Generation, release and uptake of the NAD precursor nicotinic acid riboside by human cells [Enzymology]

September 18th, 2015 by

NAD is essential for cellular metabolism and has also a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide (Nam) and nicotinic acid (NA) as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides NMN and NAMN and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of NMN or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from NA. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

Structural Insights into Mitochondrial Antiviral-Signaling Protein (MAVS)-Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Signaling [Signal Transduction]

September 18th, 2015 by

In response to viral infection, cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) sense viral RNA and promote oligomerization of mitochondrial antiviral-signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins including TRAF6 to activate antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood lacking atomic details. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2, amino acids 455-460). Further, we solved the crystal structure of MAVS T6BM2 in complex with TRAF6 TRAF_C domain at 2.95-Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted, but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS-/- mouse embryonic fibroblast (MEF) cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.
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The RNA-binding Complexes, NF45-NF90 and NF45-NF110, Associate Dynamically with the c-fos Gene and Function as Transcriptional Coactivators [Gene Regulation]

September 17th, 2015 by

The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators. The transcriptional activities of the NF complexes (NF45-NF90 and NF45-NF110) are mediated by both the upstream enhancer and core promoter regions of the c-fos gene and do not require their double-stranded RNA-binding activities. The NF complexes cooperate with general coactivators, PC4 and Mediator, to elicit a high level of transcription and display multiple interactions with activators and the components of the general transcriptional machinery. Knockdown of the endogenous NF90/NF110 in mouse cells shows an important role for the NF complexes in inducing c-fos transcription. Chromatin immunoprecipitation assays demonstrate that the NF complexes occupy the c-fos enhancer/promoter region before and after serum induction and that their occupancies within the coding region of the c-fos gene increase in parallel to that of RNAPII upon serum induction. In light of their dynamic occupancy on the c-fos gene as well as direct functions in both transcription and posttranscriptional processes, the NF complexes appear to serve as multifunctional coactivators that coordinate different steps of gene expression to facilitate rapid response of inducible genes.
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Globins Scavenge Sulfur Trioxide Anion Radical [Metabolism]

September 17th, 2015 by Gardner, P. R., Gardner, D. P., Gardner, A. P.

Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600 and ≥ 7,500 x 106 M-1 s-1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate and NAD(P)H are also large at approximately 100, 10, 130 and 30 x 106 M-1 s-1, respectively, but nevertheless allow for favorable competition by globins, and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with non-fermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP+-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.

Multiple Structural and Epigenetic Defects in the Human Leukocyte Antigen class I Antigen Presentation Pathway in a Recurrent Metastatic Melanoma Following Immunotherapy [Immunology]

September 17th, 2015 by

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study, we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis following immunotherapy. These defects include loss of tapasin and one HLA haplotype, as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germline frame-shift mutation in exon 3 (TAPBP684delA) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible following DNA methyltransferase 1 depletion. This treatment, combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.
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Structural basis for antigenic peptide recognition and processing by ER aminopeptidase 2 [Protein Structure and Folding]

September 17th, 2015 by

Endoplasmic reticulum aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5Å and 2.7Å respectively and compared them to the apo form structure determined to 3.0Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center towards the distal end of the internal cavity making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product although decreasing electron density towards its C-terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C-terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.

Integrin {alpha}6{beta}4 Promotes Autocrine EGFR Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF) [Gene Regulation]

September 17th, 2015 by

Integrin α6β4 is upregulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 upregulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in the TCGA pancreatic cancer database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on HGF-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways including MAPK, PI3K and PKC. Moreover, MMP activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional (3D) culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional upregulation of key EGFR family members, and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.
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Oligomerization and Membrane-Binding Properties of Covalent Adducts Formed by the Interaction of Alpha-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL) [Molecular Bases of Disease]

September 17th, 2015 by

Oxidative deamination of dopamine (DA) produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post mortem brains of Parkinson′s disease (PD) patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for DA metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers which may be crosslinked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipild-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons.
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ATDC (Ataxia Telangectasia Group D Complementing) Promotes Radioresistance Through an Interaction with the RNF8 Ubiquitin Ligase [DNA and Chromosomes]

September 17th, 2015 by

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The Ataxia-Telangiectasia group D Complementing gene (ATDC, also called TRIM29), is highly expressed in many malignancies. It participates in the DNA damage response (DDR) downstream of ATM and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (aa 348-588) of ATDC and the ring domain of RNF8 and was disrupted by mutation of ATDC S550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.
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Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-Molecule Chromatin Immunoprecipitation Imaging [DNA and Chromosomes]

September 17th, 2015 by

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical to appreciate the initiation, propagation and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied, but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem (mES) cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Further, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric while PRC2 can dimerize in the nucleoplasm. We showed that ES cell-differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin, but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.
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