Introduction of H2C2-type zinc-binding residues into HIV-2 Vpr increases its expression level.
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ABSTRACT:
SUBMITTER: Koga R
PROVIDER: S-EPMC5757179 | biostudies-literature | 2018 Jan
REPOSITORIES: biostudies-literature
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Project description:HIV infection and its therapy are associated with disorders of lipid metabolism and bioenergetics. Previous work has suggested that viral protein R (Vpr) may contribute to the development of lipodystrophy and insulin resistance observed in HIV-1-infected patients. In adipocytes, Vpr suppresses mRNA expression of peroxisomal proliferator-activating receptor-γ (PPARγ)-responsive genes and inhibits differentiation. We investigated whether Vpr might interact with PPARβ/δ and influence its transcriptional activity. In the presence of PPARβ/δ, Vpr induced a 3.3-fold increase in PPAR response element-driven transcriptional activity, a 1.9-fold increase in pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and a 1.6-fold increase in the phosphorylated pyruvate dehydrogenase subunit E1α leading to a 47% decrease in the activity of the pyruvate dehydrogenase complex in HepG2 cells. PPARβ/δ knockdown attenuated Vpr-induced enhancement of endogenous PPARβ/δ-responsive PDK4 mRNA expression. Vpr induced a 1.3-fold increase in mRNA expression of both carnitine palmitoyltransferase I (CPT1) and acetyl-coenzyme A acyltransferase 2 (ACAA2) and doubled the activity of β-hydroxylacyl coenzyme A dehydrogenase (HADH). Vpr physically interacted with the ligand-binding domain of PPARβ/δ in vitro and in vivo. Consistent with a role in energy expenditure, Vpr increased state-3 respiration in isolated mitochondria (1.16-fold) and basal oxygen consumption rate in intact HepG2 cells (1.2-fold) in an etomoxir-sensitive manner, indicating that the oxygen consumption rate increase is β-oxidation-dependent. The effects of Vpr on PPAR response element activation, pyruvate dehydrogenase complex activity, and β-oxidation were reversed by specific PPARβ/δ antagonists. These results support the hypothesis that Vpr contributes to impaired energy metabolism and increased energy expenditure in HIV patients.
| S-EPMC3753422 | biostudies-literature
Project description:The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.
| S-EPMC6038386 | biostudies-literature
Project description:BackgroundThe hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr.Principal findingsExperiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain.ConclusionTogether our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.
| S-EPMC2967473 | biostudies-literature
Project description:The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.
| S-EPMC4100767 | biostudies-literature
Project description:BackgroundMetalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, for regulation of their activities or for structural purposes. Metal-binding properties remain difficult to predict as well as to investigate experimentally at the whole-proteome level. Consequently, the current knowledge about metalloproteins is only partial.ResultsThe present work reports on the development of a machine learning method for the prediction of the zinc-binding state of pairs of nearby amino-acids, using predictors based on support vector machines. The predictor was trained using chains containing zinc-binding sites and non-metalloproteins in order to provide positive and negative examples. Results based on strong non-redundancy tests prove that (1) zinc-binding residues can be predicted and (2) modelling the correlation between the binding state of nearby residues significantly improves performance. The trained predictor was then applied to the human proteome. The present results were in good agreement with the outcomes of previous, highly manually curated, efforts for the identification of human zinc-binding proteins. Some unprecedented zinc-binding sites could be identified, and were further validated through structural modelling. The software implementing the predictor is freely available at: http://zincfinder.dsi.unifi.itConclusionThe proposed approach constitutes a highly automated tool for the identification of metalloproteins, which provides results of comparable quality with respect to highly manually refined predictions. The ability to model correlations between pairwise residues allows it to obtain a significant improvement over standard 1D based approaches. In addition, the method permits the identification of unprecedented metal sites, providing important hints for the work of experimentalists.
| S-EPMC1800866 | biostudies-literature
Project description:Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
| S-EPMC4582045 | biostudies-literature
Project description:The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin ? interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54-74 within the C-terminal ?-helical domain (?H3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.
| S-EPMC4689350 | biostudies-literature
Project description:HIV-1 encodes four "accessory proteins" (Vif, Vpr, Vpu, and Nef), dispensable for viral replication in vitro but essential for viral pathogenesis in vivo. Well characterized cellular targets have been associated with Vif, Vpu, and Nef, which counteract host restriction and promote viral replication. Conversely, although several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for the DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.
| S-EPMC6506760 | biostudies-literature
Project description:BackgroundSince HIV-1 Tat and Vpr genes are involved in promoter transactivation, apoptosis, etc, we carried out studies to find out nature and extent of natural variation in the two genes from seropositive patients from Northern India and determined their functional implications.MethodsHIV-1 tat exon 1 and vpr were amplified from the genomic DNA isolated from the blood of HIV-1 infected individuals using specific primers by Polymerase Chain reaction (PCR) and subjected to extensive genetic analysis (CLUSTAL W, Simplot etc). Their expression was monitored by generating myc fusion clones. Tat exon 1 and Vpr variants were co-transfected with the reporter gene construct (LTR-luc) and their transactivation potential was monitored by measuring luciferase activity. Apoptosis and cell cycle analysis was done by Propidium Iodide (PI) staining followed by FACS.ResultsExon 1 of tat was amplified from 21 samples and vpr was amplified from 16 samples. One of the Tat exon 1 variants showed phylogenetic relatedness to subtype B & C and turned out to be a unique recombinant. Two of the Vpr variants were B/C/D recombinants. These natural variations were found to have no impact on the stability of Tat and Vpr. These variants differed in their ability to transactivate B LTR and C LTR promoters. B/C recombinant Tat showed better co-operative interaction with Vpr. B/C/D recombination in Vpr was found to have no effect on its co-operativity with Tat. Recombinant Tat (B/C) induced more apoptosis than wild type B and C Tat. The B/C/D recombination in Vpr did not affect its G2 arrest induction potential but reduced its apoptosis induction ability.ConclusionsExtensive sequence and region-specific variations were observed in Tat and Vpr genes from HIV-1 infected individuals from Northern India. These variations have functional implications & therefore important for the pathogenicity of virus.
| S-EPMC3868622 | biostudies-literature
Project description:Viruses target host proteins for degradation to enhance their replication and transmission, and identifying these targets has provided key insights into the host-pathogen interaction1-3. Here, we use complementary unbiased mass spectrometry-based approaches to dissect the widespread proteomeic remodelling seen in HIV-1 infected T-cells. Remarkably, the HIV accessory protein Vpr is both necessary and sufficient to cause the vast majority of these changes. Protein regulation requires recruitment of the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex, and pulsed-Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC) and immunoprecipitation-mass spectrometry (IP-MS) identified at least 38 cellular proteins directly targeted for degradation by Vpr. Whilst other HIV-1 accessory proteins downregulate a small number of specific host factors, Vpr depletes multiple protein targets, causing systems-level changes to the cellular proteome. A subset of the novel cellular targets identified in this study are depleted by Vpr variants from across HIV-1/SIVcpz and other primate lentiviral lineages, confirming their biological importance in vivo.
2019-05-01 | PXD013221 | Pride