RESUMO
LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Proteínas de Ligação às Penicilinas/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Masculino , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/genética , Polímeros/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) is thought to lipidate apolipoprotein A-I (apoA-I) at the plasma membrane, with endosomal cholesterol contributing as substrate. The mechanisms of ABCA1 surface delivery are not well understood. We have shown that Rab8 regulates endosomal cholesterol removal to apoA-I in human fibroblasts. Here, we investigated whether Rab8 plays a role in ABCA1 plasma membrane expression and cholesterol removal in primary human macrophages. METHODS AND RESULTS: We found that Rab8 was abundantly expressed in human atherosclerotic lesional macrophages and upregulated on lipid loading of macrophages in vitro. Adenoviral overexpression of Rab8 increased ABCA1 protein levels and reduced cholesterol deposition in macrophage foam cells incubated with apoA-I. Depletion of Rab8 decreased the fraction of ABCA1 at the plasma membrane and inhibited the efflux of lipoprotein-derived endosomal cholesterol to apoA-I. In Rab8-depleted cells, ABCA1-GFP localized in beta1 integrin and transferrin receptor containing recycling organelles. CONCLUSIONS: Rab8 reduces foam cell formation by facilitating ABCA1 surface expression and stimulating endosomal cholesterol efflux to apoA-I in primary human macrophages.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Doença da Artéria Coronariana/metabolismo , Células Espumosas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células Cultivadas , Endossomos/metabolismo , Humanos , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.
Assuntos
Colesterol/isolamento & purificação , Colesterol/metabolismo , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/patologia , Doenças de Niemann-Pick/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Linhagem Celular , Proteínas de Ligação a DNA/agonistas , Expressão Gênica , Humanos , Receptores X do Fígado , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Esfingolipídeos/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/isolamento & purificação , Proteínas rab4 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Chemical modification of mitochondria with the arginine-specific reagents phenylglyoxal (PGO) and 2,3-butanedione (BAD) decreases the Ca(2+) sensitivity of the permeability transition pore (PTP) and stabilizes it in the closed conformation (Eriksson, O., Fontaine, E., and Bernardi, P. (1998) J. Biol. Chem. 273, 12669-12674). Unexpectedly, modification of mitochondria with the arginine-specific reagent p-hydroxyphenylglyoxal (OH-PGO) resulted instead in PTP opening. Sequential modification with OH-PGO and PGO (or BAD) revealed that the effects on the PTP depended on the order of the additions. PTP opening was observed when OH-PGO preceded, and PTP closing was observed when OH-PGO followed, the addition of PGO (or BAD). The differential effects of OH-PGO and PGO on the PTP open probability (i) were not modified by the conformation-specific ligands of the adenine nucleotide translocase bongkrekate and atractylate; and (ii) were also observed in de-energized mitochondria, indicating that the effect is exerted directly on the PTP. OH-PGO dramatically sensitized PTP opening, which was triggered by depolarization even in the presence of EGTA. These data show that arginine modification modulates the PTP conformation in a ligand-selective fashion and suggest that the effects of OH-PGO, PGO, and BAD are mediated by the same arginine residues. We analyzed the structure of the arginine adducts by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry using a test peptide and N-acetylarginine. The results indicate that both OH-PGO and PGO react with arginine at a stoichiometry of 2:1 and form stable adducts that may be feasible to identify the PTP at the molecular level.
Assuntos
Arginina/química , Canais Iônicos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Fenilglioxal/farmacologia , Animais , Arginina/metabolismo , Cálcio/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Fígado/química , Fígado/metabolismo , Masculino , Proteínas de Membrana/química , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Estrutura Molecular , Fenilglioxal/química , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Astrocytes secrete cholesterol in lipoprotein particles. Here we show that primary murine embryonic astrocytes secrete endogenously synthesized cholesterol but also the cholesterol precursors desmosterol and lathosterol. In astrocyte membranes, desmosterol and cholesterol were the predominant sterols. Astrocytes derived from Niemann-Pick type C lipidosis (NPC1-/-) mice displayed late endosomal cholesterol deposits, but the secretion of biosynthetic sterols from the cells was not inhibited. Both wild-type and NPC1-/- astrocytes secreted the NPC2 protein. Size-exclusion chromatography combined with electron microscopy showed that the majority of sterols were secreted separately from NPC2 in heterogeneous spherical particles with an average diameter of 20 nm. These data suggest that NPC2 and the majority of sterols secreted from astrocytes are not released together and that the secretion of neither sterols nor NPC2 requires NPC1 function. In addition, the findings reveal a complexity of sterol species in astrocytes and bring up the possibility that some of the effects assigned to astrocyte cholesterol may be attributed to its penultimate precursors.
Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Proteínas de Transporte/fisiologia , Glicoproteínas/fisiologia , Proteínas/fisiologia , Esteróis/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Células Cultivadas , Centrifugação com Gradiente de Concentração , Colesterol/metabolismo , Cromatografia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , DNA Complementar/metabolismo , Fibroblastos/metabolismo , Genótipo , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Proteína C1 de Niemann-Pick , Proteínas/genética , Proteínas/isolamento & purificação , Vírus da Floresta de Semliki/genética , Sacarose/farmacologia , Temperatura , Fatores de Tempo , Proteínas de Transporte VesicularRESUMO
Methylglyoxal (MG) (pyruvaldehyde) is a reactive carbonyl compound produced in glycolysis. MG can form covalent adducts on proteins resulting in advanced glycation end products that may alter protein function. Here we report that MG covalently modifies the mitochondrial permeability transition pore (PTP), a high conductance channel involved in the signal transduction of cell death processes. Incubation of isolated mitochondria with MG for a short period of time (5 min), followed by removal of excess free MG, prevented both ganglioside GD3- and Ca2+-induced PTP opening and the ensuing membrane depolarization, swelling, and cytochrome c release. Under these conditions MG did not significantly interfere with mitochondrial substrate transport, respiration, or oxidative phosphorylation. The suppression of permeability transition was reversible following extended incubation in MG-free medium. Of the 29 physiological carbonyl and dicarbonyl compounds tested only MG and its analogue glyoxal were able to specifically alter the behavior of the PTP. Using a set of arginine-containing peptides, we found that the major MG-derived arginine adduct formed, following a short time exposure to MG, was the 5-hydro-5-methylimidazol-4-one derivative. These findings demonstrate that MG rapidly modifies the PTP covalently and stabilizes the PTP in the closed conformation. This is probably due to the formation of an imidazolone adduct on an arginine residue involved in the control of PTP conformation (Linder, M. D., Morkunaite-Haimi, S., Kinnunen, P. J. K., Bernardi, P., and Eriksson, O. (2002) J. Biol. Chem. 277, 937-942). We deduce that the permeability transition constitutes a potentially important physiological target of MG.
Assuntos
Mitocôndrias Hepáticas/fisiologia , Aldeído Pirúvico/farmacologia , Animais , Grupo dos Citocromos c/análise , Cinética , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Permeabilidade , Ratos , Ratos WistarRESUMO
Niemann-Pick type C (NPC) disease is a fatal recessively inherited lysosomal cholesterol-sphingolipidosis. Mutations in the NPC1 gene cause approximately 95% of the cases, the rest being caused by NPC2 mutations. Here the molecular basis of a severe infantile form of the disease was dissected. The level of NPC1 protein in the patient fibroblasts was similar to that in control cells. However, the protein was partially mislocalized from late endocytic organelles diffusely to the cell periphery. In contrast, NPC2 was upregulated and accumulated in cholesterol storing late endocytic organelles. Two point mutations and a four-nucleotide deletion were identified in the NPC1 gene, leading to the amino acid substitutions C113R, P237S and deletion of 37 C-terminal amino acids (delC). Overexpression of individual NPC1 mutations revealed that delC produced an unstable protein, wild-type and NPC1-P237S colocalized with Rab7-positive late endosomes whereas NPC1-C113R localized to the ER, Rab7-negative endosomes and the cell surface. Expression of wild-type or NPC1-P237S cleared the lysosomal cholesterol accumulation in NPC1-deficient cells whereas C113R or delC did not. In the Finnish and Swedish population samples, alleles carrying C113R or delC were not identified, whereas approximately 5% of the alleles carried P237S. Our studies identify P237S as a prevalent NPC1 polymorphism and delC and C113R as deleterious NPC1 mutations. Moreover, they show that delC leads to rapid degradation of NPC1 and C113R to endocytic missorting of the protein. These changes are accompanied by lysosomal accumulation of NPC2, suggesting that NPC1 governs the endocytic transport of NPC2.