KLF6 activates Sp1-mediated prolidase transcription during TGF-ß1 signaling.

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Emerging role of cyclophilin A in HIV-1 infection: from producer cell to the target cell nucleus.

The HIV-1 genome encodes a small number of proteins with structural, enzymatic, regulatory, and accessory functions. These viral proteins interact with a number of host factors to promote the early and late stages of HIV-1 infection. During the early stages of infection, interactions between the viral proteins and host factors enable HIV-1 to enter the target cell, traverse the cytosol, dock at the nuclear pore, gain access to the nucleus, and integrate into the host genome. Similarly, the viral proteins recruit another set of host factors during the late stages of infection to orchestrate HIV-1 transcription, translation, assembly, and release of progeny virions. Among the host factors implicated in HIV-1 infection, Cyclophilin A (CypA) was identified as the first host factor to be packaged within HIV-1 particles. It is now well established that CypA promotes HIV-1 infection by directly binding to the viral capsid. Mechanistic models to pinpoint CypA's role have spanned from an effect in the producer cell to the early steps of infection in the target cell. In this review, we will describe our understanding of the role(s) of CypA in HIV-1 infection, highlight the current knowledge gaps, and discuss the potential role of this host factor in the post-nuclear entry steps of HIV-1 infection.

HIV-1 Preintegration Complex Preferentially Integrates the Viral DNA into Nucleosomes Containing Trimethylated Histone 3-Lysine 36 Modification and Flanking Linker DNA.

HIV-1 DNA is preferentially integrated into chromosomal hot spots by the preintegration complex (PIC). To understand the mechanism, we measured the DNA integration activity of PICs-extracted from infected cells-and intasomes, biochemically assembled PIC substructures using a number of relevant target substrates. We observed that PIC-mediated integration into human chromatin is preferred compared to genomic DNA. Surprisingly, nucleosomes lacking histone modifications were not preferred integration compared to the analogous naked DNA. Nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to active transcription, significantly stimulated integration, but the levels remained lower than the naked DNA. Notably, H3K36me3-modified nucleosomes with linker DNA optimally supported integration mediated by the PIC but not by the intasome. Interestingly, optimal intasome-mediated integration required the cellular cofactor LEDGF. Unexpectedly, LEDGF minimally affected PIC-mediated integration into naked DNA but blocked integration into nucleosomes. The block for the PIC-mediated integration was significantly relieved by H3K36me3 modification. Mapping the integration sites in the preferred substrates revealed that specific features of the nucleosome-bound DNA are preferred for integration, whereas integration into naked DNA was random. Finally, biochemical and genetic studies demonstrate that DNA condensation by the H1 protein dramatically reduces integration, providing further evidence that features inherent to the open chromatin are preferred for HIV-1 integration. Collectively, these results identify the optimal target substrate for HIV-1 integration, report a mechanistic link between H3K36me3 and integration preference, and importantly, reveal distinct mechanisms utilized by the PIC for integration compared to the intasomes. IMPORTANCE HIV-1 infection is dependent on integration of the viral DNA into the host chromosomes. The preintegration complex (PIC) containing the viral DNA, the virally encoded integrase (IN) enzyme, and other viral/host factors carries out HIV-1 integration. HIV-1 integration is not dependent on the target DNA sequence, and yet the viral DNA is selectively inserted into specific "hot spots" of human chromosomes. A growing body of literature indicates that structural features of the human chromatin are important for integration targeting. However, the mechanisms that guide the PIC and enable insertion of the PIC-associated viral DNA into specific hot spots of the human chromosomes are not fully understood. In this study, we describe a biochemical mechanism for the preference of the HIV-1 DNA integration into open chromatin. Furthermore, our study defines a direct role for the histone epigenetic mark H3K36me3 in HIV-1 integration preference and identify an optimal substrate for HIV-1 PIC-mediated viral DNA integration.

Human Three Prime Repair Exonuclease 1 Promotes HIV-1 Integration by Preferentially Degrading Unprocessed Viral DNA.

Three prime repair exonuclease 1 (TREX1) is the most abundant 3'→5' exonuclease in mammalian cells. It has been suggested that TREX1 degrades HIV-1 DNA to enable the virus to evade the innate immune system. However, the exact role of TREX1 during early steps of HIV-1 infection is not clearly understood. In this study, we report that HIV-1 infection is associated with upregulation, perinuclear accumulation, and nuclear localization of TREX1. However, TREX1 overexpression did not affect reverse transcription or nuclear entry of the virus. Surprisingly, HIV-1 DNA integration was increased in TREX1-overexpressing cells, suggesting a role of the exonuclease in the post-nuclear entry step of infection. Accordingly, preintegration complexes (PICs) extracted from TREX1-overexpressing cells retained higher levels of DNA integration activity. TREX1 depletion resulted in reduced levels of proviral integration, and PICs formed in TREX1-depleted cells retained lower DNA integration activity. Addition of purified TREX1 to PICs also enhanced DNA integration activity, suggesting that TREX1 promotes HIV-1 integration by stimulating PIC activity. To understand the mechanism, we measured TREX1 exonuclease activity on substrates containing viral DNA ends. These studies revealed that TREX1 preferentially degrades the unprocessed viral DNA, but the integration-competent 3'-processed viral DNA remains resistant to degradation. Finally, we observed that TREX1 addition stimulates the activity of HIV-1 intasomes assembled with the unprocessed viral DNA but not that of intasomes containing the 3'-processed viral DNA. These biochemical analyses provide a mechanism by which TREX1 directly promotes HIV-1 integration. Collectively, our study demonstrates that HIV-1 infection upregulates TREX1 to facilitate viral DNA integration. IMPORTANCE Productive HIV-1 infection is dependent on a number of cellular factors. Therefore, a clear understanding of how the virus exploits the cellular machinery will identify new targets for inhibiting HIV-1 infection. The three prime repair exonuclease 1 (TREX1) is the most active cellular exonuclease in mammalian cells. It has been reported that TREX1 prevents accumulation of HIV-1 DNA and enables the virus to evade the host innate immune response. Here, we show that HIV-1 infection results in the upregulation, perinuclear accumulation, and nuclear localization of TREX1. We also provide evidence that TREX1 promotes HIV-1 integration by preferentially degrading viral DNAs that are incompatible with chromosomal insertion. These observations identify a novel role of TREX1 in a post-nuclear entry step of HIV-1 infection.

Proteomics Profiling of Autologous Blood and Semen Exosomes from HIV-infected and Uninfected Individuals Reveals Compositional and Functional Variabilities.

Blood and semen are important body-fluids that carry exosomes for bioinformation transmission. Therefore, characterization of their proteomes is necessary for understanding body-fluid-specific physiologic and pathophysiologic functions. Using systematic multifactorial proteomic profiling, we characterized the proteomes of exosomes and exosome-free fractions from autologous blood and semen from three HIV-uninfected and three HIV-infected participants (total of 24 samples). We identified exosome-based protein signatures specific to blood and semen along with HIV-induced tissue-dependent proteomic perturbations. We validated our findings with samples from 16 additional donors and showed that unlike blood exosomes (BE), semen exosomes (SE) are enriched in clusterin. SE but not BE promote Protein·Nucleic acid binding and increase cell adhesion irrespective of HIV infection. This is the first comparative study of the proteome of autologous BE and SE. The proteins identified may be developed as biomarkers applicable to different fields of medicine, including reproduction and infectious diseases.

The HIV-1 capsid-binding host factor CPSF6 is post-transcriptionally regulated by the cellular microRNA miR-125b.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in mRNA processing. Emerging evidence suggests that CPSF6 also plays key roles in HIV-1 infection, specifically during nuclear import and integration targeting. However, the cellular and molecular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post-transcriptional mechanism that regulates CPSF6 via the cellular microRNA miR-125b. An in silico analysis revealed that the 3'UTR of CPSF6 contains a miR-125b-binding site that is conserved across several mammalian species. Because miRNAs repress protein expression, we tested the effects of miR-125b expression on CPSF6 levels in miR-125b knockdown and over-expression experiments, revealing that miR-125b and CPSF6 levels are inversely correlated. To determine whether miR-125b post-transcriptionally regulates CPSF6, we introduced the 3'UTR of CPSF6 mRNA into a luciferase reporter and found that miR-125b negatively regulates CPSF6 3'UTR-driven luciferase activity. Accordingly, mutations in the miR-125b seed sequence abrogated the regulatory effect of the miRNA on the CPSF6 3'UTR. Finally, pulldown experiments demonstrated that miR-125b physically interacts with CPSF6 3'UTR. Interestingly, HIV-1 infection down-regulated miR-125b expression concurrent with up-regulation of CPSF6. Notably, miR-125b down-regulation in infected cells was not due to reduced pri-miRNA or pre-miRNA levels. However, miR-125b down-regulation depended on HIV-1 reverse transcription but not viral DNA integration. These findings establish a post-transcriptional mechanism that controls CPSF6 expression and highlight a novel function of miR-125b during HIV-host interaction.

Activation of proline metabolism maintains ATP levels during cocaine-induced polyADP-ribosylation.

Cocaine is a commonly abused drug worldwide. Acute as well as repeated exposure to cocaine activates persistent cellular and molecular changes in the brain reward regions. The effects of cocaine are predominantly mediated via alterations in neuronal gene expression by chromatin remodeling. Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation of chromatin has been reported as an important regulator of cocaine-mediated gene expression. PARP-1 dependent ADP-ribosylation is an energy-dependent process. In this study, we investigated the cellular energy response to cocaine-induced upregulation of PARP-1 expression. Exposure of differentiated SH-SY5Y cells to varying concentrations of cocaine resulted in the induction of PARP-1 dependent PARylation of p53 tumor suppressor. Further analysis revealed that PARylation of p53 by cocaine treatment resulted in nuclear accumulation of p53. However, induction and nuclear accumulation of p53 did not correlate with neuronal apoptosis/cell death upon cocaine exposure. Interestingly, cocaine-induced p53 PARylation resulted in the induction of proline oxidase (POX)-a p53 responsive gene involved in cellular metabolism. Given that cocaine-induced p53 PARylation is an energy-dependent process, we observed that cocaine-induced PARP-1/p53/POX axes alters cellular energy metabolism. Accordingly, using pharmacological and genetic studies of PARP-1, p53, and POX, we demonstrated the contribution of POX in maintaining cellular energy during neuronal function. Collectively, these studies highlight activation of a novel metabolic pathway in response to cocaine treatment.

PF74 Inhibits HIV-1 Integration by Altering the Composition of the Preintegration Complex.

The HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA's role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74's inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74's effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74's targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCE Antiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

A Novel Role of Proline Oxidase in HIV-1 Envelope Glycoprotein-induced Neuronal Autophagy.

Proline oxidase (POX) catalytically converts proline to pyrroline-5-carboxylate. This catabolic conversion generates reactive oxygen species (ROS) that triggers cellular signaling cascades including autophagy and apoptosis. This study for the first time demonstrates a role of POX in HIV-1 envelope glycoprotein (gp120)-induced neuronal autophagy. HIV-1 gp120 is a neurotoxic factor and is involved in HIV-1-associated neurological disorders. However, the mechanism of gp120-mediated neurotoxicity remains unclear. Using SH-SY5Y neuroblastoma cells as a model, this study demonstrates that gp120 treatment induced POX expression and catalytic activity. Concurrently, gp120 also increased intracellular ROS levels. However, increased ROS had a minimal effect on neuronal apoptosis. Further investigation indicated that the immediate cellular response to increased ROS paralleled with induction of autophagy markers, beclin-1 and LC3-II. These data lead to the hypothesis that neuronal autophagy is activated as a cellular protective response to the toxic effects of gp120. A direct and functional role of POX in gp120-mediated neuronal autophagy was examined by inhibition and overexpression studies. Inhibition of POX activity by a competitive inhibitor "dehydroproline" decreased ROS levels concomitant with reduced neuronal autophagy. Conversely, overexpression of POX in neuronal cells increased ROS levels and activated ROS-dependent autophagy. Mechanistic studies suggest that gp120 induces POX by targeting p53. Luciferase reporter assays confirm that p53 drives POX transcription. Furthermore, data demonstrate that gp120 induces p53 via binding to the CXCR4 co-receptor. Collectively, these results demonstrate a novel role of POX as a stress response metabolic regulator in HIV-1 gp120-associated neuronal autophagy.

Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 µmol/L. However, at concentrations >100 µmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.

Cocaine enhances HIV-1-induced CD4(+) T-cell apoptosis: implications in disease progression in cocaine-abusing HIV-1 patients.

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 µmol/L concentrations) did not induce apoptosis, but 200 to 1000 µmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.

Cyclophilin A Facilitates HIV-1 DNA Integration.

Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5α. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA +/+ ) and CypA-depleted (CypA -/- ) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypA's effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5α. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA +/+ cells but not in CypA -/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA +/+ cells but not in CypA -/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA -/- cells had lower activity in vitro compared to those from CypA +/+ cells. PICs from cells depleted for CypA and TRIM5α also had lower activity, suggesting that CypA's effect on PIC activity is independent of TRIM5α. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA +/+ and CypA -/- cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. Importance: HIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3'-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3'-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

HIV-1 mutants that escape the cytotoxic T-lymphocytes are defective in viral DNA integration.

HIV-1 replication is durably controlled without antiretroviral therapy (ART) in certain infected individuals called elite controllers (ECs). These individuals express specific human leukocyte antigens (HLA) that tag HIV-infected cells for elimination by presenting viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In HIV-infected individuals expressing HLA-B27, CTLs primarily target the viral capsid protein (CA)-derived KK10 epitope. While selection of CA mutation R264K helps HIV-1 escape this potent CTL response, the accompanying fitness cost severely diminishes virus infectivity. Interestingly, selection of a compensatory CA mutation S173A restores HIV-1 replication. However, the molecular mechanism(s) underlying HIV-1 escape from this ART-free virus control by CTLs is not fully understood. Here, we report that the R264K mutation-associated infectivity defect arises primarily from impaired HIV-1 DNA integration, which is restored by the S173A mutation. Unexpectedly, the integration defect of the R264K variant was also restored upon depletion of the host cyclophilin A. These findings reveal a nuclear crosstalk between CA and HIV-1 integration as well as identify a previously unknown role of cyclophilin A in viral DNA integration. Finally, our study identifies a novel immune escape mechanism of an HIV-1 variant escaping a CA-directed CTL response.

Inhibition of multi-drug resistant HIV-1 reverse transcriptase by nucleoside ß-triphosphates.

Despite the success of potent reverse transcriptase (RT) inhibitors against human immunodeficiency virus type 1 (HIV-1) in combination regimens, the development of drug resistant RTs constitutes a major hurdle for the long-term efficacy of current antiretroviral therapy. Nucleoside ß-triphosphate analogs of adenosine and nucleoside reverse transcriptase inhibitors (NRTIs) (3'-azido-2',3'-dideoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine (FLT), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) were synthesized and their inhibitory activities were evaluated against wild-type and multidrug resistant HIV-1 RTs. Adenosine ß-triphosphate (1) and AZT ß-triphosphate (2) completely inhibited the DNA polymerase activity of wild type, the NRTI multi resistant, and nonnucleoside RT inhibitors (NNRTI) resistant HIV-1 RT at 10nM, 10 and 100 µM, respectively.

Synthesis and anti-HIV activities of bis-(cycloSaligenyl) pronucleotides derivatives of 3'-fluoro-3'-deoxythymidine and 3'-azido-3'-deoxythymidine.

Anti-HIV nucleoside monophosphates have limited cellular uptake due to the presence of negatively-charged phosphate group. Bis-(cycloSaligenyl) derivatives containing two anti-HIV nucleosides, 3'-fluoro-3'-deoxythymidine (FLT) and 3'-azido-3'-deoxythymidine (AZT) were synthesized to increase intracellular delivery of nucleoside monophosphates. 2,5-Bis(hydroxymethylene)benzene-1,4-diol was selected as a monocyclic bidentate scaffold and synthesized by three different methods from bis(hydroxymethylene)cyclohexan-1,4-diene-1,4-diol, or diethyl 2,5-dihydroxyterephthalate. The reaction of the tetraol with diisopropylphosphoramidous dichloride in the presence of 2,6-lutidine, followed by conjugation reactions with nucleosides (i.e., FLT and AZT) and oxidation afforded symmetrical and unsymmetrical bis-(cycloSaligenyl) diphosphate triester products, AZT-AZT, FLT-FLT, and FLT-AZT conjugates, in 63-74% overall yields and modest anti-HIV activities (IC50 = 2.8-69.6 µM).

Therapeutic Significance of microRNA-Mediated Regulation of PARP-1 in SARS-CoV-2 Infection.

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 (2019-nCoV) has devastated global healthcare and economies. Despite the stabilization of infectivity rates in some developed nations, several countries are still under the grip of the pathogenic viral mutants that are causing a significant increase in infections and hospitalization. Given this urgency, targeting of key host factors regulating SARS-CoV-2 life cycle is postulated as a novel strategy to counter the virus and its associated pathological outcomes. In this regard, Poly (ADP)-ribose polymerase-1 (PARP-1) is being increasingly recognized as a possible target. PARP-1 is well studied in human diseases such as cancer, central nervous system (CNS) disorders and pathology of RNA viruses. Emerging evidence indicates that regulation of PARP-1 by non-coding RNAs such as microRNAs is integral to cell survival, redox balance, DNA damage response, energy homeostasis, and several other cellular processes. In this short perspective, we summarize the recent findings on the microRNA/PARP-1 axis and its therapeutic potential for COVID-19 pathologies.

PROLIDASE: A Review from Discovery to its Role in Health and Disease.

Prolidase (peptidase D), encoded by the PEPD gene, is a ubiquitously expressed cytosolic metalloproteinase, the only enzyme capable of cleaving imidodipeptides containing C-terminal proline or hydroxyproline. Prolidase catalyzes the rate-limiting step during collagen recycling and is essential in protein metabolism, collagen turnover, and matrix remodeling. Prolidase, therefore plays a crucial role in several physiological processes such as wound healing, inflammation, angiogenesis, cell proliferation, and carcinogenesis. Accordingly, mutations leading to loss of prolidase catalytic activity result in prolidase deficiency a rare autosomal recessive metabolic disorder characterized by defective wound healing. In addition, alterations in prolidase enzyme activity have been documented in numerous pathological conditions, making prolidase a useful biochemical marker to measure disease severity. Furthermore, recent studies underscore the importance of a non-enzymatic role of prolidase in cell regulation and infectious disease. This review aims to provide comprehensive information on prolidase, from its discovery to its role in health and disease, while addressing the current knowledge gaps.

Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure.

Methamphetamine (METH) is a highly addictive psychostimulant that causes long-lasting effects in the brain and increases the risk of developing neurodegenerative diseases. The cellular and molecular effects of METH in the brain are functionally linked to alterations in glutamate levels. Despite the well-documented effects of METH on glutamate neurotransmission, the underlying mechanism by which METH alters glutamate levels is not clearly understood. In this study, we report an essential role of proline biosynthesis in maintaining METH-induced glutamate homeostasis. We observed that acute METH exposure resulted in the induction of proline biosynthetic enzymes in both undifferentiated and differentiated neuronal cells. Proline level was also increased in these cells after METH exposure. Surprisingly, METH treatment did not increase glutamate levels nor caused neuronal excitotoxicity. However, METH exposure resulted in a significant upregulation of pyrroline-5-carboxylate synthase (P5CS), the key enzyme that catalyzes synthesis of proline from glutamate. Interestingly, depletion of P5CS by CRISPR/Cas9 resulted in a significant increase in glutamate levels upon METH exposure. METH exposure also increased glutamate levels in P5CS-deficient proline-auxotropic cells. Conversely, restoration of P5CS expression in P5CS-deficient cells abrogated the effect of METH on glutamate levels. Consistent with these findings, P5CS expression was significantly enhanced in the cortical brain region of mice administered with METH and in the slices of cortical brain tissues treated with METH. Collectively, these results uncover a key role of P5CS for the molecular effects of METH and highlight that excess glutamate can be sequestered for proline biosynthesis as a protective mechanism to maintain glutamate homeostasis during drug exposure.

Bortezomib Sustains T Cell Function by Inducing miR-155-Mediated Downregulation of SOCS1 and SHIP1.

Suppressive mechanisms operating within T cells are linked to immune dysfunction in the tumor microenvironment. We have previously reported using adoptive T cell immunotherapy models that tumor-bearing mice treated with a regimen of proteasome inhibitor, bortezomib - a dipeptidyl boronate, show increased antitumor lymphocyte effector function and survival. Here, we identify a mechanism for the improved antitumor CD8+ T cell function following bortezomib treatment. Intravenous administration of bortezomib at a low dose (1 mg/kg body weight) in wild-type or tumor-bearing mice altered the expression of a number of miRNAs in CD8+ T cells. Specifically, the effect of bortezomib was prominent on miR-155 - a key cellular miRNA involved in T cell function. Importantly, bortezomib-induced upregulation of miR-155 was associated with the downregulation of its targets, the suppressor of cytokine signaling 1 (SOCS1) and inositol polyphosphate-5-phosphatase (SHIP1). Genetic and biochemical analysis confirmed a functional link between miR-155 and these targets. Moreover, activated CD8+ T cells treated with bortezomib exhibited a significant reduction in programmed cell death-1 (PD-1) expressing SHIP1+ phenotype. These data underscore a mechanism of action by which bortezomib induces miR-155-dependent downregulation of SOCS1 and SHIP1 negative regulatory proteins, leading to a suppressed PD-1-mediated T cell exhaustion. Collectively, data provide novel molecular insights into bortezomib-mediated lymphocyte-stimulatory effects that could overcome immunosuppressive actions of tumor on antitumor T cell functions. The findings support the approach that bortezomib combined with other immunotherapies would lead to improved therapeutic outcomes by overcoming T cell exhaustion in the tumor microenvironment.