RESUMO
CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTß catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.
Assuntos
Colina-Fosfato Citidililtransferase/metabolismo , Gotículas Lipídicas/metabolismo , Membrana Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Colina/metabolismo , Colina-Fosfato Citidililtransferase/química , Células HeLa , Humanos , Modelos Biológicos , Ácido Oleico/metabolismo , Fosforilação , Transporte Proteico , RatosRESUMO
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.
Assuntos
Proteína Quinase C/metabolismo , Receptores de Esteroides/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosfatidilinositóis/metabolismo , Fosforilação , Ligação ProteicaRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico , Retículo Endoplasmático/enzimologia , Inativação Gênica , Complexo de Golgi/enzimologia , Células HeLa , Humanos , Hidroxicolesteróis/metabolismo , Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol , Colesterol/metabolismo , Mitocôndrias , Membranas Mitocondriais/enzimologia , Proteínas Recombinantes de Fusão , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Rede trans-Golgi/metabolismo , Substituição de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetulus , Deleção de Genes , Humanos , Hidroxicolesteróis/metabolismo , Mutação , Transporte Proteico , Interferência de RNA , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Pele/citologiaRESUMO
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.
Assuntos
Apoptose/fisiologia , Proliferação de Células , Receptores de Esteroides/metabolismo , Linhagem Celular Transformada , Sobrevivência Celular/fisiologia , Genes ras/genética , Células HEK293 , Células HeLa , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipídeos/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Esteroides/química , Receptores de Esteroides/genética , Esteróis/metabolismo , Vimentina/metabolismoRESUMO
Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Endossomos/química , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Células CHO , Proteínas de Transporte/genética , Cricetinae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/deficiência , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteína C1 de Niemann-Pick , Proteínas de Transporte Vesicular , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
Rainbow smelt (Osmerus mordax) is an anadromous teleost that, beginning in late fall, accumulates plasma glycerol in excess of 200 mM, which subsequently decreases in the spring. The activity of cytosolic glycerol-3-phosphate dehydrogenase (cGPDH) is higher (i) in liver of smelt than in that of Atlantic salmon and capelin (nonglycerol accumulators), (ii) in liver of smelt maintained at 1°C than in that of smelt held at 8°-10°C, and (iii) in smelt liver than in smelt muscle, heart, brain, or kidney. In addition, transcript levels of cGPDH in liver peak in December during the onset of glycerol production and then decline over the remainder of the season. There are four cGPDH protein isoforms in smelt liver that are present regardless of glycerol production status. A minimum of four cGPDH gene copies identified by Southern blotting provide adequate genetic potential to yield multiple protein isoforms. A full-length cDNA for smelt mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) was cloned and characterized. The 2,790-bp cDNA contains a 109-bp 5'UTR, a 2,193-bp open reading frame, and a 488-bp 3'UTR; transcripts are ubiquitously expressed in both warm- and cold-acclimated smelt tissues. Smelt mGPDH encodes a 730-aa protein that clusters with that of zebrafish and frog and contains several common structural motifs. mGPDH transcript levels generally increase late in the seasonal glycerol cycle, and mGPDH enzyme activity increases significantly during the glycerol decrease phase. Taken together, these findings suggest that liver cGPDH and mGPDH play a key role in the glycerol accumulation and decrease phases, respectively.
Assuntos
Regulação Enzimológica da Expressão Gênica , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Osmeriformes/genética , Osmeriformes/metabolismo , Aclimatação , Sequência de Aminoácidos , Animais , Citosol/metabolismo , DNA Complementar/genética , Eletroforese , Congelamento , Dosagem de Genes , Perfilação da Expressão Gênica , Glicerol/sangue , Glicerol/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Terra Nova e Labrador , Filogenia , Reação em Cadeia da Polimerase , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Salmão/metabolismo , Estações do Ano , Especificidade da EspécieRESUMO
Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. However, mitochondrial cholesterol content was higher in NPC1-deficient than in wild-type cells. Cholesterol transport to the mitochondrial inner membrane increased markedly upon exposure of cholesterol-deprived cells to lipoproteins, indicating transport of endosomal cholesterol to mitochondria. Reduction of endosomal metastatic lymph node protein 64 (MLN64) by RNA interference decreased cholesterol transport to the mitochondrial inner membrane and reduced mitochondrial cholesterol levels in NPC1-deficient cells, suggesting that MLN64 transported cholesterol to mitochondria even in the absence of NPC1. In summary, this study describes a transport pathway for endosomal cholesterol to mitochondria that requires MLN64, but not NPC1, and that may be responsible for increased mitochondrial cholesterol in NPC disease.