Type 2 transglutaminase in the nucleus: the new epigenetic face of a cytoplasmic enzyme.

One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Interaction between AIF and CHCHD4 Regulates Respiratory Chain Biogenesis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.

Proteomic analysis identifies a signature of disease severity in the plasma of COVID-19 pneumonia patients associated to neutrophil, platelet and complement activation.

Most patients infected with SARS-CoV-2 display mild symptoms with good prognosis, while 20% of patients suffer from severe viral pneumonia and up to 5% may require intensive care unit (ICU) admission due to severe acute respiratory syndrome, which could be accompanied by multiorgan failure.Plasma proteomics provide valuable and unbiased information about disease progression and therapeutic candidates. Recent proteomic studies have identified molecular changes in plasma of COVID-19 patients that implied significant dysregulation of several aspects of the inflammatory response accompanied by a general metabolic suppression. However, which of these plasma alterations are associated with disease severity remains only partly characterized.A known limitation of proteomic studies of plasma samples is the large difference in the macromolecule abundance, with concentration spanning at least 10 orders of magnitude. To improve the coverage of plasma contents, we performed a deep proteomic analysis of plasma from 10 COVID-19 patients with severe/fatal pneumonia compared to 10 COVID-19 patients with pneumonia who did not require ICU admission (non-ICU). To this aim, plasma samples were first depleted of the most abundant proteins, trypsin digested and peptides subjected to a high pH reversed-phase peptide fractionation before LC-MS analysis.These results highlighted an increase of proteins involved in neutrophil and platelet activity and acute phase response, which is significantly higher in severe/fatal COVID-19 patients when compared to non-ICU ones. Importantly, these changes are associated with a selective induction of complement cascade factors in severe/fatal COVID-19 patients. Data are available via ProteomeXchange with identifier PXD036491. Among these alterations, we confirmed by ELISA that higher levels of the neutrophil granule proteins DEFA3 and LCN2 are present in COVID-19 patients requiring ICU admission when compared to non-ICU and healthy donors.Altogether, our study provided an in-depth view of plasma proteome changes that occur in COVID-19 patients in relation to disease severity, which can be helpful to identify therapeutic strategies to improve the disease outcome.

Multi-omics approach to COVID-19: a domain-based literature review.

BACKGROUND: Omics data, driven by rapid advances in laboratory techniques, have been generated very quickly during the COVID-19 pandemic. Our aim is to use omics data to highlight the involvement of specific pathways, as well as that of cell types and organs, in the pathophysiology of COVID-19, and to highlight their links with clinical phenotypes of SARS-CoV-2 infection. METHODS: The analysis was based on the domain model, where for domain it is intended a conceptual repository, useful to summarize multiple biological pathways involved at different levels. The relevant domains considered in the analysis were: virus, pathways and phenotypes. An interdisciplinary expert working group was defined for each domain, to carry out an independent literature scoping review. RESULTS: The analysis revealed that dysregulated pathways of innate immune responses, (i.e., complement activation, inflammatory responses, neutrophil activation and degranulation, platelet degranulation) can affect COVID-19 progression and outcomes. These results are consistent with several clinical studies. CONCLUSIONS: Multi-omics approach may help to further investigate unknown aspects of the disease. However, the disease mechanisms are too complex to be explained by a single molecular signature and it is necessary to consider an integrated approach to identify hallmarks of severity.

Proteomic analysis identifies deregulated metabolic and oxidative-associated proteins in Italian intrahepatic cholangiocarcinoma patients.

BACKGROUND: Cholangiocarcinoma (CCA) is an aggressive disease with poor prognosis. A molecular classification based on mutational, methylation and transcriptomic features could allow identifying tailored therapies to improve CCA patient outcome. Proteomic remains partially unexplored; here, we analyzed the proteomic profile of five intrahepatic cholangiocarcinoma (ICC) derived from Italian patients undergone surgery and one normal bile duct cell line. METHODS: Proteome profile was investigated by using 2D electrophoresis followed by Mass Spectrometry (MS). To validate proteomic data, the expression of four overexpressed proteins (CAT, SOD, PRDX6, DBI/ACBP) was evaluated by immunohistochemistry in an independent cohort of formalin fixed, paraffin-embedded (FFPE) ICC tissues. We also compared proteomic data with those obtained by transcriptomic profile evaluated by microarray analysis of the same tissues. RESULTS: We identified 19 differentially expressed protein spots, which were further characterized by MS; 13 of them were up- and 6 were down-regulated in ICC. These proteins are mainly involved in redox processes (CAT, SODM, PRDX2, PRDX6), in metabolism (ACBP, ACY1, UCRI, FTCD, HCMS2), and cell structure and organization (TUB2, ACTB). CAT is overexpressed in 86% of patients, PRDX6 in 73%, SODM in 100%, and DBI/ACBP in 81% compared to normal adjacent tissues. A concordance of 50% between proteomic and transcriptomic data was observed. CONCLUSIONS: This study pointed out that the impairment of the metabolic and antioxidant systems, with a subsequent accumulation of free radicals, might be a key step in CCA development and progression.

Negative Regulation of Mitochondrial Antiviral Signaling Protein-Mediated Antiviral Signaling by the Mitochondrial Protein LRPPRC During Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is highly efficient in establishing a chronic infection, having evolved multiple strategies to suppress the host antiviral responses. The HCV nonstructural 5A (NS5A) protein, in addition to its role in viral replication and assembly, has long been known to hamper the interferon (IFN) response. However, the mechanism of this inhibitory activity of NS5A remains partly characterized. In a functional proteomic screening carried out in HCV replicon cells, we identified the mitochondrial protein LRPPRC as an NS5A binding factor. Notably, we found that downregulation of LRPPRC expression results in a significant inhibition of HCV infection, which is associated with an increased activation of the IFN response. Moreover, we showed that LRPPRC acts as a negative regulator of the mitochondrial-mediated antiviral immunity, by interacting with mitochondrial antiviral signaling protein (MAVS) and inhibiting its association with TRAF3 and TRAF6. Finally, we demonstrated that NS5A is able to interfere with MAVS activity in a LRPPRC-dependent manner. Conclusion: Overall, our results indicate that NS5A contributes to the inhibition of innate immune pathways during HCV infection by exploiting the ability of LRPPRC to inhibit MAVS-regulated antiviral signaling.

IP-10 contributes to the inhibition of mycobacterial growth in an ex vivo whole blood assay.

Interferon-γ inducible protein 10 (IP-10), is a potent chemoattractant that promotes migration of monocytes and activated T-cells to inflammation foci. IP-10 is elevated in serum of patients with chronic hepatitis C virus (HCV) and tuberculosis (TB) infections, although it remains to be determined the contribution of IP-10 in restricting Mycobacterium tuberculosis (Mtb) replication. Here, we investigated the impact of IP-10 on mycobacteria replication using the ex vivo model of human whole-blood (WB) assay. In particular, we compared the levels of IP-10 upon infection with different Mtb clinical strains and species of non-tuberculous mycobacteria (NTM) and evaluated how IP-10 may contain bacterial replication. Interestingly, we observed that the inhibition of the host enzyme dipeptidyl peptidase IV (DPP-IV), which inactivates IP-10 through cleavage of two amino acids at the chemokine N-terminus, restricted mycobacterial persistence in WB, supporting the critical role of full length IP-10 in mediating an anti-Mtb response. Addition of recombinant IP-10 expressed in eukaryotic cells enhanced the anti-mycobacterial activity in WB, although no differences were observed when IP-10 containing different proportions of cleaved and non-cleaved forms of the chemokine were added. Moreover, recombinant IP-10 did not exert a direct anti-mycobacterial effect. Our results underscore the clinical relevance of IP-10 in mycobacteria pathogenesis and support the potential outcomes that may derive by targeting the IP-10/CXCR3 pathway as host directed therapies for the treatment of Mtb or NTM infections.

Dendritic cells activation is associated with sustained virological response to telaprevir treatment of HCV-infected patients.

First anti-HCV treatments, that include protease inhibitors in conjunction with IFN-α and Ribavirin, increase the sustained virological response (SVR) up to 80% in patients infected with HCV genotype 1. The effects of triple therapies on dendritic cell (DC) compartment have not been investigated. In this study we evaluated the effect of telaprevir-based triple therapy on DC phenotype and function, and their possible association with treatment outcome. HCV+ patients eligible for telaprevir-based therapy were enrolled, and circulating DC frequency, phenotype, and function were evaluated by flow-cytometry. The antiviral activity of plasmacytoid DC was also tested. In SVR patients, myeloid DC frequency transiently decreased, and returned to baseline level when telaprevir was stopped. Moreover, an up-regulation of CD80 and CD86 on mDC was observed in SVR patients as well as an improvement of IFN-α production by plasmacytoid DC, able to inhibit in vitro HCV replication.

Iron overload down-regulates the expression of the HIV-1 Rev cofactor eIF5A in infected T lymphocytes.

BACKGROUND: Changes in iron metabolism frequently accompany HIV-1 infection. However, while many clinical and in vitro studies report iron overload exacerbates the development of infection, many others have found no correlation. Therefore, the multi-faceted role of iron in HIV-1 infection remains enigmatic. METHODS: RT-qPCR targeting the LTR region, gag, Tat and Rev were performed to measure the levels of viral RNAs in response to iron overload. Spike-in SILAC proteomics comparing i) iron-treated, ii) HIV-1-infected and iii) HIV-1-infected/iron treated T lymphocytes was performed to define modifications in the host cell proteome. Data from quantitative proteomics were integrated with the HIV-1 Human Interaction Database for assessing any viral cofactors modulated by iron overload in infected T lymphocytes. RESULTS: Here, we demonstrate that the iron overload down-regulates HIV-1 gene expression by decreasing the levels of viral RNAs. In addition, we found that iron overload modulates the expression of many viral cofactors. Among them, the downregulation of the REV cofactor eIF5A may correlate with the iron-induced inhibition of HIV-1 gene expression. Therefore, we demonstrated that eiF5A downregulation by shRNA resulted in a significant decrease of Nef levels, thus hampering HIV-1 replication. CONCLUSIONS: Our study indicates that HIV-1 cofactors influenced by iron metabolism represent potential targets for antiretroviral therapy and suggests eIF5A as a selective target for drug development.

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.

Calreticulin exposure dictates the immunogenicity of cancer cell death.

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Autophagy protects cells from HCV-induced defects in lipid metabolism.

BACKGROUND & AIMS: Autophagy is a lysosome-mediated catabolic process that mediates degradation and recycling of all major components of eukaryotic cells. Different stresses, including viral and bacterial infection, induce autophagy, which can promote cell survival by removing the stress inducer or by attenuating its dangerous effects. High levels of autophagy occur during infection of cells with hepatitis C virus (HCV), but the clinical relevance of this process is not clear. METHODS: Levels of autophagy were analyzed in liver biopsy samples from 22 patients with HCV infection using microtubule-associated protein-1 light chain 3 immunoblotting; associations with histological and metabolic parameters were evaluated by Pearson correlation analysis. We investigated the role of HCV-induced autophagy in lipid degradation in cells infected with the virus or replicons, and analyzed autophagosome contents by confocal microscopy and by measuring lipid levels after inhibition of autophagy by Beclin 1 knockdown or lysosome inhibitors. RESULTS: In liver biopsy samples from patients with HCV, there was an inverse correlation between microvesicular steatosis and level of autophagy (r = -0.617; P = .002). HCV selectively induced autophagy of lipids in virus-infected and replicon cells. In each system, autophagosomes frequently colocalized with lipid deposits, mainly formed by unesterified cholesterol. Inhibition of the autophagic process in these cells significantly increased the induction of cholesterol accumulation by HCV. CONCLUSIONS: Autophagy counteracts the alterations in lipid metabolism induced by HCV. Disruption of the autophagic process might contribute to development of steatosis in patients with HCV.

Interplay between autophagy and apoptosis in the development of Danio rerio follicles and the effects of a probiotic.

The present study investigated autophagic processes in Danio rerio preovulatory follicles (Stage III and IV). There were more autophagosomes, as revealed by electron microscopy, in follicles from females fed the probiotic Lactobacillus rhamnosus IMC 501. This was confirmed by increased expression of genes involved in the autophagic process, namely ambra1, becn1, lc3 and uvrag. In addition, preovulatory follicles from females fed the probiotic contained more microtubule-associated protein 1 light chain 3 isoform II (LC3-II) and less p62 protein. The increased autophagy in preovulatory follicles from females fed the probiotic was concomitant with a decrease in the apoptotic process in the ovary, as evidenced by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling analysis and confirmed by lower expression of genes involved in apoptosis (i.e., p53, bax, apaf and cas3) and higher expression as igfII and igf1r. The results of the present study provide preliminary evidence of the involvement of autophagy during follicle development in the zebrafish ovary. In addition, we have demonstrated for the first time that a functional food, such as L. rhamnosus IMC 501, can modulate the balance between apoptosis and autophagy that regulates ovary physiology in zebrafish by inhibiting follicular apoptosis and improving follicular survival.

A microRNA Arising from the Negative Strand of SARS-CoV-2 Genome Targets FOS to Reduce AP-1 Activity.

Virus-encoded microRNAs were first reported in the Epstein-Barr virus in 2004. Subsequently, a few hundred viral miRNAs have been identified, mainly in DNA viruses belonging to the herpesviridae family. To date, only 30 viral miRNAs encoded by RNA viruses are reported by miRBase. Since the outbreak of the SARS-CoV-2 pandemic, several studies have predicted and, in some cases, experimentally validated miRNAs originating from the positive strand of the SARS-CoV-2 genome. By integrating NGS data analysis and qRT-PCR approaches, we found that SARS-CoV-2 also encodes for a viral miRNA arising from the minus (antisense) strand of the viral genome, in the region encoding for ORF1ab, herein referred to as SARS-CoV-2-miR-AS1. Our data show that the expression of this microRNA increases in a time course analysis of SARS-CoV-2 infected cells. Furthermore, enoxacin treatment enhances the accumulation of the mature SARS-CoV-2-miR-AS1 in SARS-CoV-2 infected cells, arguing for a Dicer-dependent processing of this small RNA. In silico analysis suggests that SARS-CoV-2-miR-AS1 targets a set of genes which are translationally repressed during SARS-CoV-2 infection. We experimentally validated that SARS-CoV-2-miR-AS1 targets FOS, thus repressing the AP-1 transcription factor activity in human cells.

Liver protein profiling in chronic hepatitis C: identification of potential predictive markers for interferon therapy outcome.

The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH.

It is well known that secreted and exosomal proteins are associated with a broad range of physiological processes involving tissue homeostasis and differentiation. In the present paper, our purpose was to characterize the proteome of the culture medium in which the oocytes within the primordial/primary follicles underwent apoptosis induced by cisplatin (CIS) or were, for the most part, protected by LH against the drug. To this aim, prepubertal ovarian tissues were cultured under control and in the presence of CIS, LH, and CIS + LH. The culture media were harvested after 2, 12, and 24 h from chemotherapeutic drug treatment and analyzed by liquid chromatography-mass spectrometry (LC-MS). We found that apoptotic conditions generated by CIS in the cultured ovarian tissues and/or oocytes are reflected in distinct changes in the extracellular microenvironment in which they were cultured. These changes became evident mainly from 12 h onwards and were characterized by the inhibition or decreased release of a variety of compounds, such as the proteases Htra1 and Prss23, the antioxidants Prdx2 and Hbat1, the metabolic regulators Ldha and Pkm, and regulators of apoptotic pathways such as Tmsb4x. Altogether, these results confirm the biological relevance of the LH action on prepuberal ovaries and provide novel information about the proteins released by the ovarian tissues exposed to CIS and LH in the surrounding microenvironment. These data might represent a valuable resource for future studies aimed to clarify the effects and identify biomarkers of these compounds' action on the developing ovary.

AMBRA1 regulates mitophagy by interacting with ATAD3A and promoting PINK1 stability.

PINK1 accumulation at the outer mitochondrial membrane (OMM) is a key event required to signal depolarized mitochondria to the autophagy machinery. How this early step is, in turn, modulated by autophagy proteins remains less characterized. Here, we show that, upon mitochondrial depolarization, the proautophagic protein AMBRA1 is recruited to the OMM and interacts with PINK1 and ATAD3A, a transmembrane protein that mediates mitochondrial import and degradation of PINK1. Downregulation of AMBRA1 expression results in reduced levels of PINK1 due to its enhanced degradation by the mitochondrial protease LONP1, which leads to a decrease in PINK1-mediated ubiquitin phosphorylation and mitochondrial PRKN/PARKIN recruitment. Notably, ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells upon mitochondrial damage. Overall, our findings underline an upstream contribution of AMBRA1 in the control of PINK1-PRKN mitophagy by interacting with ATAD3A and promoting PINK1 stability. This novel regulatory element may account for changes of PINK1 levels in neuropathological conditions.Abbreviations: ACTB/ß-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATAD3A: ATPase family AAA domain containing 3A; BCL2L1/BCL-xL: BCL2 like 1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMA1: OMA1 zinc metallopeptidase; OMM: outer mitochondrial membrane; PARL: presenilin associated rhomboid like; PARP: poly(ADP-ribose) polymerase; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; SDHA: succinate dehydrogenase complex flavoprotein subunit A; TOMM70: translocase of outer mitochondrial membrane 70.

Peroxiredoxin 6 Modulates Insulin Secretion and Beta Cell Death via a Mitochondrial Dynamic Network.

In pancreatic beta cells, mitochondrial metabolism controls glucose-stimulated insulin secretion (GSIS) by ATP production, redox signaling, and calcium (Ca2+) handling. Previously, we demonstrated that knockout mice for peroxiredoxin 6 (Prdx6-/- ), an antioxidant enzyme with both peroxidase and phospholipase A2 activity, develop a mild form of diabetes mellitus with a reduction in GSIS and in peripheral insulin sensitivity. However, whether the defect of GSIS present in these mice is directly modulated by Prdx6 is unknown. Therefore, the main goal of the present study was to evaluate if depletion of Prdx6 affects directly GSIS and pancreatic beta ß-cell function. Murine pancreatic ß-cell line (ßTC6) knockdown for Prdx6 (Prdx6KD) was employed, and insulin secretion, ATP, and intracellular Ca2+ content were assessed in response to glucose stimulation. Mitochondrial morphology and function were also evaluated through electron microscopy, and by testing mitochondrial membrane potential, oxygen consumption, and mitochondrial mass. Prdx6KD cells showed a significant reduction in GSIS as confirmed by decrease in both ATP release and Ca2+ influx. GSIS alteration was also demonstrated by a marked impairment of mitochondrial morphology and function. These latest are mainly linked to mitofusin downregulation, which are, in turn, strictly related to mitochondrial homeostasis (by regulating autophagy) and cell fate (by modulating apoptosis). Following a pro-inflammatory stimulus (typical of diabetic subjects), and in agreement with the deregulation of mitofusin steady-state levels, we also observed an enhancement in apoptotic death in Prdx6KD compared to control cells. We analyzed molecular mechanisms leading to apoptosis, and we further demonstrated that Prdx6 suppression activates both intrinsic and extrinsic apoptotic pathways, ultimately leading to caspase 3 and PARP-1 activation. In conclusion, Prdx6 is the first antioxidant enzyme, in pancreatic ß-cells, that by controlling mitochondrial homeostasis plays a pivotal role in GSIS modulation.

Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope.

The proapoptotic activity of the transcription factor p53 critically depends on the phosphorylation of serine 46 (p53S46P). Here, we show that syncytia containing p53S46P could be detected in lymph node biopsies from human immunodeficiency virus (HIV)-1 carriers, in the brain of patients with HIV-1-associated dementia and in cocultures of HeLa expressing the HIV-1 envelope glycoprotein complex (Env) with HeLa cells expressing CD4. In this latter model, cell death was the result of a sequential process involving cell fusion, nuclear fusion (karyogamy), phosphorylation of serine 15 (p53S15P), later on serine 46 (p53S46P), and transcription of p53 target genes. Cytoplasmic p38 mitogen-activated protein kinase (MAPK) was found to undergo an activating phosphorylation (p38T180/Y182P [p38 with phosphorylated threonine 180 and tyrosine 182]) before karyogamy and to translocate into karyogamic nuclei. p38T180/Y182P colocalized and coimmunoprecipitated with p53S46P. Recombinant p38 phosphorylated recombinant p53 on serine 46 in vitro. Inhibition of p38 MAPK by pharmacological inhibitors, dominant-negative p38, or small interfering RNA, suppressed p53S46P (but not p53S15P), the expression of p53-inducible genes, the conformational activation of proapoptotic Bax and Bak, the release of cytochrome c from mitochondria, and consequent apoptosis. p38T180/Y182P was also detected in HIV-1-induced syncytia, in vivo, in patients' lymph nodes and brains. Dominant-negative MKK3 or MKK6 inhibited syncytial activation of p38, p53S46P, and apoptosis. Altogether, these findings indicate that p38 MAPK-mediated p53 phosphorylation constitutes a critical step of Env-induced apoptosis.