The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase is Palmitoylated and Contains Tightly Bound Zinc [Membrane Biology]

October 20th, 2015 by Gottlieb, C. D., Zhang, S., Linder, M. E.

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 moles of zinc per mole DHHC3 protein. Taken together, our data demonstrate coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.

Genetic screen reveals link between maternal-effect sterile gene mes-1 and P. aeruginosa-Induced neurodegeneration in C. elegans [Neurobiology]

October 16th, 2015 by Wu, Q., Cao, X., Yan, D., Wang, D., Aballay, A.

Increasing evidence indicates that immune responses to microbial infections may contribute to neurodegenerative diseases. Here, we show that Pseudomonas aeruginosa infection of Caenorhabditis elegans causes a number of neural changes that are hallmarks of neurodegeneration. Using an unbiased genetic screen to identify genes involved in the control of P. aeruginosa-induced neurodegeneration, we identified mes-1, which encodes a receptor tyrosine kinase-like protein that is required for unequal cell divisions in the early embryonic germline. We showed that sterile but not fertile mes-1 animals were resistant to neurodegeneration induced by P. aeruginosa infection. Similar results were observed using animals carrying a mutation in the maternal-effect gene pgl-1, which is required for postembryonic germline development, and the germline-deficient strains glp-1 and glp-4. Additional studies indicated that the FOXO transcription factor DAF-16 is required for resistance to P. aeruginosa-induced neurodegeneration in germline-deficient strains. Thus, our results demonstrate that P. aeruginosa infection results in neurodegeneration phenotypes in C. elegans that are controlled by the germline in a cell-nonautonomous manner.
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Advanced Glycation End Products Affect Osteoblast Proliferation and Function by Modulating Autophagy Via the RAGE/Raf/MEK/ERK Pathway. [Molecular Bases of Disease]

October 15th, 2015 by Meng, H.-Z., Zhang, W.-L., Liu, F., Yang, M.-W.

The interaction between advanced glycation end products (AGEs) and receptor of AGEs (RAGE) is associated with the development and progression of diabetes-associated osteoporosis, but the mechanisms involved are still poorly understood. In this study, we found that AGE-modified bovine serum albumin (AGE-BSA) induced a biphasic effect on the viability of hFOB1.19 cells: cell proliferation was stimulated after exposure to low-dose AGE-BSA, but cell apoptosis was stimulated after exposure to high-dose AGE-BSA. The low-dose AGE-BSA facilitates proliferation of hFOB1.19 cells by concomitantly promoting autophagy, RAGE production, and the Raf / MEK / ERK signaling pathway activation. Furthermore, we investigated the effects of AGE-BSA on the function of hFOB1.19 cells. Interestingly, the results suggest that the short-term effects of low-dose AGE-BSA increase osteogenic function and decrease osteoclastogenic function, which are likely mediated by autophagy and the RAGE/Raf/MEK/ERK signal pathway. In contrast, with increased treatment time, the opposite effects were observed. Collectively, AGE-BSA had a biphasic effect on the viability of hFOB1.19 cells in vitro, which was determined by the concentration of AGE-BSA and treatment time. A low concentration of AGE-BSA activated the Raf/MEK/ERK signal pathway through the interaction with RAGE, induced autophagy and regulated the proliferation and function of hFOB1.19 cells.
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BMP-2 Activates NFATc1 via an Autoregulatory Loop Involving Smad/Akt/Ca2+ Signaling [Gene Regulation]

October 15th, 2015 by

Bone remodeling is controlled by dual actions of osteoclasts (OC) and osteoblasts (OB). The calcium sensitive NFATc1 transcription factor, as an OC signature gene, regulates differentiation of OC downstream of BMP-2-stimulated osteoblast-coded factors. To analyze a functional link between BMP-2 and NFATc1 we analyzed bones from the OB-specific BMP-2 knock out (BMP-2 cKO) mice for NFATc1 expression by immunohistochemical staining and found significant reduction in NFATc1 expression. This indicated a requirement of BMP-2 for NFATc1 expression in OB. We showed that BMP-2, via receptor-specific Smad pathway, regulates expression of NFATc1 in OB. PI 3 kinase/Akt signaling acting downstream of BMP-2 also drives NFATc1 expression and transcriptional activation. Under basal condition, NFATc1 is phosphorylated. Activation of NFAT requires dephosphorylation by calcium-dependent serine threonine phosphatase, calcineurin. We examined the role of calcium in BMP-2-stimulated regulation of NFATc1 in osteoblasts. BAPTA-AM, an inhibitor of intracellular calcium abundance blocked BMP-2-induced transcription of NFATc1. Interestingly, BMP-2 induced calcium release from intracellular stores and increased calcineurin phosphatase activity resulting in NFATc1 nuclear translocation. Cyclosporine A (CSA), which inhibits calcineurin upstream of NFATc1, blocked BMP-2-induced NFATc1 mRNA and protein expression. Expression of NFATc1 directly increased its transcription and VIVIT peptide, an inhibitor of NFATc1, suppressed BMP-2-stimulated NFATc1 transcription confirming its autoregulation. Together these data show a role of NFATc1 downstream of BMP-2 in mouse bone development and provide novel evidence for the presence of a cross-talk between Smad, PI 3 kinase/Akt and Ca2+ signaling for BMP-2-induced NFATc1 expression through an autoregulatory loop.

Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis [Enzymology]

October 15th, 2015 by

ATP-binding cassette (ABC)-type ATPases are chemo-mechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome (SMC) proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.
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Tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis by upregulating NKp30 ligand B7-H6 [Cell Biology]

October 15th, 2015 by Cao, G., Wang, J., Zheng, X., Wei, H., Tian, Z., Sun, R.

Immune cells are believed to participate in initiating anti-tumor effects during regular tumor therapy such as chemotherapy, radiation, hyperthermia and cytokine injection. One of the mechanisms underlying this process is the expression of so-called stress-inducible immunostimulating ligands. Although the activating receptor NKG2D has been proven to play roles in tumor therapy through targeting its ligands, the role of NKp30, another key activating receptor, is seldom addressed. In this study, we found that the NKp30 ligand B7-H6 was widely expressed in tumor cells and closely correlated to their susceptibility to NK cell lysis. Further studies showed that treatment of tumor cells with almost all standard tumor therapeutics, including chemotherapy (cisplatin, 5-fluorouracil), radiation therapy, non-lethal heat shock, and cytokine therapy (TNF-α), could upregulate the expression of B7-H6 in tumor cells and enhance tumor sensitivity to NK cell cytolysis. B7-H6 shRNA treatment effectively dampened sensitization of tumor cells to NK-mediated lysis. Our study not only reveals the possibility that tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis but also suggests that B7-H6 could be a potential target for tumor therapy in the future.
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Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-Homology Domains [Protein Structure and Folding]

October 15th, 2015 by

Many multi-component protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors.

ABC Transport System Solute Binding Protein-Guided Identification of Novel D-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciencs C58 [Metabolism]

October 15th, 2015 by

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute binding proteins (SBPs) for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic (TRAP) transporters. Here, we apply this strategy to the ATP-binding cassette (ABC) transporters, and report the discovery of novel catabolic pathways for D-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions: 1) oxidation of D-altritol to D-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of D-tagatose to D-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of D-tagatose 6-phosphate C-4 to D-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool (EFI-EST) and the EFI-Genome Neighborhood Tool (EFI-GNT).
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The Nogo/ Nogo-66 receptor (NgR) signal is involved in neuroinflammation through the regulation of microglia inflammatory mediator expression [Immunology]

October 15th, 2015 by

Microglia has been proposed to play a pivotal role in the inflammation response of the central nervous system (CNS) by expressing a range of proinflammatory enzymes and cytokines under pathological stimulus. Our previous study has confirmed that Nogo receptor (NgR), an axon outgrowth inhibition receptor, is also expressed on microglia and regulate cell adhesion and migration behavior in vitro. In the present study, we further investigated the pro-inflammatory effects and possible mechanisms of Nogo on microglia in vitro. In this study, Nogo peptide, Nogo-P4, a 25-aa core inhibitory peptide sequence of Nogo-66, was used. We found that Nogo-P4 was able to induce the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the release of proinflammatory cytokines including interleukin-1β (IL-1β), Tumor necrosis factor-α (TNF-α), nitric oxide (NO) and Prostaglandin E2 (PGE2) in microglia, which could be reversed by Nogo-66(1-40) antagonist peptide (NEP1-40), phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR siRNA treatment. After Nogo-P4 stimulated microglia the phosphorylation levels of NF-κB and STAT3 were increased obviously, which further mediated microglia expressing proinflammatory factors induced by Nogo-P4. Taken together, we concluded that Nogo peptide could directly take part in CNS inflammatory process by influencing the expression of proinflammatory factors in microglia, which was related to the NF-κB and STAT3 signal pathways. Besides neurite outgrowth restriction, the Nogo/NgR signal might be involved in multiple processes in various inflammation-associated CNS diseases.
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Glycogen Synthase Kinase-3{beta}-mediated Phosphorylation in the Most C-terminal Region of Protein Interacting with C Kinase 1 (PICK1) Regulates the Binding of PICK1 to Glutamate Receptor Subunit GluA2 [Molecular Bases of Disease]

October 15th, 2015 by

Protein interacting with C kinase 1 (PICK1) is a synaptic protein interacting with the AMPA receptor subunits GluA2/3. The interaction between GluA2 and PICK1 is required for the removal of GluA2 from the synaptic plasma membrane during long-term depression (LTD). It has been suggested that glycogen synthase kinase-3β(GSK-3β) is activated during LTD, but the relationships between GluA2, PICK1, and GSK-3β are not well understood. In particular, the substrate(s) of GSK-3β have not been determined yet. Here, we showed that PICK1 was a substrate of GSK-3β. We found that Ser339, Ser342, Ser412, and Ser416 of PICK1 were putative GSK-3β-mediated phosphorylation sites. Among these sites, Ser416 played a crucial role in regulating the interaction between GluA2 and PICK1. We showed that replacing Ser416 with Ala disrupted the GluA2-PICK1 interaction, while substituting Ser416 with Glu or Asp retained this interaction. However, the deletion of Ser416 did not affect the GluA2-PICK1 interaction, and the substitution of Ser416 with Ala did not alter the PICK1-PICK1 interaction. Using image analysis in COS-7 cells with AcGFP1-fused PICK1, we showed that substitution of Ser416 with Ala increased the formation of AcGFP1-positive clusters, suggesting an increase in the association of PICK1 with the membrane. This may have resulted in the dissociation of the GluA2-PICK1 complexes. Our results indicated that GSK-3β-mediated phosphorylation of PICK1 at Ser416 was required for its association with the AMPA receptor subunit. Thus, the GSK-3β-mediated phosphorylation of PICK1 may be a regulating factor during LTD induction.
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