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The inflammasomes are multiprotein complexes sensing tissue damage and infectious agents to initiate innate immune responses. Different inflammasomes containing distinct sensor molecules exist. The NLRP3 inflammasome is unique as it detects a variety of danger signals. It has been reported that NLRP3 is recruited to mitochondria-associated endoplasmic reticulum membranes (MAMs) and is activated by MAM-derived effectors. Here, we show that in response to inflammasome activators, MAMs localize adjacent to Golgi membranes. Diacylglycerol (DAG) at the Golgi rapidly increases, recruiting protein kinase D (PKD), a key effector of DAG. Upon PKD inactivation, self-oligomerized NLRP3 is retained at MAMs adjacent to Golgi, blocking assembly of the active inflammasome. Importantly, phosphorylation of NLRP3 by PKD at the Golgi is sufficient to release NLRP3 from MAMs, resulting in assembly of the active inflammasome. Moreover, PKD inhibition prevents inflammasome autoactivation in peripheral blood mononuclear cells from patients carrying NLRP3 mutations. Hence, Golgi-mediated PKD signaling is required and sufficient for NLRP3 inflammasome activation. ISSN:0022-1007 ISSN:1540-0069 ISSN:1540-9538

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

SummaryProteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.

Autophagy is an intracellular catabolic transport route conserved among all eukaryotic cells. It has multiple important physiological functions, one of which is to act as an immune mechanism against intracellular microbes.(1,2) Bacteria and viruses targeted for destruction are sequestered into large double-membrane vesicles called autophagosomes and subsequently delivered to the lysosomes where they are consumed by resident hydrolases. Unfortunately, conserved cellular pathways are often exploited by pathogens to facilitate their entry or replication, and autophagy is no exception. It has become clear that certain bacteria and viruses subvert this process to their advantage.(3) Nidoviruses, which comprise coronaviruses and arteriviruses, might be among the successful. Long recognized as the cause of veterinary infections, this group of enveloped, positive-stranded RNA viruses is currently of considerable interest due to the emergence of new human coronaviruses, especially the severe acute respiratory syndrome (SARS)-coronavirus. In this mini-review, we will summarize the so far limited number of studies that have demonstrated that double-membrane vesicles resembling autophagosomes are the sites of nidovirus replication. In addition, we will discuss how the formation of these large vesicles might be induced.