ADP Ribosylation Factors 1 and 4 and Group VIA Phospholipase A2 Regulate Morphology and Intraorganellar Traffic in the Endoplasmic Reticulum–Golgi Intermediate Compartment

In search of morphological determinants for the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), we found that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC by regulating tubular carriers. This is predicted to impact the rate of transport and destination of cargos in the ERGIC

CK2 Phosphorylates Sec31 and Regulates ER-To-Golgi Trafficking

Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking

NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins towards Virus Assembly

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly

Tissue and subcellular localization of mammalian renalase, a FAD-containing protein involved in the pathogenesis of cardiovascular diseases

Renalase is a secretory protein and flavoenzyme that is ubiquitous in vertebrates and conserved in some other phyla. In mammals it has been shown to modulate cardiovascular responses, being particularly active in decreasing catecholaminergic tone, lowering blood pressure, and in protecting the heart against ischemic damage (1). Lowered renalase levels in tissue and plasma might be the basis of the cardiovascular complications observed in chronic kidney disease patients (1). Renalase secretion into the circulation is enhanced in response to stressors such as hypotension, but the molecular mechanism regulating its basal or stimulated secretion are unknown(2). We find that renalase has a signal-sequence, but this sequence is not cleaved prior to its secretion, suggesting that it may traffic in an atypical secretory pathway. In pig kidney, our immunofluorescence studies showed that renalase is exclusively expressed in the proximal tubule. Similar studies in human immortalized HK-2 cells, as well as on pig and mouse primary cell lines, indicated that renalase is preferentially localized in the cytoplasm, where it shows a punctate distribution, suggestive of an organelle association. The identification of these subcellular compartment(s), mechanism of association, and renalase\u2019s mechanism of secretion are underway. This knowledge could lead to novel therapies for cardiovascular and kidney diseases (3). This work has been supported by travel fellowships granted by the Italian Society of Biochemistry and Molecular Biology (SIB) and by the Consorzio Interuniversitario di Biotecnologie to S. Baroni. 1. Desir GV. Curr Opin Nephrol Hypertens. 2011; 20: 31-6. 2. Milani M, et al. J Mol Biol. 2011; 411: 463-73. 3. Unger T, et al. Eur Heart J. 2011; 32: 2739-47

Activation of soluble adenylyl cyclase protects against secretagogue stimulated zymogen activation in rat pancreaic acinar cells.

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis

Bicarbonate treatment reduces acinar cell vacuole formation but not blebbing.

<p>Acini were treated with or without bicarbonate (25 mM) either alone or in the presence of either CER 100 nM or CARB 1 mM for 1 hour. Cells were collected and fixed in PLP fixative followed by embedding in EPON. Sections were cut and stained with hematoxylin and examined microscopically. Control (A), CER (B), CARB (C), Bicarbonate (D), CER+Bicarbonate (E) and CARB+Bicarbonate (F). Each is a representative photograph (40x magnification). Vacuoles are indicated by arrowheads.</p