Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions.

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT.

Autophagosome formation in relation to the endoplasmic reticulum.

Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

Measuring Phosphatidylinositol Generation on Biological Membranes.

Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.

Dysregulated phosphatidylinositol signaling promotes endoplasmic-reticulum-stress-mediated intestinal mucosal injury and inflammation in zebrafish.

Dysregulated phosphatidylinositol (PI) signaling has been implicated in human gastrointestinal (GI) malignancies and inflammatory states, underlining the need to study pathophysiological roles of PI in an in vivo genetic model. Here, we study the significance of PI in GI pathophysiology using the zebrafish mutant cdipt(hi559), which lacks PI synthesis, and unravel a crucial role of PI in intestinal mucosal integrity and inflammation. The cdipt(hi559) mutants exhibit abnormal villous architecture and disorganized proliferation of intestinal epithelial cells (IECs), with pathologies reminiscent of inflammatory bowel disease (IBD), including apoptosis of goblet cells, abnormal mucosecretion, bacterial overgrowth and leukocyte infiltration. The mutant IECs exhibit vacuolation, microvillus atrophy and impaired proliferation. The cdipt(hi559) gene expression profile shows enrichment of acute phase response signaling, and the endoplasmic reticulum (ER) stress factors hspa5 and xbp1 are robustly activated in the mutant GI tissue. Temporal electron micrographic analyses reveal that PI-deficient IECs undergo sequential ER-Golgi disruption, mitochondrial depletion, macroautophagy and cell death, consistent with chronic ER-stress-mediated cytopathology. Furthermore, pharmacological induction of ER stress by inhibiting protein glycosylation or PI synthase inhibition in leukocyte-specific reporter lines replicates the cdipt(hi559) inflammatory phenotype, suggesting a fundamental role of PI metabolism and ER stress in mucosal inflammation. Antibiotics and anti-inflammatory drugs resolved the inflammation, but not the autophagic necroapoptosis of IECs, suggesting that bacterial overgrowth can exacerbate ER stress pathology, whereas persistent ER stress is sufficient to trigger inflammation. Interestingly, the intestinal phenotype was partially alleviated by chemical chaperones, suggesting their therapeutic potential. Using zebrafish genetic and pharmacological models, this study demonstrates a newly identified link between intracellular PI signaling and ER-stress-mediated mucosal inflammation. The zebrafish cdipt mutants provide a powerful tool for dissecting the fundamental mechanisms of ER-stress-mediated human GI diseases and a platform to develop molecularly targeted therapies.