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

Human cytomegalovirus encodes two glycoproteins, US2 and US11, that target major histocompatibility complex (MHC) class I heavy chains for proteasomal degradation. We have developed a mRNA-dependent cell-free system that recapitulates US2- and US11-mediated degradation of MHC class I heavy chains. Microsomes support the degradation of MHC class I heavy chains in the presence of US2 or US11 in a cytosol-dependent manner. In vitro, the glycosylated heavy chain is exported from the microsomes. A deglycosylated breakdown intermediate of the heavy chain identical to that generated in intact cells accumulates in soluble form in the presence of proteasome inhibitors. Microsomes derived from the U373 astrocytoma cell line are far more effective than canine-derived membranes in supporting this US2- or US11-dependent reaction. In contrast, the HIV-encoded Vpu membrane protein can cause the destruction of CD4 from either human- or canine-derived membranes. Using the in vitro system, we show that a truncation mutant of US2 that lacks the cytosolic domain is unable to catalyze degradation, whereas a similar truncation of US11 continues to catalyze degradation of class I heavy chains. Therefore, US2 requires both transmembrane and cytosolic interactions to trigger dislocation of heavy chains, whereas US11 relies on the transmembrane domain to target heavy chains. US2 and US11 thus utilize different targeting mechanisms for class I degradation.
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PMID:Membrane-specific, host-derived factors are required for US2- and US11-mediated degradation of major histocompatibility complex class I molecules. 1171 8

HIV-1 is a fundamentally difficult target for vaccines due to its high mutation rate and its repertoire of immunoevasive strategies. To address these difficulties, a multivalent, proteasome-targeted, live genetic vaccine was recently developed against HIV-1 using the expression library immunization approach. In this HIV-1 vaccine all open reading frames of HIV-1 are expressed from 32 plasmids as Ag fragments fused to the ubiquitin protein to increase Ag targeting to the proteasome to enhance CTL responses. In this work we demonstrate the ability of the HIV-1 library vaccine to simultaneously provoke robust HLA-A*0201-restricted T cell responses against all 32 HIV-1 library vaccine Ags after single immunization by gene gun. These CD8 T cell responses included HLA-A*0201-restricted CTL activity, CD8/IFN-gamma T cell responses, and HLA tetramer binding against defined immunodominant epitopes in gag, pol, env, and nef as well as potent CD8/IFN-gamma responses against undefined HLA-A*0201-restricted epitopes in all remaining Ags of the library. CD8 responses mediated by single gag, pol, env, and nef plasmids from the vaccine demonstrated little reduction in specific T cell responses when these plasmids were diluted into the context of the full 32-plasmid library, suggesting that Ag dominance or immune interference is not an overt problem to limit the efficacy of this complex vaccine. Therefore, this work demonstrates the ability of the HIV-1 library vaccine to generate robust multivalent genome-wide T cell responses as one approach to control the highly mutable and immunoevasive HIV-1 virus.
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PMID:Generation of genome-wide CD8 T cell responses in HLA-A*0201 transgenic mice by an HIV-1 ubiquitin expression library immunization vaccine. 1175 84

Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.
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PMID:HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56. 1189 91

Cytotoxic T lymphocytes (CTLs) play a key role in the control of persistent viral infections. Differences in the quality of this cellular immune response influence the long-term outcome of such infections, but the factors that determine which virus-derived peptide epitopes are targeted by CTLs remain poorly understood. Here, we examine the antigen-processing requirements of three human leukocyte antigen (HLA) A*0201-restricted HIV-1 CTL epitopes. Each of these three peptides appears to be generated by a distinct proteolytic pathway, despite presentation on the cell surface in association with the same HLA class I molecule. Presentation of the commonly immunodominant SLYNTVATL (HIV-1 p17 Gag; residues 77-85) epitope was unaffected by inhibition of the proteasome with lactacystin, but was dependent on the presence of the beta-subunit LMP7. These findings are consistent with emerging data on the complexity of peptide epitope generation, and suggest that differences in antigen processing might contribute to patterns of CTL recognition in vivo.
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PMID:Differential processing of HLA A2-restricted HIV type 1 cytotoxic T lymphocyte epitopes. 1195 41

We present a predictive method that can simulate an essential step in the antigen presentation in higher vertebrates, namely the step involving the proteasomal degradation of polypeptides into fragments which have the potential to bind to MHC Class I molecules. Proteasomal cleavage prediction algorithms published so far were trained on data from in vitro digestion experiments with constitutive proteasomes. As a result, they did not take into account the characteristics of the structurally modified proteasomes--often called immunoproteasomes--found in cells stimulated by gamma-interferon under physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC Class I ligands, i.e. the specificity of the immunoproteasome is different than the constitutive proteasome. The tools developed in this study in combination with a predictor of MHC and TAP binding capacity should give a more complete prediction of the generation and presentation of peptides on MHC Class I molecules. Here we demonstrate that such an approach produces an accurate prediction of the CTL the epitopes in HIV Nef. The method is available at www.cbs.dtu.dk/services/NetChop/.
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PMID:Prediction of proteasome cleavage motifs by neural networks. 1198 29

UBA domains are a commonly occurring sequence motif of approximately 45 amino acid residues that are found in diverse proteins involved in the ubiquitin/proteasome pathway, DNA excision-repair, and cell signaling via protein kinases. The human homologue of yeast Rad23A (HHR23A) is one example of a nucleotide excision-repair protein that contains both an internal and a C-terminal UBA domain. The solution structure of HHR23A UBA(2) showed that the domain forms a compact three-helix bundle. We report the structure of the internal UBA(1) domain of HHR23A. Comparison of the structures of UBA(1) and UBA(2) reveals that both form very similar folds and have a conserved large hydrophobic surface patch. The structural similarity between UBA(1) and UBA(2), in spite of their low level of sequence conservation, leads us to conclude that the structural variability of UBA domains in general is likely to be rather small. On the basis of the structural similarities as well as analysis of sequence conservation, we predict that this hydrophobic surface patch is a common protein-interacting surface present in diverse UBA domains. Furthermore, accumulating evidence that ubiquitin binds to UBA domains leads us to the prediction that the hydrophobic surface patch of UBA domains interacts with the hydrophobic surface on the five-stranded beta-sheet of ubiquitin. Detailed comparison of the structures of the two UBA domains, combined with previous mutagenesis studies, indicates that the binding site of HIV-1 Vpr on UBA(2) does not completely overlap the ubiquitin binding site.
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PMID:Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. 1207 61

Hypoxia-inducible factor-1 (HIF) is a transcription factor central to oxygen homeostasis. It is regulated via its alpha isoforms. In normoxia they are ubiquitinated by the von Hippel-Lindau E3 ligase complex and destroyed by the proteasome, thereby preventing the formation of an active transcriptional complex. Oxygen-dependent enzymatic hydroxylation of either of two critical prolyl residues in each HIFalpha chain has recently been identified as the modification necessary for targeting by the von Hippel-Lindau E3 ligase complex. Here we demonstrate that polypeptides bearing either of these prolyl residues interfere with the degradative pathway, resulting in stabilization of endogenous HIFalpha chains and consequent up-regulation of HIF target genes. Similar peptides in which the prolyl residues are mutated are inactive. Induction of peptide expression in cell cultures affects physiologically important functions such as glucose transport and leads cocultured endothelial cells to form tubules. Coupling of these HIFalpha sequences to the HIV tat translocation domain allows delivery of recombinant peptide to cells with resultant induction of HIF-dependent genes. Injection of tat-HIF polypeptides in a murine sponge angiogenesis assay causes a markedly accelerated local angiogenic response and induction of glucose transporter-1 gene expression. These results demonstrate the feasibility of using these polypeptides to enhance HIF activity, opening additional therapeutic avenues for ischemic diseases.
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PMID:Peptide blockade of HIFalpha degradation modulates cellular metabolism and angiogenesis. 1214 54

The human cytotoxic T-lymphocyte (CTL) response to human immunodeficiency virus type 1 (HIV-1) has been intensely studied, and hundreds of CTL epitopes have been experimentally defined, published, and compiled in the HIV Molecular Immunology Database. Maps of CTL epitopes on HIV-1 protein sequences reveal that defined epitopes tend to cluster. Here we integrate the global sequence and immunology databases to systematically explore the relationship between HIV-1 amino acid sequences and CTL epitope distributions. CTL responses to five HIV-1 proteins, Gag p17, Gag p24, reverse transcriptase (RT), Env, and Nef, have been particularly well characterized in the literature to date. Through comparing CTL epitope distributions in these five proteins to global protein sequence alignments, we identified distinct characteristics of HIV amino acid sequences that correlate with CTL epitope localization. First, experimentally defined HIV CTL epitopes are concentrated in relatively conserved regions. Second, the highly variable regions that lack epitopes bear cumulative evidence of past immune escape that may make them relatively refractive to CTLs: a paucity of predicted proteasome processing sites and an enrichment for amino acids that do not serve as C-terminal anchor residues. Finally, CTL epitopes are more highly concentrated in alpha-helical regions of proteins. Based on amino acid sequence characteristics, in a blinded fashion, we predicted regions in HIV regulatory and accessory proteins that would be likely to contain CTL epitopes; these predictions were then validated by comparison to new sets of experimentally defined epitopes in HIV-1 Rev, Tat, Vif, and Vpr.
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PMID:Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation. 1216 96

Cancer cells frequently show high constitutive activity of the antiapoptotic transcription factor nuclear factor kappaB (NF-kappaB), which results in their enhanced survival. Activation of NF-kappaB classically depends on degradation of its inhibitor IkappaBalpha by the 26s proteasome. Specific proteasome inhibitors induce apoptosis in cancer cells and, at nonlethal concentrations, sensitize cells to the cytotoxic effects of ionizing radiation and chemotherapeutic drugs. Recently, the protease coded by the HIV-I virus has been shown to share cleavage activities with the proteasome. For this reason, we investigated whether the HIV-I protease inhibitor saquinavir can inhibit NF-kappaB activation, block 26s proteasome activity in prostate cancer cells, and promote their apoptosis. The effect of saquinavir on LPS/IFN-gamma-induced activation of NF-kappaB was assessed by gel-shift assays and by Western analysis of corresponding IkappaBalpha-levels. Its effect on 20s and 26s proteasome activity was analyzed with a fluorogenic peptide assay using whole cell lysates from LnCaP, DU-145, and PC-3 prostate cancer cells pretreated with saquinavir for 9 h. Proteasome inhibition in living cells was assessed using ECV 304 cells stably transfected with an expression plasmid for an ubiquitin/green fluorescence protein fusion protein (ECV 304/10). Apoptosis was monitored morphologically and by flow cytometry. Saquinavir treatment prevented LPS/IFN-gamma-induced activation of NF-kappaB in RAW cells and stabilized expression of IkappaBalpha. It inhibited 20s and 26s proteasome activity in lysates from LnCaP, DU-145, and PC-3 prostate cancer cells with an IC(50) of 10 micro M and caused the accumulation of an ubiquitin/green fluorescence protein fusion protein in living ECV 304/10 cells. Incubation of PC-3 and DU-145 prostate cancer, U373 glioblastoma, and K562 and Jurkat leukemia cells with saquinavir caused a concentration-dependent induction of apoptosis. In the case of PC-3 and DU-145, saquinavir sensitized the surviving cells to ionizing radiation. We conclude that saquinavir inhibits proteasome activity in mammalian cells as well as acting on the HIV-I protease. Because saquinavir induced apoptosis in human cancer cells, HIV-I protease inhibitors might become a new class of cytotoxic drugs, alone or in combination with radiation or chemotherapy.
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PMID:The human immunodeficiency virus (HIV)-1 protease inhibitor saquinavir inhibits proteasome function and causes apoptosis and radiosensitization in non-HIV-associated human cancer cells. 1223 89

Ritonavir is an HIV protease inhibitor used in the therapy of HIV infection. Ritonavir has also been shown to inhibit the chymotrypsin-like activity of isolated 20S proteasomes. Here, we demonstrate that ritonavir, like classical proteasome inhibitors, has antitumoral activities. In vitro, ritonavir strongly reduced the rate of proliferation of several tumor cell lines and induced their apoptosis. Nontransformed cell lines and terminally differentiated bone-marrow macrophages were comparatively resistant to the apoptosis-inducing effect. In vivo, ritonavir, administered p.o. for a week at doses of 6-8.8 mg/mouse/day, caused significant growth inhibition (76-79% after 7 days of treatment) of established EL4-T cell thymomas growing s.c. in syngeneic C57BL/6 mice. Unexpectedly, we found that ritonavir activates the chymotrypsin-like activity of isolated 26S proteasomes, in strong contrast to its effect on isolated 20S proteasomes. The net effect of low micromolar concentrations of ritonavir on the chymotrypsin-like activity in cells and cell lysates was a weak inhibition, consistent with marginal alterations of polyubiquitinated proteins, marginal alterations in acid-soluble proteolytic peptide levels, and a small accumulation of the tumor suppressor protein p53, in cells treated with ritonavir. In contrast, we found a relatively strong accumulation of the cyclin-dependent kinase inhibitor p21(WAF-1), a sign of deregulation of cell-cycle progression typical for apoptosis induction in transformed cells by classical proteasome inhibitors. We demonstrate that p21 accumulation in the presence of ritonavir is attributable to the inhibition of proteolytic degradation. Accumulation of p21 most likely reflects a selective inhibition of proteasomes, in line with the atypical degradation of p21, which does not require ubiquitination. These findings suggest that selective perturbation of proteasomal protein degradation may play a role in the antitumoral activities of ritonavir.
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PMID:Antitumor effect of the human immunodeficiency virus protease inhibitor ritonavir: induction of tumor-cell apoptosis associated with perturbation of proteasomal proteolysis. 1246 Sep 5

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