Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related ...to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.
•Macrophages could serve as long-lived compartments for HIV-1 infection.•Numerous host factors can modulate the life cycle of HIV-1.•Thymidylate synthase can be seen as a regulator of HIV-1 replication in human macrophages.
Metabolic reprogramming occurs in cancer cells and is regulated partly by the opposing actions of tyrosine kinases and tyrosine phosphatases. Several members of the protein tyrosine phosphatase (PTP) ...superfamily have been linked to cancer as either pro‐oncogenic or tumor‐suppressive enzymes. In order to investigate which PTPs can modulate the metabolic state of cancer cells, we performed an shRNA screen of PTPs in HCT116 human colorectal cancer cells. Among the 72 PTPs efficiently targeted, 24 were found to regulate mitochondrial respiration, 8 as negative and 16 as positive regulators. Of the latter, we selected TC‐PTP (PTPN2) for further characterization since inhibition of this PTP resulted in major functional defects in oxidative metabolism without affecting glycolytic flux. Transmission electron microscopy revealed an increase in the number of damaged mitochondria in TC‐PTP‐null cells, demonstrating the potential role of this PTP in regulating mitochondrial homeostasis. Downregulation of STAT3 by siRNA‐mediated silencing partially rescued the mitochondrial respiration defect observed in TC‐PTP‐deficient cells, supporting the role of this signaling axis in regulating mitochondrial activity. In addition, mitochondrial stress prevented an increased expression of electron transport chain‐related genes in cells with TC‐PTP silencing, correlating with decreased ATP production, cellular proliferation, and migration. Our shRNA‐based metabolic screen revealed that PTPs can serve as either positive or negative regulators of cancer cell metabolism. Taken together, our findings uncover a new role for TC‐PTP as an activator of mitochondrial metabolism, validating this PTP as a key target for cancer therapeutics.
The “shock and kill” HIV‐1 cure strategy proposes eradication of stable cellular reservoirs by clinical treatment with latency‐reversing agents (LRAs). Although resting CD4+ T cells latently infected ...with HIV‐1 constitute the main reservoir that is targeted by these approaches, their consequences on other reservoirs such as the central nervous system are still unknown and should be taken into consideration. We performed experiments aimed at defining the possible role of astrocytes in HIV‐1 persistence in the brain and the effect of LRA treatments on this viral sanctuary. We first demonstrate that the diminished HIV‐1 production in a proliferating astrocyte culture is due to a reduced proliferative capacity of virus‐infected cells compared with uninfected astrocytes. In contrast, infection of non‐proliferating astrocytes led to a robust HIV‐1 infection that was sustained for over 60 days. To identify astrocytes latently infected with HIV‐1, we designed a new dual‐color reporter virus called NL4.3 eGFP‐IRES‐Crimson that is fully infectious and encodes for all viral proteins. Although we detected a small fraction of astrocytes carrying silent HIV‐1 proviruses, we did not observe any reactivation using various LRAs and even strong inducers such as tumor necrosis factor, thus suggesting that these proviruses were either not transcriptionally competent or in a state of deep latency. Our findings imply that astrocytes might not constitute a latent reservoir per se but that relentless virus production by this brain cell population could contribute to the neurological disorders seen in HIV‐1‐infected persons subjected to combination antiretroviral therapy.
Main Points
Latency reversing agents (LRA) and their effect on productive and latent/defective HIV‐1 infection of human astrocytes.
Phosphatases of regenerating liver (PRL-1, PRL-2, and PRL-3, also known as PTP4A1, PTP4A2, and PTP4A3) control magnesium homeostasis through an association with the CNNM magnesium transport ...regulators. Although high PRL levels have been linked to cancer progression, regulation of their expression is poorly understood. Here we show that modulating intracellular magnesium levels correlates with a rapid change of PRL expression by a mechanism involving its 5′UTR mRNA region. Mutations or CRISPR-Cas9 targeting of the conserved upstream ORF present in the mRNA leader derepress PRL protein synthesis and attenuate the translational response to magnesium levels. Mechanistically, magnesium depletion reduces intracellular ATP but up-regulates PRL protein expression via activation of the AMPK/mTORC2 pathway, which controls cellular energy status. Hence, altered PRL-2 expression leads to metabolic reprogramming of the cells. These findings uncover a magnesium-sensitive mechanism controlling PRL expression, which plays a role in cellular bioenergetics.
HIV-1 is extremely specialized since, even amongst CD4(+) T lymphocytes (its major natural reservoir in peripheral blood), the virus productively infects only a small proportion of cells under an ...activated state. As the percentage of HIV-1-infected cells is very low, most studies have so far failed to capture the precise transcriptomic profile at the whole-genome scale of cells highly susceptible to virus infection. Using Affymetrix Exon array technology and a reporter virus allowing the magnetic isolation of HIV-1-infected cells, we describe the host cell factors most favorable for virus establishment and replication along with an overview of virus-induced changes in host gene expression occurring exclusively in target cells productively infected with HIV-1. We also establish that within a population of activated CD4(+) T cells, HIV-1 has no detectable effect on the transcriptome of uninfected bystander cells at early time points following infection. The data gathered in this study provides unique insights into the biology of HIV-1-infected CD4(+) T cells and identifies genes thought to play a determinant role in the interplay between the virus and its host. Furthermore, it provides the first catalogue of alternative splicing events found in primary human CD4(+) T cells productively infected with HIV-1.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein tyrosine phosphatases (PTPs) play a critical role in co-ordinating the signaling networks that maintain lymphocyte homeostasis and direct lymphocyte activation. By dephosphorylating tyrosine ...residues, PTPs have been shown to modulate enzyme activity and both mediate and disrupt protein-protein interactions. Through these molecular mechanisms, PTPs ultimately impact lymphocyte responses to environmental cues such as inflammatory cytokines and chemokines, as well as antigenic stimulation. Mouse models of acute and chronic intestinal inflammation have been shown to be exacerbated in the absence of PTPs such as PTPN2 and PTPN22. This increase in disease severity is due in part to hyper-activation of lymphocytes in the absence of PTP activity. In accordance, human PTPs have been linked to intestinal inflammation. Genome wide association studies (GWAS) identified several PTPs within risk loci for inflammatory bowel disease (IBD). Therapeutically targeting PTP substrates and their associated signaling pathways, such as those implicated in CD4
T cell responses, has demonstrated clinical efficacy. The current review focuses on the role of PTPs in controlling CD4
T cell activity in the intestinal mucosa and how disruption of PTP activity in CD4
T cells can contribute to intestinal inflammation.
Receptor tyrosine kinases (RTKs) control the cellular response to a range of stimuli by binding extracellular factors and transmitting appropriate signals to intracellular sites. Protein tyrosine ...phosphatase 1B (PTP1B) modulates the activity of several RTKs by directly targeting the phosphorylated tyrosine residues that dictate their signaling output. Interestingly, the phenotypes of PTP1B deficiency in different contexts point to a more complex role in regulating RTK signaling. Exciting recent results indicate that the endocytic down-regulation of RTKs could be directly controlled by PTP1B. Microscopy studies have demonstrated an effect of PTP1B on post-endocytic internalization of RTKs into multivesicular bodies, and specific substrates that could influence their endosomal trafficking have been identified. These findings reveal a novel link between two important mechanisms of RTK signal attenuation and highlight the multifaceted impact of PTP1B on cell signaling.
Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the ...degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.
The molecular basis for HIV-1 susceptibility in primary human monocyte-derived macrophages (MDMs) was previously evaluated by comparing the transcriptome of infected and bystander populations. ...Careful analysis of the data suggested that the ubiquitin ligase MDM2 acted as a positive regulator of HIV-1 replication in MDMs. In this study, MDM2 silencing through transcript-specific small interfering RNAs in MDMs induced a reduction in HIV-1 reverse transcription and integration along with an increase in the expression of p53-induced genes, including
Experiments with Nutlin-3, a pharmacological inhibitor of MDM2 p53-binding activity, showed a similar effect on HIV-1 infection, suggesting that the observed restriction in HIV-1 production results from the release/activation of p53 and not the absence of MDM2
Knockdown and inhibition of MDM2 also both correlate with a decrease in the Thr592-phosphorylated inactive form of SAMHD1. The expression level of MDM2 and the p53 activation status are therefore important factors in the overall susceptibility of macrophages to HIV-1 infection, bringing a new understanding of signaling events controlling the process of virus replication in this cell type.
Macrophages, with their long life span
and their resistance to HIV-1-mediated cytopathic effect, might serve as viral reservoirs, contributing to virus persistence in an infected individual. Identification of host factors that increase the overall susceptibility of macrophages to HIV-1 might provide new therapeutic targets for the efficient control of viral replication in these cells and limit the formation of reservoirs in exposed individuals. In this study, we demonstrate the importance of p53 regulation by MDM2, which creates a cellular environment more favorable to the early steps of HIV-1 replication. Moreover, we show that p53 stabilization reduces virus infection in human macrophages, highlighting the important role of p53 in antiviral immunity.
Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain ...incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-β, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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