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

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

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

Risk factors for pancreatic cancer: underlying mechanisms and potential targets

Purpose of the review: Pancreatic cancer is extremely aggressive, forming highly chemo-resistant tumors, and has one of the worst prognoses. The evolution of this cancer is multi-factorial. Repeated acute pancreatic injury and inflammation are important contributing factors in the development of pancreatic cancer. This article attempts to understand the common pathways linking pancreatitis to pancreatic cancer. Recent findings: Intracellular activation of both pancreatic enzymes and the transcription factor NF-κB are important mechanisms that induce acute pancreatitis (AP). Recurrent pancreatic injury due to genetic susceptibility, environmental factors such as smoking, alcohol intake, and conditions such as obesity lead to increases in oxidative stress, impaired autophagy and constitutive activation of inflammatory pathways. These processes can stimulate pancreatic stellate cells, thereby increasing fibrosis and encouraging chronic disease development. Activation of oncogenic Kras mutations through inflammation, coupled with altered levels of tumor suppressor proteins (p53 and p16) can ultimately lead to development of pancreatic cancer. Summary: Although our understanding of pancreatitis and pancreatic cancer has tremendously increased over many years, much remains to be elucidated in terms of common pathways linking these conditions

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