Pools In Cells Research Articles

Viola phlebovirus is a novel Phlebotomus fever serogroup member identified in Lutzomyia (Lutzomyia) longipalpis from Brazilian Pantanal

BackgroundHigh throughput sequencing (HTS) boosted the discovery of novel viruses and new variants of known viruses. Here we investigated the presence of viruses in 12 pools of sand flies captured in three climatic periods in RAPELD grids at Rio Claro, Chapada dos Guimarães and at Pirizal, North Pantanal, Mato Grosso State, Midwestern Brazil by HTS, viral isolation of a putative Phlebovirus positive pool in Vero cells, RT-PCR and transmission electron microscopy (TEM).ResultsOne pool containing three Lutzomyia (Lutzomyia) longipalpis sand flies captured in the transitional climatic period in North Pantanal showed a tripartite genomic sequence of a putative novel Phlebovirus belonging to the phlebotomus fever serogroup. Phylogenetic analysis revealed this virus is closely related and share a common ancestor with phleboviruses included in the same clade: Chagres, Urucuri and Uriurana virus. RNA-dependent RNA polymerase (RdRP) presented 60%, 59% and 58% of amino-acid (aa) similarity with these phleboviruses, respectively. Similarity of Nucleoprotein and NSs protein codified by ambissense strategy of segment S was of 49% and 37%, respectively, with the proteins of the closest phlebovirus, Uriurana virus. Glycoproteins (G1, G2) and NSm protein presented 49% and 48% aa similarity with Chagres and Uriurana virus, respectively. Uriurana virus was isolated from sand flies in Brazilian Amazon and Urucuri from rodents in Utinga forest, Pará State. Chagres virus is an arbovirus responsible for outbreaks of febrile illness in Panama. This phlebovirus was isolated in Vero cells, confirmed by TEM and RT-PCR for the L segment of the virus, and named Viola phlebovirus.ConclusionsHTS, viral isolation, RT-PCR and TEM showed the presence of one virus in sand flies from North Pantanal with identity to a putative novel Phlebovirus from phlebotomus fever serogroup, named Viola phlebovirus.

Avian Influenza Virus PB1 Gene in H3N2 Viruses Evolved in Humans To Reduce Interferon Inhibition by Skewing Codon Usage toward Interferon-Altered tRNA Pools.

ABSTRACTInfluenza A viruses cause an annual contagious respiratory disease in humans and are responsible for periodic high-mortality human pandemics. Pandemic influenza A viruses usually result from the reassortment of gene segments between human and avian influenza viruses. These avian influenza virus gene segments need to adapt to humans. Here we focus on the human adaptation of the synonymous codons of the avian influenza virus PB1 gene of the 1968 H3N2 pandemic virus. We generated recombinant H3N2 viruses differing only in codon usage of PB1 mRNA and demonstrated that codon usage of the PB1 mRNA of recent H3N2 virus isolates enhances replication in interferon (IFN)-treated human cells without affecting replication in untreated cells, thereby partially alleviating the interferon-induced antiviral state. High-throughput sequencing of tRNA pools explains the reduced inhibition of replication by interferon: the levels of some tRNAs differ between interferon-treated and untreated human cells, and evolution of the codon usage of H3N2 PB1 mRNA is skewed toward interferon-altered human tRNA pools. Consequently, the avian influenza virus-derived PB1 mRNAs of modern H3N2 viruses have acquired codon usages that better reflect tRNA availabilities in IFN-treated cells. Our results indicate that the change in tRNA availabilities resulting from interferon treatment is a previously unknown aspect of the antiviral action of interferon, which has been partially overcome by human-adapted H3N2 viruses.

Queuosine modification protects cognate tRNAs against ribonuclease cleavage.

Eukaryotic transfer RNAs (tRNA) contain on average 13 modifications that perform a wide range of roles in translation and in the generation of tRNA fragments that regulate gene expression. Queuosine (Q) modification occurs in the wobble anticodon position of tRNAs for amino acids His, Asn, Tyr, and Asp. In eukaryotes, Q modification is fully dependent on diet or on gut microbiome in multicellular organisms. Despite decades of study, cellular roles of Q modification remain to be fully elucidated. Here we show that in human cells, Q modification specifically protects its cognate tRNAHis and tRNAAsn against cleavage by ribonucleases. We generated cell lines that contain completely depleted or fully Q-modified tRNAs. Using these resources, we found that Q modification significantly reduces angiogenin cleavage of its cognate tRNAs in vitro. Q modification does not change the cellular abundance of the cognate full-length tRNAs, but alters the cellular content of their fragments in vivo in the absence and presence of stress. Our results provide a new biological aspect of Q modification and a mechanism of how Q modification alters small RNA pools in human cells.

Utilisation of 10-formyldihydrofolate as substrate by dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) tranformylase/IMP cyclohydrolase (PurH) in Escherichia coli.

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.

Abstract 4971: Identification of new modulators of nucleotide metabolism and replication stress in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with an overall survival of less than one year. An improved knowledge of PDAC biology, to uncover vulnerabilities specific to cancer cells, is needed to develop more effective therapeutic options. We are investigating the intersection between three aspects of PDAC biology that can, ultimately, be developed for therapeutics: (i) cytokine signaling, with a particular focus on the metabolic effects of interferons (IFNs), which are present in the highly inflamed and dense PDAC stromal microenvironment; (ii) nucleotide metabolism, a network of tightly regulated biochemical pathways that produce deoxyribonucleotide triphosphates (dNTPs), which are required for DNA replication; and (iii) the replication stress response pathway, an intracellular signaling mechanism that is activated by perturbations in DNA replication, and has been recently shown to govern key aspects of nucleotide metabolism. We hypothesize that IFN signaling reduces the levels of already limited dNTP pools in PDAC cancer cells, which respond by initiating metabolic and signaling mechanisms that coordinately function to increase dNTP recycling and biosynthetic mechanisms while simultaneously reducing their consumption. Our results, which include integrated metabolic, transcriptomic, and proteomic analyses, indicate that IFN signaling in PDAC cells induces a switch in nucleotide metabolism from a biosynthetic to a predominantly dNTP catabolic phenotype. This switch appears to be mediated by dNTP phosphohydrolysis catalyzed by the Sterile Alpha Motif and Histidine/aspartic acid Domain-containing protein (SAMHD1). Furthermore, we have investigated the effects of IFN signaling on dNTP phosphohydrolysis to deoxyribonucleosides across a panel of PDAC cancer cells, resembling the spectrum of the human disease. We also examined the biochemical fates of the deoxyribonucleoside products of SAMHD1 using targeted LC-MS/MS metabolic tracing experiments. We have also demonstrated a role for the replication stress response kinase Ataxia Telangiectasia and Rad3-related protein (ATR) in regulating dNTP levels in PDAC cells exposed to IFN. Furthermore, we have demonstrated that ATR activity is an actionable co-dependency of IFN-exposed cells and that pharmacologic inhibition of ATR eradicates PDAC cells exposed to IFN. Collectively, these studies increase our understanding of the interplay between cell extrinsic (IFN signaling) and intrinsic (replication stress) signal transduction networks, and the regulation of nucleotide metabolism in PDAC, and uncovered critical vulnerabilities to be exploited by new therapeutic approaches against this extremely aggressive and difficult to treat malignancy. Citation Format: Evan R. Abt, Amanda Dann, Thuc M. Le, Joe R. Capri, Chloe M. Cheng, Juna Yi, Soumya Poddar, Woosuk Kim, Timothy R. Donahue, Caius G. Radu. Identification of new modulators of nucleotide metabolism and replication stress in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4971.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca²⁺ in Cat Esophageal Smooth Muscle Cells.

A comprehensive collection of proteins senses local changes in intracellular Ca2+ concentrations ([Ca2+]i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca2+ concentrations in cat esophageal smooth muscle cells. To measure [Ca2+]i levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca2+]i in the cells. Pretreatment with EGTA, an extracellular Ca2+ chelator, decreased the S1P-induced increase in [Ca2+]i, and an L-type Ca2+-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca2+ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP3 receptor blocker, the S1P-evoked increase in [Ca2+]i was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of Gi-protein, suppressed the increase in [Ca2+]i evoked by S1P. These results suggest that the S1P-induced increase in [Ca2+]i in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca2+ from the InsP3-sensitive Ca2+ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca2+ via the L type Ca2+ channel, which was dependent on activation of the S1P4 receptor coupled to PTX-sensitive Gi protein, via phospholipase C-mediated Ca2+ release from the InsP3-sensitive Ca2+ pool in cat esophageal smooth muscle cells.

08 Inhibition of autophagy by mevalonate pathway inhibitors, a new therapeutic approach to sensitize glioblastoma cells to temozolomide induced apoptosis

Glioblastoma multiforme (GBM) is the deadliest brain tumor with an approximate 14 month survival rate after diagnosis and treatment. Temozolomide (TMZ), the chemotherapeutic drug of choice for GBM, is an alkylating agent that causes DNA damage. TMZ treatment results in the induction of apoptosis in GBM cells, however, it induces autophagy and consequently chemoresistance. Statins are mevalonate (MEV) cascade inhibitors with beneficial effects on the enhancement of the survival rate of patients with different types of cancer. Here, we determined the effect of simvastatin (Simva), a blood brain barrier permeable statin, on the sensitization of GBM cells to TMZ induced apoptosis through inhibition of autophagy flux. We pretreated two GMB cell lines, U251 and U87 cells, with low doses of Simva (1 and 2.5 M, respectively) with or without different intermediates of the mevalonate cascade and then treated cells with TMZ (100 M) for 48-96 hrs. A signficiantly reduced viability and increased in the population of apoptotic dead cells were observed in GBM cells treated with the Simva-TMZ compared to cells treated with TMZ alone. Addition of MEV, Farnesyl pyrophosphate, Geranylgeranyl pyrophosphate and cholesterol did not attenuate these effects significantly. Sima-TMZ treatment did not alter the total cholesterol pool in U87 and U251 cells compared to controls. Western blot analysis, immunocytochemistry and transmission electron microscopy revealed that Simva-TMZ inhibited autophagic flux. Overall, the results suggest that sensitization of GBM cells to TMZ-induced apoptosis by Simva is independent on the cholesterol biosynthetic pathway but may involve inhibition of autophagy.

Calix[4]arene Based Redox Sensitive Molecular Probe for SERS Guided Recognition of Labile Iron Pool in Tumor Cells.

Targeting the intracellular "labile" iron pool is turned as a key modulator for cancer progression since the former is responsible for several pathological processes in tumor cells. Herein, we report a nonfluorescent calix[4]arene based triazole appended molecular probe (PTBC) for redox-specific detection of Fe3+ under physiological condition by UV-vis, FT-IR, 1H NMR, HR-MS spectroscopies, ITC, and the binding strategy between Calix[4]arene and Fe3+ was modeled by DFT calculations. As a new insight PTBC probe showed significant Raman fingerprint through surface enhanced Raman scattering (SERS) modality revealing the ultrasensitive detection of Fe3+ with a LOD of 2 nM. Interestingly, intracellular "iron pool" has been recognized in human lung adenocarcinoma cells (A549) by the PTBC illustrating the distinct Raman mapping. Finally, PTBC imparted cytotoxicity via reactive oxygen species (ROS) generation in cellular milieu signifies its capability as a theranostic molecular probe.

D NTP metabolism links mechanical cues and YAP / TAZ to cell growth and oncogene‐induced senescence

YAP/TAZ, downstream transducers of the Hippo pathway, are powerful regulators of cancer growth. How these factors control proliferation remains poorly defined. Here, we found that YAP/TAZ directly regulate expression of key enzymes involved in deoxynucleotide biosynthesis and maintain dNTP precursor pools in human cancer cells. Regulation of deoxynucleotide metabolism is required for YAP-induced cell growth and underlies the resistance of YAP-addicted cells to chemotherapeutics targeting dNTP synthesis. During RAS-induced senescence, YAP/TAZ bypass RAS-mediated inhibition of nucleotide metabolism and control senescence. Endogenous YAP/TAZ targets and signatures are inhibited by RAS/MEK1 during senescence, and depletion of YAP/TAZ is sufficient to cause senescence-associated phenotypes, suggesting a role for YAP/TAZ in suppression of senescence. Finally, mechanical cues, such as ECM stiffness and cell geometry, regulate senescence in a YAP-dependent manner. This study indicates that YAP/TAZ couples cell proliferation with a metabolism suited for DNA replication and facilitates escape from oncogene-induced senescence. We speculate that this activity might be relevant during the initial phases of tumour progression or during experimental stem cell reprogramming induced by YAP.

Inhibition of Autophagy by Mevalonate Pathway Inhibitors, a New Therapeutic Approach to sensitize Glioblastoma Cells to Temozolomide Induced Apoptosis

Glioblastoma multiforme (GBM) is the deadliest brain tumor with an approximate 20 month survival rate after diagnosis and treatment. Temozolomide (TMZ), the chemotherapeutic drug of choice for GBM, is an alkylating agent that causes DNA damage. TMZ treatment results in the induction of apoptosis in GBM cells, however, it induces autophagy and consequently chemoresistance. Statins are mevalonate (MEV) cascade inhibitors with beneficial effects on the enhancement of the survival rate of patients with different types of cancer, particulary if consumed long term before diagnosis of cancer. Here, we determined the effect of simvastatin (Simva), a blood brain barrier permeable statin, on the sensitization of GBM cells to TMZ induced apoptosis through inhibition of autophagy flux. We pretreated two GMB cell lines, U251 and U87 cells, with low doses of Simva (1 and 2.5 μM, respectively) with or without different intermediates of the mevalonate cascade: MEV (2.5 mM), Farnesyl pyrophosphate (FPP) (15 μM), Geranylgeranyl pyrophosphate (GGPP) (15 μM) and cholesterol (50 μM) for 4 h and then treated cells with TMZ (100 μM) for 48–96 h. A signficiantly reduced viability and increase in the population of apoptotic dead cells were observed in GBM cells treated with the combination of Simva‐TMZ compared to cells treated with TMZ alone. Addition of MEV, FPP, GGPP and cholesterol did not attenuate these effects significantly. Sima‐TMZ treatment did not alter the total cholesterol pool in U87 and U251 cells compared to controls. Western blot analysis, immunocytochemistry and transmission electron microscopy revealed that the combination of Simva with TMZ inhibited autophagic flux. Overall, the results suggest that sensitization of GBM cells to TMZ‐induced apoptosis by Simva is independent on the cholesterol biosynthetic pathway but may involve inhibition of autophagy flux.Support or Funding InformationSupported by funding from a Mitacs Accelerate PDF Award & a Health Science Center Foundation Operating Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Cyclin-Binding Motif in Human SAMHD1 Is Required for Its HIV-1 Restriction, dNTPase Activity, Tetramer Formation, and Efficient Phosphorylation.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) regulates intracellular deoxynucleoside triphosphate (dNTP) levels and functions as a retroviral restriction factor through its dNTP triphosphohydrolase (dNTPase) activity. Human SAMHD1 interacts with cell cycle regulatory proteins cyclin A2, cyclin-dependent kinase 1 (CDK1), and CDK2. This interaction mediates phosphorylation of SAMHD1 at threonine 592 (T592), which negatively regulates HIV-1 restriction. We previously reported that the interaction is mediated, at least in part, through a cyclin-binding motif (RXL, amino acids [aa] 451 to 453). To understand the role of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of RXL mutants and the effect on HIV-1 restriction. We found that the RXL mutation (R451A and L453A, termed RL/AA) disrupted SAMHD1 tetramer formation and abolished its dNTPase activity in vitro and in cells. Compared to wild-type (WT) SAMHD1, the RL/AA mutant failed to restrict HIV-1 infection and had reduced binding to cyclin A2. WT SAMHD1 and RL/AA mutant proteins were degraded by Vpx from HIV-2 but were not spontaneously ubiquitinated in the absence of Vpx. Analysis of proteasomal and autophagy degradation revealed that WT and RL/AA SAMHD1 protein levels were enhanced only when both pathways of degradation were simultaneously inhibited. Our results demonstrate that the RXL motif of human SAMHD1 is required for its HIV-1 restriction, tetramer formation, dNTPase activity, and efficient phosphorylation at T592. These findings identify a new functional domain of SAMHD1 important for its structural integrity, enzyme activity, phosphorylation, and HIV-1 restriction.IMPORTANCE SAMHD1 is the first mammalian dNTPase identified as a restriction factor that inhibits HIV-1 replication by decreasing the intracellular dNTP pool in nondividing cells, although the critical mechanisms regulating SAMHD1 function remain unclear. We previously reported that mutations of a cyclin-binding RXL motif in human SAMHD1 significantly affect protein expression levels, half-life, nuclear localization, and phosphorylation, suggesting an important role of this motif in modulating SAMHD1 functions in cells. To further understand the significance and mechanisms of the RXL motif in regulating SAMHD1 activity, we performed structural and functional analyses of the RXL motif mutation and its effect on HIV-1 restriction. Our results indicate that the RXL motif is critical for tetramer formation, dNTPase activity, and HIV-1 restriction. These findings help us understand SAMHD1 interactions with other host proteins and the mechanisms regulating SAMHD1 structure and functions in cells.

Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa.

Spermatogenesis starts within the seminiferous tubules of the testis by mitotic division of spermatogonia that produces spermatocytes. Meiotic division of these spermatocytes produces haploid spermatids that differentiate into spermatozoa. In this study, we examined the expression of ENaC and CFTR (a Cl- channel) in rat testicular sections using confocal microscopic immunofluorescence. The structural integrity of the seminiferous tubule sections was verified by precise phalloidin staining of the actin fibers located abundantly at both basal and adluminal tight junctions. The acrosome forming regions in the round spermatids were stained using an FITC coupled lectin (wheat germ agglutinin). In all phases of the germ cells (spermatogonia, spermatocytes, and spermatids) ENaC was localized in cytoplasmic pools. Prior to spermiation, ENaC immunofluorescence appeared along the tails of the spermatids. In spermatozoa isolated from the epididymis, ENaC was localized at the acrosome and a central region of the sperm flagellum. The mature sperm are transcriptionally silent. Hence, we suggest that ENaC subunits in cytoplasmic pools in germ cells serve as the source of ENaC subunits located along the tail of spermatozoa. The locations of ENaC is compatible with a possible role in the acrosomal reaction and sperm mobility. In contrast to ENaC, CFTR immunofluorescence was most strongly observed specifically within the Sertoli cell nuclei. Based on the nuclear localization of CFTR we suggest that, in addition to its role as an ion channel, CFTR may have an independent role in gene regulation within the nuclei.

Chromis-1, a Ratiometric Fluorescent Probe Optimized for Two-Photon Microscopy Reveals Dynamic Changes in Labile Zn(II) in Differentiating Oligodendrocytes.

Despite the significant advantages of two-photon excitation microscopy (TPEM) over traditional confocal fluorescence microscopy in live-cell imaging applications, including reduced phototoxicity and photobleaching, increased depth penetration, and minimized autofluorescence, only a few metal ion-selective fluorescent probes have been designed and optimized specifically for this technique. Building upon a donor-acceptor fluorophore architecture, we developed a membrane-permeant, Zn(II)-selective fluorescent probe, chromis-1, that exhibits a balanced two-photon cross section between its free and Zn(II)-bound form and responds with a large spectral shift suitable for emission-ratiometric imaging. With a Kd of 1.5 nM and wide dynamic range, the probe is well suited for visualizing temporal changes in buffered Zn(II) levels in live cells as demonstrated with mouse fibroblast cell cultures. Moreover, given the importance of zinc in the physiology and pathophysiology of the brain, we employed chromis-1 to monitor cytoplasmic concentrations of labile Zn(II) in oligodendrocytes, an important cellular constituent of the brain, at different stages of development in cell culture. These studies revealed a decrease in probe saturation upon differentiation to mature oligodendrocytes, implying significant changes to cellular zinc homeostasis during maturation with an overall reduction in cellular zinc availability. Optimized for TPEM, chromis-1 is especially well-suited for exploring the role of labile zinc pools in live cells under a broad range of physiological and pathological conditions.

TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole

Amyotrophic lateral sclerosis (ALS) represents a fatal neurodegenerative disease, which is characterized by a rapid loss of lower and upper motor neurons. As a major neuropathological hallmark, protein aggregates containing the Transactivating Response Region (TAR) DNA Binding Protein (TDP-43) are detectable in about 95% of sporadic ALS patients. TDP-43 interacts with itself physiologically to form liquid droplets, which may progress to pathological aggregates. In this study, we established the NanoBit luciferase complementation assay to measure TDP-43 self-interaction and found the fusion of the split luciferase subunits to the N-terminus of the protein as the strongest interacting partners. A screen of pharmacologically active compounds from the LOPAC®1280 library identified auranofin, chelerythrine and riluzole as dose-dependent inhibitors of TDP-43 self-interaction. Further analysis of drug action of the gold-containing thioredoxin reductase inhibitor auranofin revealed a redistribution from insoluble TDP-43 protein pool to PBS-soluble protein pool in N2a cells. In addition, auranofin treatment diminished reduced glutathione as a sign for oxidative modulation.

SESN2 facilitates mitophagy by helping Parkin translocation through ULK1 mediated Beclin1 phosphorylation

Mitophagy, the selective degradation of mitochondria by autophagy, is crucial for the maintenance of healthy mitochondrial pool in cells. The critical event in mitophagy is the translocation of cytosolic Parkin, a ubiquitin ligase, to the surface of defective mitochondria. This study elucidates a novel role of SESN2/Sestrin2, a stress inducible protein, in mitochondrial translocation of PARK2/Parkin during mitophagy. The data demonstrates that SESN2 downregulation inhibits BECN1/Beclin1 and Parkin interaction, thereby preventing optimum mitochondrial accumulation of Parkin. SESN2 interacts with ULK1 (unc-51 like kinase 1) and assists ULK1 mediated phosphorylation of Beclin1 at serine-14 position required for binding with Parkin prior to mitochondrial translocation. The trigger for SESN2 activation and regulation of Parkin translocation is the generation of mitochondrial superoxide. Scavenging of mitochondrial superoxide lower the levels of SESN2, resulting in retardation of Parkin translocation. Importantly, we observe that SESN2 mediated cytosolic interaction of Parkin and Beclin1 is PINK1 independent but mitochondrial translocation of Parkin is PINK1 dependent. Together, these findings suggest the role of SESN2 as a positive regulator of Parkin mediated mitophagy.

Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803.

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Salmonella escapes antigen presentation through K63 ubiquitination mediated endosomal proteolysis of MHC II via modulation of endosomal acidification in dendritic cells.

CD4+ T-cell response is vital for successful clearance of Salmonella Typhimurium infection. Efficient antigen presentation is crucial for effective CD4+ T-cell response. Previous study has reported that Salmonella abrogates antigen presentation capacity of dendritic cells in order to escape host adaptive immune response. In this study, we have elucidated the mechanism of Salmonella-mediated downregulation of the total cellular Major Histocompatibility Complex (MHC) II pool in dendritic cells. Infected dendritic cells show upregulation of E3 ubiquitin ligase, MARCH1 expression and K63-linked ubiquitination of MHCII. Salmonella infection also enhances the internalisation of ubiquitin-tagged MHC II molecules that are subsequently degraded by endosomal proteases. In addition, Salmonella regulates the activation of endosomal proteases by lowering the pH of endosomes. In infected dendritic cells, Salmonella delays NOX2 recruitment to the phagosomes thereby preventing its alkalinisation. NOX2 is a significant part of innate immune response against pathogens as it is responsible for Reactive Oxygen Species (ROS) production. In this study, we have demonstrated how Salmonella evades MHC II-mediated adaptive immune response in dendritic cells through enhanced endosomal proteolysis. To escape host CD4+T response, Salmonella delays NOX2 recruitment, an innate immune response element to the phagosomes.

B-cell Receptor Signaling Regulates Metabolism in Chronic Lymphocytic Leukemia.

Peripheral blood chronic lymphocytic leukemia (CLL) cells are quiescent but have active transcription and translation processes, suggesting that these lymphocytes are metabolically active. Based on this premise, the metabolic phenotype of CLL lymphocytes was investigated by evaluating the two intracellular ATP-generating pathways. Metabolic flux was assessed by measuring glycolysis as extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation as oxygen consumption rate (OCR) and then correlated with prognostic factors. Further, the impact of B-cell receptor signaling (BCR) on metabolism was determined by genetic ablation and pharmacological inhibitors. Compared with proliferative B-cell lines, metabolic fluxes of oxygen and lactate were low in CLL cells. ECAR was consistently low, but OCR varied considerably in human patient samples (n = 45). Higher OCR was associated with poor prognostic factors such as ZAP 70 positivity, unmutated IGHV, high β2M levels, and higher Rai stage. Consistent with the association of ZAP 70 and IGHV unmutated status with active BCR signaling, genetic ablation of BCR mitigated OCR in malignant B cells. Similarly, knocking out PI3Kδ, a critical component of the BCR pathway, decreased OCR and ECAR. In concert, PI3K pathway inhibitors dramatically reduced OCR and ECAR. In harmony with a decline in metabolic activity, the ribonucleotide pools in CLL cells were reduced with duvelisib treatment. Collectively, these data demonstrate that CLL metabolism, especially OCR, is linked to prognostic factors and is curbed by BCR and PI3K pathway inhibition.Implications: This study identifies a relationship between oxidative phosphorylation in CLL and prognostic factors providing a rationale to therapeutically target these processes. Mol Cancer Res; 15(12); 1692-703. ©2017 AACR.

Supraoptimal activity of CHLOROPHYLL DEPHYTYLASE1 results in an increase in tocopherol level in mature arabidopsis seeds

ABSTRACTTocopherols are synthesized in photosynthetic organisms, playing a role in plant stress tolerance. Recent studies showed that the phytol moiety of tocopherols comes from the salvaged phytol chain during chlorophyll degradation. However, the enzyme(s) responsible for chlorophyll dephytylation remains unclear. Recently, we reported the identification and characterization of CHLOROPHYLL DEPHYTYLASE1 (CLD1) of Arabidopsis, suggesting its role in chlorophyll turnover at steady state. In this addendum to the report, we presented and discussed the results related to the function of CLD1 in tocopherol biosynthesis. The tocopherol levels in the mature seeds were not altered in the transgenic lines with reduced CLD1 expression but were moderately increased in the plants with supraoptimal CLD1 activity compared to wild type. These results suggest that manipulating CLD1 activity could affect tocopherol biosynthesis to a certain extent and that other dephytylating enzymes are sharing redundant function in contributing the phytol pool in plant cells.

Synthesis and Bioevaluation of Macrocycle-Polyamine Conjugates as Cell Migration Inhibitors.

The motuporamines are natural products isolated from the New Guinea sea sponge Xestospongia exigua. Dihydromotuporamine C contains a large macrocycle and an appended polyamine component and was shown to be both antimetastatic and cytotoxic to human L3.6pl pancreatic cancer cells. A series of macrocycle-polyamine conjugates were prepared, and the sequence of the polyamine component was varied to optimize the antimigration properties (as measured in L3.6pl cells) of this molecular class. A one-carbon spacer between the 15-membered carbocycle and the appended polyamine showed improved antimigration properties. A survey of different polyamine sequences containing two, three, or four carbon spacers revealed that the natural polyamine sequence (norspermidine, a 3,3-triamine) was superior in terms of inhibiting the migration of L3.6pl cells in vitro. An investigation of the respective ceramide and sphingomyelin populations in L3.6pl cells revealed that these molecules can modulate both ceramide and sphingomyelin pools in cells and inhibit cell migration.