Mechanisms for assembly of the nucleoplasmic reticulum.

The nuclear envelope consists of an outer membrane connected to the endoplasmic reticulum, an inner membrane facing the nucleoplasm and a perinuclear space separating the two bilayers. The inner and outer nuclear membranes are physically connected at nuclear pore complexes that mediate selective communication and transfer of materials between the cytoplasm and nucleus. The spherical shape of the nuclear envelope is maintained by counterbalancing internal and external forces applied by cyto- and nucleo-skeletal networks, and the nuclear lamina and chromatin that underly the inner nuclear membrane. Despite its apparent rigidity, the nuclear envelope can invaginate to form an intranuclear membrane network termed the nucleoplasmic reticulum (NR) consisting of Type-I NR contiguous with the inner nuclear membrane and Type-II NR containing both the inner and outer nuclear membranes. The NR extends deep into the nuclear interior potentially facilitating communication and exchanges between the nuclear interior and the cytoplasm. This review details the evidence that NR intrusions that regulate cytoplasmic communication and genome maintenance are the result of a dynamic interplay between membrane biogenesis and remodelling, and physical forces exerted on the nuclear lamina derived from the cyto- and nucleo-skeletal networks.

Nuclear lipid droplets in Caco2 cells originate from nascent precursors and in situ at the nuclear envelope.

Intestinal epithelial cells convert excess fatty acids into triglyceride (TAG) for storage in cytoplasmic lipid droplets and secretion in chylomicrons. Nuclear lipid droplets (nLDs) are present in intestinal cells but their origin and relationship to cytoplasmic TAG synthesis and secretion is unknown. nLDs and related lipid-associated promyelocytic leukemia structures (LAPS) were abundant in oleate-treated Caco2 but less frequent in other human colorectal cancer cell lines and mouse intestinal organoids. nLDs and LAPS in undifferentiated oleate-treated Caco2 cells harbored the phosphatidate phosphatase Lipin1, its product diacylglycerol, and CTP:phosphocholine cytidylyltransferase (CCT)α. CCTα knockout Caco2 cells had fewer but larger nLDs, indicating a reliance on de novo PC synthesis for assembly. Differentiation of Caco2 cells caused large nLDs and LAPS to form regardless of oleate treatment or CCTα expression. nLDs and LAPS in Caco2 cells did not associate with apoCIII and apoAI and formed dependently of microsomal triglyceride transfer protein expression and activity, indicating they are not derived from endoplasmic reticulum luminal LDs precursors. Instead, undifferentiated Caco2 cells harbored a constitutive pool of nLDs and LAPS in proximity to the nuclear envelope that expanded in size and number with oleate treatment. Inhibition of TAG synthesis did affect the number of nascent nLDs and LAPS but prevented their association with promyelocytic leukemia protein, Lipin1α, and diacylglycerol, which instead accumulated on the nuclear membranes. Thus, nLD and LAPS biogenesis in Caco2 cells is not linked to lipoprotein secretion but involves biogenesis and/or expansion of nascent nLDs by de novo lipid synthesis.

Differential contributions of phosphotransferases CEPT1 and CHPT1 to phosphatidylcholine homeostasis and lipid droplet biogenesis.

The cytidine diphosphate-choline (Kennedy) pathway culminates with the synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by choline/ethanolamine phosphotransferase 1 (CEPT1) in the endoplasmic reticulum (ER), and PC synthesis by choline phosphotransferase 1 (CHPT1) in the Golgi apparatus. Whether the PC and PE synthesized by CEPT1 and CHPT1 in the ER and Golgi apparatus has different cellular functions has not been formally addressed. Here, we used CRISPR editing to generate CEPT1-and CHPT1-KO U2OS cells to assess the differential contribution of the enzymes to feedback regulation of nuclear CTP:phosphocholine cytidylyltransferase (CCT)α, the rate-limiting enzyme in PC synthesis, and lipid droplet (LD) biogenesis. We found that CEPT1-KO cells had a 50 and 80% reduction in PC and PE synthesis, respectively, while PC synthesis in CHPT1-KO cells was also reduced by 50%. CEPT1 KO caused the posttranscriptional induction of CCTα protein expression as well as its dephosphorylation and constitutive localization on the inner nuclear membrane and nucleoplasmic reticulum. This activated CCTα phenotype was prevented by incubating CEPT1-KO cells with PC liposomes to restore end-product inhibition. Additionally, we determined that CEPT1 was in close proximity to cytoplasmic LDs and CEPT1 KO resulted in the accumulation of small cytoplasmic LDs, as well as increased nuclear LDs enriched in CCTα. In contrast, CHPT1 KO had no effect on CCTα regulation or LD biogenesis. Thus, CEPT1 and CHPT1 contribute equally to PC synthesis; however, only PC synthesized by CEPT1 in the ER regulates CCTα and the biogenesis of cytoplasmic and nuclear LDs.

Mechanisms by Which Probiotic Bacteria Attenuate the Risk of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second leading cause of cancer-related deaths worldwide. Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV), alcoholic liver disease (ALD), and non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) are the major extrinsic risk factors of HCC development. Genetic background is pivotal in HCC pathogenesis, and both germline mutations and single nucleotide polymorphism (SNP) are intrinsic risk factors of HCC. These HCC risk factors predispose to hepatic injury and subsequent activation of fibrogenesis that progresses into cirrhosis and HCC. Probiotic bacteria can mitigate HCC risk by modulating host gut microbiota (GM) to promote growth of beneficial microbes and inhibit HCC-associated dysbiosis, thus preventing pathogen-associated molecular patterns (PAMPs)-mediated hepatic inflammation. Probiotics have antiviral activities against HBV and HCV infections, ameliorate obesity and risk of NAFLD/NASH, and their antioxidant, anti-proliferative, anti-angiogenic, and anti-metastatic effects can prevent the HCC pathogenesis. Probiotics also upregulate the expression of tumor suppressor genes and downregulate oncogene expression. Moreover, metabolites generated by probiotics through degradation of dietary phytochemicals may mitigate the risk of HCC development. These multiple anticancer mechanisms illustrate the potential of probiotics as an adjuvant strategy for HCC risk management and treatment.

Protein kinase D1 and oxysterol-binding protein form a regulatory complex independent of phosphorylation.

Protein kinase D (PKD) controls secretion from the trans-Golgi network (TGN) by phosphorylating phosphatidylinositol 4-kinase IIIß and proteins that bind and/or transfer phosphatidylinositol 4-phosphate (PtdIns-4P), such as oxysterol-binding protein (OSBP) and ceramide transfer protein. Here, we investigated the consequences of PKD phosphorylation of OSBP at endoplasmic reticulum (ER)-Golgi membrane contact sites (MCS). Results with OSBP phospho-mutants revealed that PKD phosphorylation did not affect sterol and PtdIns-4P binding, activation of sphingomyelin (SM) synthesis at Golgi-ER MCS or other OSBP phospho-sites. Instead, an interaction was identified between the N-terminal region of OSBP and PKD1 that was independent of kinase activity and OSBP phosphorylation status. S916 autophosphorylation of PKD1 was inhibited by OSBP expression suggesting the interaction negatively regulates PKD1 activity. Stimulation of PKD1 activity by phorbol ester promoted the Golgi-localization of wild-type and phospho-mutants of OSBP but did not affect OSBP-dependent SM synthesis. Only when wild-type or kinase-dead PKD1 was overexpressed was 25-hydroxycholesterol-activated SM synthesis inhibited. We conclude that OSBP and PKD1 form a complex that inhibits both the oxysterol-dependent activity of OSBP at the ER-Golgi and activation of PKD1. Formation of the complex was independent of PKD1 activity and phosphorylation of OSBP.

Golgi localization of oxysterol binding protein-related protein 4L (ORP4L) is regulated by ligand binding.

Oxysterol binding protein (OSBP)-related protein 4L (ORP4L, also known as OSBPL2), a closely related paralogue and interacting partner of OSBP, binds sterols and phosphatidylinositol 4-phosphate [PI(4)P] and regulates cell proliferative signalling at the plasma membrane (PM). Here, we report that ORP4L also interacts with the trans-Golgi network (TGN) in an OSBP-, sterol- and PI(4)P-dependent manner. Characterization of ORP4L lipid and VAP binding mutants indicated an indirect mechanism for translocation to ER-Golgi contact sites in response to 25-hydroxycholesterol that was dependent on OSBP and PI(4)P. shRNA silencing revealed that ORP4L was required to maintain the organization and PI(4)P content of the Golgi and TGN. In contrast, the interaction of ORP4L with the PM was not dependent on its sterol, PI(4)P or VAP binding activities. At the PM, ORP4L partially localized with a genetically encoded probe for PI(4)P but not with a probe for phosphatidylinositol 4,5-bisphosphate. We conclude that ORP4L is differentially localized to the PM and ER-Golgi contacts sites. OSBP-, lipid- and VAP-regulated interactions of ORP4L with ER-Golgi contact sites are involved in the maintenance of Golgi and TGN structure.

Oxysterol-binding protein recruitment and activity at the endoplasmic reticulum-Golgi interface are independent of Sac1.

Oxysterol-binding protein (OSBP) localizes to endoplasmic reticulum (ER)-Golgi contact sites where it transports cholesterol and phosphatidylinositol 4-phosphate (PI-4P), and activates lipid transport and biosynthetic activities. The PI-4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER-Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co-localized at apparent ER-Golgi contact sites in response to 25-hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER-Golgi contact sites, OSBP-dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP-dependent activation of sphingomyelin synthesis at ER-Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI-4P that regulates OSBP activity or recruitment to contact sites.

How CCTα puts a leash on phospholipid synthesis.

The proportion of phosphatidylcholine (PC) in the membrane is controlled by CTP:phosphocholine cytidylyltransferase α (CCTα), which is known to be regulated by a dual auto-inhibitory and membrane-binding domain. However, the detailed mechanism by which this domain regulates CCTα activity is not clear. Ramezanpour et al. use a combined computational and biochemical approach to define new details of this mechanism, providing an elegant illustration of how the lipid-sensing domain of a phospholipid biosynthetic enzyme controls membrane homeostasis.

Bridging the molecular and biological functions of the oxysterol-binding protein family.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.

Cholesterol transfer at endosomal-organelle membrane contact sites.

PURPOSE OF REVIEW: Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). RECENT FINDINGS: LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. SUMMARY: Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

Phosphatidylcholine synthesis regulates triglyceride storage and chylomicron secretion by Caco2 cells.

Intracellular lipid droplets (LDs) supply fatty acids for energy, membrane biogenesis, and lipoprotein secretion. The surface monolayer of LDs is composed of phospholipids, primarily phosphatidylcholine (PC), that stabilize the neutral lipid core of triglyceride (TG). To determine the relationship between PC synthesis and TG storage and secretion in chylomicrons, we used a model of intestinal-derived human epithelial colorectal adenocarcinoma (Caco2) cells with knockout of PCYT1A, which encodes the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT)α in the CDP-choline pathway, that were treated with the fatty acid oleate. CRISPR/Cas9 knockout of CCTα in Caco2 cells (Caco2-KO cells) reduced PC synthesis by 50%. Compared with Caco2 cells, Caco2-KO cells exposed to oleate had fewer and larger LDs and greater TG accumulation as a result. The addition of exogenous lysophosphatidylcholine to Caco2-KO cells reversed the LD morphology defect. Caco2-KO cells, differentiated into epithelial monolayers, accumulated intracellular TG and had deficient TG and chylomicron-associated apoB48 secretion; apoB100 secretion was unaffected by CCTα knockout or oleate. Metabolic-labeling and LD imaging of Caco2-KO cells indicated preferential shuttling of de novo synthesized TG into larger LDs rather than into chylomicrons. Thus, reduced de novo PC synthesis in Caco2 cells enhances TG storage in large LDs and inhibits apoB48 chylomicron secretion.

19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome.

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT.

The role of phosphatidylcholine and choline metabolites to cell proliferation and survival.

The reorganization of metabolic pathways in cancer facilitates the flux of carbon and reducing equivalents into anabolic pathways at the expense of oxidative phosphorylation. This provides rapidly dividing cells with the necessary precursors for membrane, protein and nucleic acid synthesis. A fundamental metabolic perturbation in cancer is the enhanced synthesis of fatty acids by channeling glucose and/or glutamine into cytosolic acetyl-CoA and upregulation of key biosynthetic genes. This lipogenic phenotype also extends to the production of complex lipids involved in membrane synthesis and lipid-based signaling. Cancer cells display sensitivity to ablation of fatty acid synthesis possibly as a result of diminished capacity to synthesize complex lipids involved in signaling or growth pathways. Evidence has accrued that phosphatidylcholine, the major phospholipid component of eukaryotic membranes, as well as choline metabolites derived from its synthesis and catabolism, contribute to both proliferative growth and programmed cell death. This review will detail our current understanding of how coordinated changes in substrate availability, gene expression and enzyme activity lead to altered phosphatidylcholine synthesis in cancer, and how these changes contribute directly or indirectly to malignant growth. Conversely, apoptosis targets key steps in phosphatidylcholine synthesis and degradation that are linked to disruption of cell cycle regulation, reinforcing the central role that phosphatidylcholine and its metabolites in determining cell fate.

Oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is essential for cell proliferation and survival.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.

ras-Induced up-regulation of CTP:phosphocholine cytidylyltransferase α contributes to malignant transformation of intestinal epithelial cells.

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

The role of phospholipids in the biological activity and structure of the endoplasmic reticulum.

The endoplasmic reticulum (ER) is an interconnected network of tubular and planar membranes that supports the synthesis and export of proteins, carbohydrates and lipids. Phospholipids, in particular phosphatidylcholine (PC), are synthesized in the ER where they have essential functions including provision of membranes required for protein synthesis and export, cholesterol homeostasis, and triacylglycerol storage and secretion. Coordination of these biological processes is essential, as highlighted by findings that link phospholipid metabolism in the ER with perturbations in lipid storage/secretion and stress responses, ultimately contributing to obesity/diabetes, atherosclerosis and neurological disorders. Phospholipid synthesis is not uniformly distributed in the ER but is localized at membrane interfaces or contact zones with other organelles, and in dynamic, proliferating ER membranes. The topology of phospholipid synthesis is an important consideration when establishing the etiology of diseases that arise from ER dysfunction. This review will highlight our current understanding of the contribution of phospholipid synthesis to proper ER function, and how alterations contribute to aberrant stress responses and disease. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Lipid- and phospho-regulation of CTP:Phosphocholine Cytidylyltransferase α association with nuclear lipid droplets.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1ß, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

A mechanism for suppression of the CDP-choline pathway during apoptosis.

Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-deficient MCF7 cells to show that caspase 3 is required for CCTα proteolysis and release from the nucleus during apoptosis. CCTα-Δ28 (a caspase-cleaved mimic) expressed in CCTα-deficient Chinese hamster ovary cells was cytosolic and had increased in vitro activity. However, [³H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCTα. In camptothecin-treated MCF7 and MCF7-C3 cells, choline kinase activity was unaffected; however, choline transport into cells was reduced by 30 and 60%, respectively. We conclude that caspase 3-mediated removal of the CCTα NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. Rather, the CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.

CTP:phosphocholine cytidylyltransferase α (CCTα) and lamins alter nuclear membrane structure without affecting phosphatidylcholine synthesis.

CTP:phosphocholine cytidylyltransferase α (CCTα) is a nuclear enzyme that catalyzes the rate-limiting step in the CDP-choline pathway for phosphatidylcholine (PC) synthesis. Lipid activation of CCTα results in its translocation to the nuclear envelope and expansion of an intranuclear membrane network termed the nucleoplasmic reticulum (NR) by a mechanism involving membrane deformation. Nuclear lamins are also required for stability and proliferation of the NR, but whether this unique structure, or the nuclear lamina in general, is required for PC synthesis is not known. To examine this relationship, the nuclear lamina was depleted by RNAi or disrupted by expression of the Hutchinson-Gilford progeria syndrome (HGPS) mutant lamin A (progerin), and the effect on CCTα and choline metabolism was analyzed. siRNA-mediated silencing of lamin A/C or lamin B1 in CHO cells to diminish the NR had no effect on PC synthesis, while double knockdown non-specifically inhibited the pathway. Confirming this minor role in PC synthesis, only 10% of transiently overexpressed choline/ethanolamine phosphotransferase was detected in the NR. In CHO cells, CCTα was nucleoplasmic and co-localized with GFP-progerin in nuclear folds and invaginations; however, HGPS fibroblasts displayed an abnormal distribution of CCTα in the cytoplasm and nuclear envelope that was accompanied by a 2-fold reduction in PC synthesis. In spite of its altered localization, choline-labeling experiments showed that CCT activity was unaffected, and inhibition of PC synthesis was traced to reduced activity of a hemicholinium-sensitive choline transporter. We conclude that CCTα and lamins specifically cooperate to form the NR, but the overall structure of the nuclear envelope has a minimal impact on CCT activity and PC synthesis.