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A novel mechanism for Ca(2+)/calmodulin-dependent protein kinase II targeting to L-type Ca(2+) channels that initiates long-range signaling to the nucleus.
Wang X, Marks CR, Perfitt TL, Nakagawa T, Lee A, Jacobson DA, Colbran RJ
(2017) J Biol Chem 292: 17324-17336
MeSH Terms: Animals, Calcium Channels, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cell Nucleus, Female, Hippocampus, Learning, Memory, Neurons, Protein Domains, Rats, Rats, Sprague-Dawley, Signal Transduction
Show Abstract · Added November 13, 2017
Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca(2+) channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca(2+)-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of CaV1 LTCC α1 subunits that is not conserved in CaV2 or CaV3 voltage-gated Ca(2+) channel subunits. Mutations in the CaV1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the in vitro interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Proteolytic processing of lysyl oxidase-like-2 in the extracellular matrix is required for crosslinking of basement membrane collagen IV.
López-Jiménez AJ, Basak T, Vanacore RM
(2017) J Biol Chem 292: 16970-16982
MeSH Terms: Amino Acid Oxidoreductases, Basement Membrane, Collagen Type IV, Extracellular Matrix, HEK293 Cells, Humans, Mutagenesis, Site-Directed, Protein Domains, Protein Processing, Post-Translational, Proteolysis
Show Abstract · Added November 3, 2017
Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
Zurawski Z, Page B, Chicka MC, Brindley RL, Wells CA, Preininger AM, Hyde K, Gilbert JA, Cruz-Rodriguez O, Currie KPM, Chapman ER, Alford S, Hamm HE
(2017) J Biol Chem :
Show Abstract · Added July 12, 2017
Gi/o-coupled GPCRs can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels(VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin(syt)1 for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently-labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently-labeled syt 1 undergoes competition with Gβγ for SNARE binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at higher affinity than wild-type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides, corresponding to regions on Gβ and Gγ shown to be important for the interaction, disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays utilizing full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction, and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
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Identification of a Novel Series of Anti-inflammatory and Anti-oxidative Phospholipid Oxidation Products Containing Cyclopentenone Moiety in vitro and in vivo: Implication in Atherosclerosis.
Lu J, Guo S, Xue X, Chen Q, Ge J, Zhuo Y, Zhong H, Chen B, Zhao M, Han W, Suzuki T, Zhu M, Xia L, Schneider C, Blackwell TS, Porter NA, Zheng L, Tsimikas S, Yin H
(2017) J Biol Chem :
Show Abstract · Added March 29, 2017
Oxidative stress and inflammation are two major contributing factors to atherosclerosis, a leading cause for cardiovascular diseases (CVDs). Oxidation of phospholipids on the surface of low density lipoprotein (LDL) particles generated under oxidative stress has been associated with the progression of atherosclerosis but the underlying molecular mechanisms remain poorly defined. We identified a novel series of oxidation products containing cyclopentenone moiety, termed deoxy-A2/J2-isoprostanes-phosphocholine (deoxy-A2/J2-IsoP-PC), from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) in vivo using mass spectrometry and by comparison to a chemically synthesized standard. Transcriptomic analysis (RNA-seq) demonstrated that these compounds affected over two hundred genes in bone marrow-derived macrophages (BMDMs) and genes associated with inflammatory and anti-oxidative responses are among the top five differentially expressed. To further investigate the biological relevance of these novel oxidized phospholipids in atherosclerosis, we chemically synthesized a representative compound 1-palmitoyl-2-15-deoxy-δ-12,14-Prostaglandin J2-sn-glycero-3-phosphocholine (15d-PGJ2-PC) and found that it induced anti-inflammatory and anti-oxidant responses in macrophages through modulation of NF-κB, PPARγ and Nrf2 pathways; this compound also showed potent anti-inflammatory properties in a mice model of LPS-induced systematic inflammatory response syndrome (SIRS). Additionally, 15d-PGJ2-PC inhibited macrophage foam cell formation, suggesting a beneficial role against atherosclerosis. These properties were consistent with decreased levels of these compounds in the plasma of patients with coronary heart disease (CHD) compared to control subjects. Our findings uncovered a novel molecular mechanism for the negative regulation of inflammation and positive enhancement of anti-oxidative responses in macrophages by these oxidized phospholipids in LDL in the context of atherosclerosis.
Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
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Expression of Cataract-linked γ-Crystallin Variants in Zebrafish Reveals a Proteostasis Network That Senses Protein Stability.
Wu SY, Zou P, Fuller AW, Mishra S, Wang Z, Schey KL, Mchaourab HS
(2016) J Biol Chem 291: 25387-25397
Show Abstract · Added May 6, 2017
The refractivity and transparency of the ocular lens is dependent on the stability and solubility of the crystallins in the fiber cells. A number of mutations of lens crystallins have been associated with dominant cataracts in humans and mice. Of particular interest were γB- and γD-crystallin mutants linked to dominant cataracts in mouse models. Although thermodynamically destabilized and aggregation-prone, these mutants were found to have weak affinity to the resident chaperone α-crystallin in vitro To better understand the mechanism of the cataract phenotype, we transgenically expressed different γD-crystallin mutants in the zebrafish lens and observed a range of lens defects that arise primarily from the aggregation of the mutant proteins. Unlike mouse models, a strong correlation was observed between the severity and penetrance of the phenotype and the level of destabilization of the mutant. We interpret this result to reflect the presence of a proteostasis network that can "sense" protein stability. In the more destabilized mutants, the capacity of this network is overwhelmed, leading to the observed increase in phenotypic penetrance. Overexpression of αA-crystallin had no significant effects on the penetrance of lens defects, suggesting that its chaperone capacity is not limiting. Although consistent with the prevailing hypothesis that a chaperone network is required for lens transparency, our results suggest that αA-crystallin may not be efficient to inhibit aggregation of lens γ-crystallin. Furthermore, our work implicates additional inputs/factors in this underlying proteostasis network and demonstrates the utility of zebrafish as a platform to delineate mechanisms of cataract.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Lysyl Oxidase-like-2 Cross-links Collagen IV of Glomerular Basement Membrane.
Añazco C, López-Jiménez AJ, Rafi M, Vega-Montoto L, Zhang MZ, Hudson BG, Vanacore RM
(2016) J Biol Chem 291: 25999-26012
Show Abstract · Added October 30, 2016
The 7S dodecamer is recognized as an important structural cross-linking domain of collagen IV networks that provide mechanical stability to basement membranes, a specialized form of extracellular matrix essential for the development and maintenance of tissue architecture. Although the 7S dodecamer is stabilized by covalent cross-linking, the molecular mechanism by which such cross-links are formed has not been revealed. Here, we aimed to identify the enzyme(s) that cross-links the 7S dodecamer and characterize its expression in the kidney glomerulus. Pharmacological inhibition of candidate extracellular matrix enzymes revealed that lysyl oxidase activity is required for cross-linking of 7S polypeptides. Among all lysyl oxidase family members, lysyl oxidase-like-2 (LOXL2) was identified as the isoform cross-linking collagen IV in mouse embryonal PFHR-9 cells. Biochemical analyses revealed that LOXL2 readily promoted the formation of lysyl-derived cross-links in the 7S dodecamer but not in the NC1 domain. We also established that LOXL2 is the main lysyl oxidase family member present in the glomerular extracellular matrix. Altogether, we demonstrate that LOXL2 is a novel component of the molecular machinery that forms cross-linked collagen IV networks, which are essential for glomerular basement membrane stability and molecular ultrafiltration function.
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Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.
Raval MH, Quintero OA, Weck ML, Unrath WC, Gallagher JW, Cui R, Kachar B, Tyska MJ, Yengo CM
(2016) J Biol Chem 291: 22781-22792
Show Abstract · Added April 7, 2017
Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Activation of Epidermal Growth Factor Receptor in Macrophages Mediates Feedback Inhibition of M2 Polarization and Gastrointestinal Tumor Cell Growth.
Zhao G, Liu L, Peek RM, Hao X, Polk DB, Li H, Yan F
(2016) J Biol Chem 291: 20462-72
Show Abstract · Added April 6, 2017
EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.
Kaya AI, Lokits AD, Gilbert JA, Iverson TM, Meiler J, Hamm HE
(2016) J Biol Chem 291: 19674-86
Show Abstract · Added April 8, 2017
G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Loss of Serotonin Transporter Function Alters ADP-mediated Glycoprotein αIIbβ3 Activation through Dysregulation of the 5-HT2A Receptor.
Oliver KH, Duvernay MT, Hamm HE, Carneiro AM
(2016) J Biol Chem 291: 20210-9
Show Abstract · Added October 26, 2016
Reduced platelet aggregation and a mild bleeding phenotype have been observed in patients chronically taking selective serotonin reuptake inhibitors (SSRIs). However, it remains unclear how SSRIs, which inhibit the plasma membrane serotonin transporter (SERT), modulate hemostasis. Here, we examine how sustained inhibition of SERT activity alters serotonergic signaling and influences platelet activation and hemostasis. Pharmaceutical blockade (citalopram dosing) or genetic ablation (SERT(-/-)) of SERT function in vivo led to reduced serotonin (5-hydroxytryptamine (5-HT)) blood levels that paralleled a mild bleeding phenotype in mice. Transfusion of wild-type platelets to SERT(-/-) mice normalized bleeding times to wild-type levels, suggesting that loss of SERTs causes a deficiency in platelet activation. Although SERT(-/-) platelets displayed no difference in P-selectin or αIIbβ3 activation upon stimulation with thrombin, ADP-mediated αIIbβ3 activation is reduced in SERT(-/-) platelets. Additionally, synergistic potentiation of αIIbβ3 activation by ADP and 5-HT is lost in SERT(-/-) platelets. Acute treatment of wild-type platelets with 5-HT2A receptor (5-HT2AR) antagonists or SSRIs revealed that functional 5-HT2ARs, not SERTs, are necessary for the synergistic activation of αIIbβ3 by dual 5-HT/ADP stimulation. Pharmacological studies using radiolabeled guanosine 5'-3-O-([(35)S]thio)triphosphate and [(3)H]ketanserin revealed that platelets isolated from SERT(-/-) or citalopram-treated mice have reduced activation of G-proteins coupled to 5-HT2ARs and receptor surface expression. Taken together, these data demonstrate that sustained SERT loss of function reduces 5-HT2AR surface expression that is critical for the synergistic activation of αIIbβ3 by 5-HT and ADP. These results highlight an antiplatelet strategy centered on blocking or desensitizing 5-HT2AR to attenuate ADP-mediated αIIbβ3 activation.
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