MMP Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling [Molecular Bases of Disease]

August 17th, 2015 by

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously-inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) is also reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5-S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP and control peptides followed by mass-spectrometry of the digests, we demonstrated that the mutant sequence is several-fold more sensitive to MMP-9 proteolysis relative to the wild-type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell-surface levels of the Nav1.7, Nav1.6 and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.
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Role of Pex21p for piggyback import of Gpd1p and Pnc1p into peroxisomes of Saccharomyces cerevisiae [Membrane Biology]

August 15th, 2015 by

Proteins designated for peroxisomal proteins import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, while Pex18p is especially important for oleate-induced import of PTS2-proteins. The glycerol-producing PTS2 protein glycerol-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol and in the nucleus under osmotic stress conditions. We show (i) that Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD salvage pathway, Pnc1p, and (ii) that Pnc1p, a nicotinamidase without functional PTS2 is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.

Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria [Molecular Bases of Disease]

August 14th, 2015 by

Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. Here, we show that M. tuberculosis RecG rescues E. coli delta recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.
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AMP-activated Protein Kinase Suppresses Arachidonate 15-Lipoxygenase Expression in Interleukin-4-polarized Human Macrophages [Lipids]

August 14th, 2015 by

Macrophages respond to the Th2 cytokine interleukin-4 (IL-4) with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK-activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) inhibited IL-4-evoked activation of signal transducer and activator of transcription (STAT) 3, while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. However, phenformin prevented IL-4-induced association of STAT6 and histone H3K9-acetylation at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by an ALOX15 knockdown. Finally, pre-treatment of macrophages with IL-4 for 48h increased the mRNA expression of pro-inflammatory cytokines IL-6, IL-12, CXCL9, CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubations with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.

Structural Basis for the ATP-Dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase [Protein Structure and Folding]

August 14th, 2015 by Chen, Y., Sun, Y., Song, H., Guo, Z.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes of the family including acyl/aryl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and inline alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.
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The IVVY Motif and Tumor Necrosis Factor Receptor Associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor kappa B (RANK) Cooperate to Induce Osteoclastogenesis [Cell Biology]

August 14th, 2015 by Jules, J., Wang, S., Shi, Z., Liu, J., Wei, S., Feng, X.

RANK activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TRAFs via three cytoplasmic motifs (Motif 1: PFQEP369-373; Motif 2: PVQEET559-564; and Motif 3: PVQEQG604-609) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY535-538) which is dispensable for the activation of TRAF-induced signaling pathways but is essential for osteoclast lineage commitment by inducing the expression of the nuclear factor of activated T-cells, c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF-/IL-1-mediated osteoclastogenesis requires RANKL assistance, and the IVVY motif is also critical for TNF-/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here, we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that while all three TRAF motifs play roles in TNF-/IL-1-mediated osteoclastogenesis, Motifs 2 and 3 are more potent than Motif 1. Accordingly, inactivation of Motifs 2 and 3 blocks TNF-/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of Motifs 2 and 3, similarly to inactivation of IVVY motif, abrogates the expression of NFATc1and osteoclast genes in assays reflecting RANK-initiated and TNF-/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of Motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provide a novel insight into the molecular mechanism of RANK signaling in osteoclastogenesis.
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Prolyl Isomerase Pin1 Negatively Regulates AMPK by Associating with the CBS Domain in the {gamma}-subunit [Signal Transduction]

August 14th, 2015 by

AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the β subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr221-Pro containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, while Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1 and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPK α subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes such as PGC-1α which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation, in the muscles of Pin1 KO mice, as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.

Unifying the DNA End Processing Roles of the Artemis Nuclease: Ku-Dependent Artemis Resection at Blunt DNA Ends [Enzymology]

August 14th, 2015 by Chang, H. H. Y., Watanabe, G., Lieber, M. R.

Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because during V(D)J recombination, the RAG complex generates hairpins when it creates the double-strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone, due to a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both DNA-PKcs and Ku accompany Artemis, but not when Ku is omitted. This is the first biochemical data demonstrating a Ku-dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT-sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis:DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single- to double-strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs.

Mechanisms of Inhibition and Potentiation of {alpha}4{beta}2 Nicotinic Acetylcholine Receptors by Members of the Ly6 Protein Family [Neurobiology]

August 14th, 2015 by Wu, M., Puddifoot, C. A., Taylor, P., Joiner, W. J.

α4β2 nicotinic acetylcholine receptors (nAChRs) are abundantly expressed throughout the central nervous system and are thought to be the primary target of nicotine, the main addictive substance in cigarette smoking. Understanding the mechanisms by which these receptors are regulated may assist in developing compounds to selectively interfere with nicotine addiction. Here we report previously unrecognized modulatory properties of members of the Ly6 protein family on α4β2 nAChRs. Using a FRET-based Ca2+ flux assay, we found that the maximum response of α4β2 receptors to agonist was strongly inhibited by Ly6h and Lynx2 but potentiated by Ly6g6e. The mechanisms underlying these opposing effects appear to be fundamentally distinct. Receptor inhibition by Lynx2 was accompanied by suppression of α4β2 expression at the cell surface, even when assays were preceded by chronic exposure of cells to an established chaperone, nicotine. Receptor inhibition by Lynx2 also was resistant to pretreatment with extracellular phospholipase C, which cleaves lipid moieties like those that attach Ly6 proteins to the plasma membrane. In contrast, potentiation of α4β2 activity by Ly6g6e was readily reversible by pretreatment with phospholipase C. Potentiation was also accompanied by slowing of receptor desensitization and an increase in peak currents. Collectively our data support roles for Lynx2 and Ly6g6e in intracellular trafficking and allosteric potentiation of α4β2 nAChRs, respectively.
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The steroid hormone 20-hydroxyecdysone upregulates Ste-20 family serine/threonine kinase Hippo to induce programmed cell death [Gene Regulation]

August 13th, 2015 by Dong, D.-J., Jing, Y.-P., Liu, W., Wang, J.-X., Zhao, X.-F.

The steroid hormone 20-hydroxyecdysone (20E) and the serine/threonine Ste20-like kinase Hippo signal promote programmed cell death (PCD) during development, although the interaction between them remains unclear. Here, we present evidence that 20E upregulates Hippo to induce PCD during the metamorphic development of insects. We found that Hippo is involved in 20E-induced metamorphosis via promoting the phosphorylation and cytoplasmic retention of Yorkie (Yki), causing suppressed expression of the inhibitor of apoptosis (IAP), thereby releasing its inhibitory effect on caspase. Furthermore, we show that 20E induced the expression of Hippo at the transcriptional level through the ecdysone receptor (EcR), ultraspiracle protein (USP) and hormone receptor 3 (HR3). We also found that Hippo suppresses the binding of Yki complex to the HR3 promoter. In summary, 20E upregulates the transcription of Hippo via EcRB1, USP1 and HR3 to induce PCD, and Hippo has negative feedback effects on HR3 expression. These two signaling pathways coordinate PCD during insect metamorphosis.
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