佐々木 桂奈江

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death. OSW-1, a natural compound with anticancer activity, potently inhibits OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the PI4P pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed by the loss of regulators of PI4P synthesis, such as PITPNB and PI4KB. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi.

Abstract The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

Abstract The Golgi stress response is a homeostatic mechanism that augments Golgi function when Golgi function becomes insufficient (Golgi stress). Glycosylation of the core proteins of proteoglycans is one of the important functions of the Golgi. If the production of core proteins is increased and the amount of glycosylation enzymes for proteoglycans becomes insufficient (PG-type Golgi stress), the proteoglycan pathway of the Golgi stress response is activated, resulting in the transcriptional induction of glycosylation enzymes, including NDST2, HS6ST1 and GLCE. The transcriptional induction of these glycosylation enzymes is regulated by the enhancer element, PGSE-A; however, transcription factors that induce transcription from PGSE-A have not yet been identified. We herein proposed KLF2 and KLF4 as candidate transcription factors for transcriptional induction from PGSE-A, and revealed that their expression was up-regulated in response to PG-type Golgi stress. These results suggest that KLF2 and KLF4 are important regulators of the proteoglycan pathways of the mammalian Golgi stress response.

OSW-1 is a plant-derived natural product proposed to selectively kill cancer cells by binding to members of the oxysterol binding protein family, thereby disrupting lipid/sterol homeostasis. However, how these protein-ligand interactions mediate cell death signaling has remained elusive. Here, we discovered that OSW-1 selectively activates the Golgi stress response leading to apoptosis, providing a mechanistic basis for the anticancer activity of OSW-1.

Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or -macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca(2+)-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using biotinylated ALG-2. Dependency of Ca(2+) for interaction is examined by inclusion of CaCl(2) or EGTA in buffers used for binding assays.