical carcinoma in human immunodeficiency virus (HIV)-infected women was suggested by the recent observation that HHV-6 can infect cervical carcinoma cells, accelerating their tumorigenicity in vivo. Therefore, the ability of four HHV-6 genomic clones derived from HHV-6 to transactivate the long terminal repeat (LTR) of HIV-1 in two cervical carcinoma cell lines and in a T-lymphoid cell line was tested. Two HHV-6 clones, pZVH-14 and pZVB-70, which were previously shown to increase the expression of human papillomavirus (HPV)-transforming genes, were, per se, weak transactivators of the HIV-1 LTR. However, an increased effect occurred when these clones were combined with the HIV-1 transactivator TAT-1. No such effect was seen with two other HHV-6 clones used as controls. Analysis with HIV-1 LTR deletion mutants indicated that this enhancing effect requires the presence of elements contained in both the enhancer region and the TAT activation region (TAR) of HIV-1. This data may have implications for the potential role of HHV-6 in AIDS and AIDS-related cervical carcinoma.
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PMID:Enhancement of TAT-induced transactivation of the HIV-1 LTR by two genomic fragments of HHV-6. 889 36

Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.
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PMID:Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. 902 Nov 81

Transfer of "anti-HIV-1 genes" into hematopoietic stem cells of human immunodeficiency virus-1 (HIV-1)-infected individuals may be a potent therapeutic approach to render mature cells arising from transduced stem cells resistant to the destructive events associated with HIV-1 infection. To determine the feasibility of gene therapy for acquired immunodeficiency syndrome in individuals already infected with HIV-1, granulocyte colony-stimulating factor mobilized peripheral blood CD34+ cells were isolated from HIV-1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes: the Rev binding domain of the Rev Responsive Element (RRE decoy) (L-RRE-neo), a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and the trans-dominant mutant of rev (M10) (L-M10-SN). As a control, a vector mediating only neomycin resistance (LN) was used. After 3 days of transduction on allogeneic stroma in the presence of stem cell factor, interleukin-6 (IL-6), and IL-3, the cultures were G418-selected, and then challenged with HIV-1(JR-FL) and a primary HIV-1 isolate. Compared with the control cultures, the L-RRE-neo-, L-TR/TAT-neo-, and L-M10-SN-transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication after challenge with HIV-1(JR-FL) and the primary HIV-1 isolate. Growth of the hematopoietic cells in long-term bone marrow culture was not perturbed by the presence of any of the anti-HIV-1 genes. This study shows that anti-HIV-1 genes can be introduced into CD34+ cells from individuals already infected with HIV-1, and strongly inhibit HIV-1 replication in primary monocytes derived from the CD34+ progenitors.
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PMID:Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes. 911 67

Human immunodeficiency virus-1 (HIV-1)-Tat, the transactivating gene product of HIV-1, has been shown to interact with different cell types, inducing gene expression, altering their growth and migratory behavior. In this study we examined whether Tat might affect functions of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphoma (NHL), relevant to the in vivo dissemination. Our results show that Tat significantly augmented the motility of the two AIDS-related Burkitt's lymphoma cell lines (AS283 and PA682PB) and AIDS-primary effusion lymphoma cell line (HBL-6-AIDS-PEL). Mutations in RGD or basic domain of Tat (KGE-MBP and LxI-MBP, respectively) sharply reduced migration compared with wild type, suggesting that both domains are required for migration. In contrast, a Tat protein mutation outside the active domains (NH(2)-TAT-GST) did not reduce lymphoma cell migration. The treatment of lymphoma cells with Tat did not influence their adhesion to matrix proteins or to human vascular endothelial cells, but endothelial cells treated with Tat became more adhesive to lymphoma cells. Flow cytometric analysis showed that treatment of endothelial cells with Tat induced the cell surface expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1) and E-selectin and increased the expression of intercellular adhesion molecule-1 (ICAM-1). Only antibodies against VCAM-1 on endothelial cells or against the VLA-4 integrin expressed on AS283 cells inhibited the increment of adhesion, indicating the relevance of this pathway in the adhesion of lymphoma cells to vascular endothelium. In our work, we show for the first time that Tat can enhance the migration of lymphoma cells and their adhesion to endothelial cells, two processes that may contribute to the malignant behavior of NHL in patients with AIDS.
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PMID:Human immunodeficiency virus-1 (HIV-1)-Tat protein promotes migration of acquired immunodeficiency syndrome-related lymphoma cells and enhances their adhesion to endothelial cells. 1047

In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.
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PMID:The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis. 1113 39

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells. A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors, and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics. Similarly, the third alpha-helix of Antennapedia homeodomain, and VP22 protein from herpes simplex virus promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD-TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein. In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein-protein interactions in living cells and for screening novel therapeutic proteins.
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PMID:A peptide carrier for the delivery of biologically active proteins into mammalian cells. 1173 88

Gene therapy of many genetic diseases requires permanent gene transfer into self-renewing stem cells and restriction of transgene expression to specific progenies. Human immunodeficiency virus (HIV)-derived lentiviral vectors are very effective in transducing rare, nondividing stem cell populations (e.g., hematopoietic stem cells) without altering their long-term repopulation and differentiation capacities. We developed a strategy for transcriptional targeting of lentiviral vectors based on replacing the viral long terminal repeat (LTR) enhancer with cell lineage-specific, genomic control elements. An upstream enhancer (HS2) of the erythroid-specific GATA-1 gene was used to replace most of the U3 region of the LTR, immediately upstream of the HIV type 1 (HIV-1) promoter. The modified LTR was used to drive the expression of a reporter gene (the green fluorescent protein [GFP] gene), while a second gene (a truncated form of the p75 nerve growth factor receptor [DeltaLNGFR]) was placed under the control of an internal constitutive promoter to monitor cell transduction, or to immunoselect transduced cells, independently from the expression of the targeted promoter. The transcriptionally targeted vectors were used to transduce cell lines, human CD34+ hematopoietic stem-progenitor cells, and murine bone marrow (BM)-repopulating stem cells. Gene expression was analyzed in the stem cell progeny in vitro and in vivo after xenotransplantation into nonobese diabetic-SCID mice or BM transplantation in coisogenic mice. The modified LTR directed high levels of transgene expression specifically in mature erythroblasts, in a TAT-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the modified LTR was higher, better restricted, and showed less position-effect variegation than that obtained by the same combination of enhancer-promoter elements placed in a conventional, internal position. Cloning of the woodchuck hepatitis virus posttranscriptional regulatory element at a defined position in the targeted vector allowed selective accumulation of the genomic transcripts with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of a spliced, major viral transcript to express a therapeutic gene and that of an internal, independently regulated promoter to express an additional gene for either cell marking or in vivo selection purposes.
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PMID:Transcriptional targeting of lentiviral vectors by long terminal repeat enhancer replacement. 1190 39

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT-GDH fusion protein. The TAT-GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence. TAT-GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT-GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.
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PMID:TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells. 1191 70

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immunofluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.
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PMID:Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes. 1238 68


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