HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages.

We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation.

The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages.

Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS.     

Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.     

Discovery of a novel class of biphenyl pyrazole sodium channel blockers for treatment of neuropathic pain

A series of novel biphenyl pyrazole dicarboxamides were identified as potential sodium channel blockers for treatment of neuropathic pain. Compound 20 had outstanding efficacy in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain.     

Regulation of Morphogenesis and Biocontrol Properties in Trichoderma virens by a VELVET Protein,Vel1

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.Trichoderma-based formulation products account for about 60% of the biofungicide market (35). Despite the use of Trichoderma-based biofungicides as an alternative and additive to chemical fungicides, the applications of these preparations are limited because their efficacy is lower than that of fungicides. A lack of understanding of the regulation of biocontrol has limited progress in enhancing the competitiveness of these fungi through genetic manipulation of desired traits. The success of a biocontrol agent also depends on the ability of researchers to develop an effective formulation based on active propagules that survive under the conditions that occur in nature and are effective against the target pathogens. Trichoderma spp. produce two types of propagules, conidia during solid-state fermentation and chlamydospores during liquid fermentation. Both types are used in commercial formulations depending on the growth conditions (17, 35). Thus, understanding how the two sporulation pathways are controlled is critical for obtaining an improved, balanced formulation product. Identification of a global regulator of morphogenesis and biocontrol properties (such as antibiosis and mycoparasitism) would provide an opportunity to manipulate the morphogenetic and antagonistic traits, leading to wider commercial acceptance of Trichoderma spp. in the long run.Trichoderma virens is a commercially formulated biocontrol agent that is effective against soilborne plant pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp.; its major direct mode of action is antibiosis and mycoparasitism (20, 36). This species has also been used as a model system for studies of biocontrol mechanisms, and the genome has recently been sequenced (http://genome.jgi-psf.org/Trive1). The role of beta-glucanases, chitinases, and proteases in biocontrol has been reported previously (2, 8, 29). Some strains of T. virens (designated Q strains) produce copious amounts of the antibiotic gliotoxin that is involved in biocontrol (10, 12, 39). In an attempt to identify regulators of biocontrol properties, the role of a mitogen-activated protein kinase (MAPK) pathway was studied previously (22, 24). Deletion of the TmkA/Tvk1 MAPK gene resulted in derepressed conidiation and different biocontrol behavior for two strains of T. virens; Mukherjee et al. (24) noted the reduced ability of these mutants to parasitize the sclerotia of S. rolfsii and R. solani, while Mendoza-Mendoza et al. (22) found that deletion of this MAPK gene improved the biocontrol activity of T. virens against R solani and P. ultimum. The production of secondary metabolites was not affected by deletion of this gene. To date, no gene that regulates the balance between conidiation or chlamydospore formation, secondary metabolism, and antagonistic or biocontrol properties has been identified in any Trichoderma sp.The Vel1 VELVET protein has been shown to be a regulator of morphogenesis and secondary metabolism in some filamentous fungi (6). In Aspergillus nidulans, VeA physically interacts with VelB and the regulator of secondary metabolism LaeA to form a complex that regulates secondary metabolism and sexual reproduction (3). Deletion of the VeA gene leads to an increase in asexual development (conidiation in the dark) and reduced biosynthesis of sterigmatocystin (the product of a polyketide synthetase [PKS]) and penicillin (the product of a nonribosomal peptide synthetase [NRPS]), while it reduces and delays sexual reproduction (15, 16). VeA is also required for the production of sclerotia and for aflatoxin biosynthesis in Aspergillus parasiticus (7). Deletion of the VeA gene in Neurospora crassa, like deletion of the VeA gene in A. nidulans, results in deregulated conidiation, while in Acremonium chrysogenum, loss of VeA leads to increased hyphal fragmentation and reduced cephalosporin production (4, 9). Deletion of the VeA gene in Fusarium verticilliodes resulted in a loss of hydrophobicity and an increased macroconidium-to-microconidium ratio; these defects could be restored by growing the organism on osmotically stabilized media (18). The mutants were also defective in production of the mycotoxins fumonisin and fusarin (25).To test the hypothesis that Vel1 is a global regulator of gene expression in T. virens, we examined the functions of Vel1 in this organism by using gene knockout and complementation. Here we report that in addition to a role in conidiation and secondary metabolism, Vel1 also regulates conidiophore aggregation, chlamydosporogenesis, mycoparasitism, and biocontrol efficacy in T. virens. Thus, we identified the first master regulator of morphogenesis and antagonistic properties in this economically important fungus.     

Quantification of in vitro malodor generation by anionic surfactant-induced fluorescent sensor property of tryptophan

In this article, we report tuning of the sensory capability of an amino acid (tryptophan) in a biomimicking anionic micellar nano cage. It has been shown that anionic surfactant concentration dictates the sensing behavior of tryptophan toward body malodor component (butyric acid) generated by bacterial degradation of tributyrin. We have proposed a fluorescence quenching mechanism that is based on short-chain fatty acid (SCFA) proximity with tryptophan present at the micelle-water interface. Anionic surfactant-induced fluorescent sensor activity of tryptophan exhibits high sensitivity (detection limit up to 10 μM) and specific selectivity (toward SCFA, < C12) in aqueous solution. We also determined antibacterial efficacy of various zinc salts based on the sensory activity of tryptophan, which has been correlated with the established resazurin assay.     

Challenges and Opportunities for Customizing Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) as an alternative to synthetic plastics have been gaining increasing attention. Being natural in their origin, PHAs are completely biodegradable and eco-friendly. However, consistent efforts to exploit this biopolymer over the last few decades have not been able to pull PHAs out of their nascent stage, inspite of being the favorite of the commercial world. The major limitations are: (1) the high production cost, which is due to the high cost of the feed and (2) poor thermal and mechanical properties of polyhydroxybutyrate (PHB), the most commonly produced PHAs. PHAs have the physicochemical properties which are quite comparable to petroleum based plastics, but PHB being homopolymers are quite brittle, less elastic and have thermal properties which are not suitable for processing them into sturdy products. These properties, including melting point (T_m), glass transition temperature (T_g), elastic modulus, tensile strength, elongation etc. can be improved by varying the monomeric composition and molecular weight. These enhanced characteristics can be achieved by modifications in the types of substrates, feeding strategies, culture conditions and/or genetic manipulations.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

Interleukin-19 (IL-19) induces heme oxygenase-1 (HO-1) expression and decreases reactive oxygen species in human vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.