ABSTRACT
Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.
Subject(s)
Endoplasmic Reticulum/metabolism , GTP Phosphohydrolases/metabolism , Membrane Fusion/physiology , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Microsomes/metabolism , Proteolipids/metabolism , Qa-SNARE Proteins/metabolism , Qb-SNARE Proteins/metabolism , R-SNARE Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/antagonists & inhibitors , Vesicular Transport Proteins/antagonists & inhibitors , Vesicular Transport Proteins/geneticsABSTRACT
Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA--a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking--protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-alpha and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.
Subject(s)
Antineoplastic Agents , Cisplatin , Drug Resistance, Neoplasm/genetics , Gene Knockdown Techniques , RNA, Small Interfering/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Cell Line , Cisplatin/therapeutic use , Female , Gene Knockdown Techniques/methods , HeLa Cells , Humans , Lentivirus/genetics , Mice , Mice, Nude , RNA Interference , RNA, Small Interfering/chemistry , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/biosynthesis , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Xenograft Model Antitumor Assays/methodsABSTRACT
Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF-stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with gamma-secretase inhibitors or Notch receptor-specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions.
