Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and alpha-SNAP) in the enlarged/circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the "disassembly" of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCT-induced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.
Am J Physiol Lung Cell Mol Physiol 2007 Jun
PMID:Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension. 1733 6

Discussions of the initiation of pulmonary arterial hypertension (PAH) in man and in experimental models have centered around intimal and medial proliferation in medium-sized pulmonary arteries. The histologic events are thought to include disordered proliferation of enlarged, vacuolated endothelial cells, neo-muscularization of the affected blood vessels, and vascular pruning. The discovery of the association of familial and sporadic PAH with mutations in BMPR2 has generated intense interest in cytokine receptor trafficking and function in the endothelial cell and how this might be disrupted to yield an enlarged proliferative cell phenotype. Nevertheless, considerations of the subcellular machinery of membrane trafficking in the endothelial cell and consequences of the disruption of this outward and inward membrane trafficking are largely absent from discussions of the pathobiology of PAH. Long-standing electron microscopy data in the PAH field has demonstrated marked disruptions of intracellular membrane trafficking in human and experimental PAH. Further, a role of the membrane-trafficking regulator Nef in simian HIV-induced PAH in macaques and in HIV-induced PAH in man is now evident. Additionally, monocrotaline and hypoxia are known to disrupt the function of Golgi tethers, SNAREs, SNAPs, and N-ethylmaleimide-sensitive factor ("the Golgi blockade hypothesis"). These results, along with recent reports demonstrating the trapping of PAH-associated human BMPR2 mutants in the Golgi, highlight the implications of disrupted intracellular membrane trafficking in the pathobiology of PAH. The purpose of this review is to present a brief overview of the molecular basis of intracellular trafficking and relate these considerations to the pathobiology of PAH.
Am J Respir Cell Mol Biol 2007 Jul
PMID:Dysfunctional intracellular trafficking in the pathobiology of pulmonary arterial hypertension. 1736 75

The SNAP25-type proteins belong to the superfamily of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and function as important components of the vesical trafficking machinery in eukaryotic cells. In this paper, we report the cloning and expression characterization of OsSNAP32 gene, and the subcellular localization of its encoded protein. The OsSNAP32 gene contains five exons and four introns, and is located between RFLP markers C12276S and S1917 on chromosome 2 in rice. The OsSNAP32 has a molecular weight of 31.3 kD, comprises 283 amino acid residues, and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal, respectively. Multiple sequence alignment of the SNARE domains indicates that OsSNAP32 protein is homologous to HvSNAP34 and HvSNAP28 (63% and 55% of amino acid identity respectively) from barley. The transient expression method in onion epidermal cells, revealed that OsSNAP32 is located in the plasma membrane, like other SNAP25-type proteins. Semi-quantitative RT-PCR assay showed that the OsSNAP32 is highly expressed in leaves and culms, and low in roots of rice, while hardly detected in immature spikes and flowering spikes. The expression of OsSNAP32 was significantly activated in rice seedlings treated with H2O2, PEG6000, and low temperature or after inoculation with rice blast (Magnaporthe grisea strain Hoku 1). The results suggest that this gene belongs to a novel member of this gene family encoding SNAP25-type proteins, involved in the rice responses to biotic and abiotic stresses.
Mol Biol Rep 2008 Jun
PMID:Molecular cloning and characterization of a novel SNAP25-type protein gene OsSNAP32 in rice (Oryza sativa L.). 1738 Apr 28

We identified in a yeast two-hybrid screen the EF-hand Ca(2+)-binding protein Cab45 as an interaction partner of Munc18b. Although the full-length Cab45 resides in Golgi lumen, we characterize a cytosolic splice variant, Cab45b, expressed in pancreatic acini. Cab45b is shown to bind (45)Ca(2+), and, of its three EF-hand motifs, EF-hand 2 is demonstrated to be crucial for the ion binding. Cab45b is shown to interact with Munc18b in an in vitro assay, and this interaction is enhanced in the presence of Ca(2+). In this assay, Cab45b also binds the Munc18a isoform in a Ca(2+)-dependent manner. The endogenous Cab45b in rat acini coimmunoprecipitates with Munc18b, syntaxin 2, and syntaxin 3, soluble N-ethylmaleimide-sensitive factor attachment protein receptors with key roles in the Ca(2+)-triggered zymogen secretion. Furthermore, we show that Munc18b bound to syntaxin 3 recruits Cab45b onto the plasma membrane. Importantly, antibodies against Cab45b are shown to inhibit in a specific and dose-dependent manner the Ca(2+)-induced amylase release from streptolysin-O-permeabilized acini. The present study identifies Cab45b as a novel protein factor involved in the exocytosis of zymogens by pancreatic acini.
Mol Biol Cell 2007 Jul
PMID:A cytosolic splice variant of Cab45 interacts with Munc18b and impacts on amylase secretion by pancreatic acini. 1744 89

Recycling of G protein-coupled receptors determines the functional resensitization of receptors and is implicated in switching beta2 adrenoceptor (beta2AR) G protein specificity in cardiomyocytes. The human beta2AR carboxyl end binds to the N-ethylmaleimide-sensitive factor (NSF), an ATPase integral to membrane trafficking machinery. It is interesting that the human beta2AR (hbeta2AR) carboxyl end pulled down NSF from mouse heart lysates, whereas the murine one did not. Despite this difference, both beta2ARs exhibited substantial agonist-induced internalization, recycling, and Gi coupling in cardiomyocytes. The hbeta2AR, however, displayed faster rates of agonist-induced internalization and recycling compared with the murine beta2AR (mbeta2AR) and a more profound Gi component in its contraction response. Replacing the mbeta2AR proline (-1) with a leucine generated a gain-of-function mutation, mbeta2AR-P417L, with a rescued ability to bind NSF, faster internalization and recycling than the mbeta2AR, and a significant enhancement in Gi signaling, which mimics the hbeta2AR. Selective disruption of the mbeta2AR-P417L binding to NSF inhibited the receptor coupling to Gi. Mean-while, inhibiting NSF with N-ethylmaleimide blocked the mbeta2AR recycling after agonist-induced endocytosis. Expressing the NSF-E329Q mutant lacking ATPase activity inhibited the mbeta2AR coupling to Gi in cardiomyocytes. Our results revealed a dual regulation on hbeta2AR trafficking and signaling by NSF through direct binding to cargo receptor and its ATPase activity and uncovered an unprecedented role for the receptor binding to NSF in regulating G protein specificity that has diverged between mouse and human beta2ARs.
Mol Pharmacol 2007 Aug
PMID:N-ethylmaleimide-sensitive factor regulates beta2 adrenoceptor trafficking and signaling in cardiomyocytes. 1751 Feb 9

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.
Mol Biol Cell 2007 Aug
PMID:Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF. 1752 84

Lung surfactant is secreted through the fusion of lamellar bodies with the plasma membrane of alveolar epithelial type II cells. Annexin A2, a Ca(2+)- and phospholipid-binding protein, promotes the fusion of lamellar bodies with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are known to have an essential role in surfactant secretion. We hypothesized that annexin A2 acts as a Ca(2+) sensor and mediates membrane fusion via its interaction with SNAREs. Both purified or endogenous annexin A2 in type II cells specifically bound with SNAP-23 in a Ca(2+)-dependent manner, as determined by pull-down experiments using recombinant glutathione S-transferase-tagged SNAP-23. A deletion study identified the cysteine-rich region (CRR) of SNAP-23 as the binding site for annexin A2. Mutations of cysteine residues in the CRR dramatically decreased the binding. SNAP-23 also co-immunoprecipitated with annexin A2; however, a SNAP-23 mutant failed to co-immunoprecipitate with annexin A2. Immunofluorescence revealed a co-localization of SNAP-23 and annexin A2 in type II cells. Furthermore, anti-SNAP-23 antibody significantly inhibited annexin A2-mediated fusion between lamellar bodies and the plasma membrane. These data suggest that annexin A2 and SNAP-23 are involved in the same pathway in the regulation of lung surfactant secretion.
Am J Respir Cell Mol Biol 2007 Oct
PMID:Physical and functional interactions of SNAP-23 with annexin A2. 1757 76

We used multiple approaches to investigate the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. We conclude that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG.
Mol Biol Cell 2007 Oct
PMID:Rab6 regulates both ZW10/RINT-1 and conserved oligomeric Golgi complex-dependent Golgi trafficking and homeostasis. 1769 96

Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of Gbetagamma with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of Gbetagamma requires the carboxyl terminus of the 25-kDa synaptosome-associated protein (SNAP25) and is downstream of the well known inhibition of Ca2+ entry through voltage-gated calcium channels. We propose a mechanism in which Gbetagamma and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the Gbetagamma interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the Gbetagamma- and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-sensitive manner with binding of Gbetagamma to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca2+ concentrations, Ca2+-synaptotagmin I can displace Gbetagamma binding and the Gbetagamma-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via Gbetagamma to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and Gbetagamma-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between Gbetagamma and synaptotagmin I for SNARE binding. This type of dynamic regulation may represent a novel mechanism for modifying transmitter release in a graded manner based on the history of action potentials that increase intracellular Ca2+ concentrations and of inhibitory signals through G(i)-coupled GPCRs.
Mol Pharmacol 2007 Nov
PMID:Gbetagamma interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. 1771 96

Lung vascular lesions in pulmonary arterial hypertension (PAH) are characterized by enlarged, vacuolated ("megalocytotic") pulmonary arterial endothelial (PAEC) and smooth muscle cells (PASMC). We have recently proposed that dysfunction of vesicle tethers, soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), and SNAP receptors (SNAREs), leading to disruptions of intracellular trafficking in the Golgi to plasma membrane (centrifugal) and the plasma membrane to cell interior (centripetal) directions is a key causal mechanism in this disease. In PAH, there was a reciprocal relationship between loss of caveolin-1 (cav-1) in PAECs and increased expression of "activated" tyrosine-phosphorylated STAT3 (PY-STAT3) associated with a block in centrifugal trafficking to/through the Golgi organelle. In the present study, we investigated 1) whether centripetal trafficking of STAT3 and PY-STAT3 in PAECs and PASMCs was membrane-associated, and 2) whether this might be affected in PAH. Immunofluorescence and live cell imaging studies showed that, in both PAEC and PASMC, STAT3 was associated with cytoplasmic vesicles partially colocalizing with markers of the endolysosomal compartments (clathrin, EEA1, Rab5, Rab11, and LAMP1). Overexpression of cav-1 increased the targeting of STAT3 to lysosomes and inhibited STAT3 transcriptional activity. Exposure of PAECs to monocrotaline (MCT) pyrrole, which causes PAH in the rat, led to a loss of caveolar STAT3 with increased sequestration of STAT3 and PY-STAT3 in endosomes. In vivo, marked cytoplasmic sequestration of activated PY-STAT3 was a common feature in PAEC in the rat/MCT model and in cells in the proliferative arterial and plexiform lesions in PAH in humans. These data highlight the epigenetic regulation of centripetal cytokine and growth-factor signaling pathways and its modulation in PAH.
Am J Physiol Lung Cell Mol Physiol 2008 Mar
PMID:Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension. 1808 67


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