RESUMO
Newly activated CD8(+) T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals that mediate metabolic reprogramming remain poorly defined. Here we demonstrate an essential role for sterol regulatory element-binding proteins (SREBPs) in the acquisition of effector-cell metabolism. Without SREBP signaling, CD8(+) T cells were unable to blast, which resulted in attenuated clonal expansion during viral infection. Mechanistic studies indicated that SREBPs were essential for meeting the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs were dispensable for homeostatic proliferation, which indicated a context-specific requirement for SREBPs in effector responses. Our studies provide insights into the molecular signals that underlie the metabolic reprogramming of CD8(+) T cells during the transition from quiescence to activation.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Imunidade Adaptativa/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Transgenes/genéticaRESUMO
Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic (13)C6-ring-labeled compounds including (13)C6-4HB, (13)C6-pABA, (13)C6-resveratrol, and (13)C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form (13)C6-Q when cultured in the presence of (13)C6-pABA. However, E. coli cells treated with (13)C6-pABA generated (13)C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of (13)C6-resveratrol or (13)C6-coumarate were able to synthesize (13)C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.
Assuntos
Ácidos Cumáricos/metabolismo , Estilbenos/metabolismo , Ubiquinona/biossíntese , Ubiquinona/química , Animais , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Humanos , Camundongos , Propionatos , Resveratrol , Saccharomyces cerevisiae/metabolismoRESUMO
Focal segmental glomerulosclerosis (FSGS) and collapsing glomerulopathy are common causes of nephrotic syndrome. Variants in >20 genes, including genes critical for mitochondrial function, have been associated with these podocyte diseases. One such gene, PDSS2, is required for synthesis of the decaprenyl tail of coenzyme Q10 (Q10) in humans. The mouse gene Pdss2 is mutated in the kd/kd mouse model of collapsing glomerulopathy. We examined the hypothesis that human PDSS2 polymorphisms are associated with podocyte diseases. We genotyped 377 patients with primary FSGS or collapsing glomerulopathy, together with 900 controls, for 9 single-nucleotide polymorphisms in the PDSS2 gene in a case-control study. Subjects included 247 African American (AA) and 130 European American (EA) patients and 641 AA and 259 EA controls. Among EAs, a pair of proxy SNPs was significantly associated with podocyte disease, and patients homozygous for one PDSS2 haplotype had a strongly increased risk for podocyte disease. By contrast, the distribution of PDSS2 genotypes and haplotypes was similar in AA patients and controls. Thus a PDSS2 haplotype, which has a frequency of 13% in the EA control population and a homozygote frequency of 1.2%, is associated with a significantly increased risk for FSGS and collapsing glomerulopathy in EAs. Lymphoblastoid cell lines from FSGS patients had significantly less Q10 than cell lines from controls; contrary to expectation, this finding was independent of PDSS2 haplotype. These results suggest that FSGS patients have Q10 deficiency and that this deficiency is manifested in patient-derived lymphoblastoid cell lines.
Assuntos
Alquil e Aril Transferases/genética , Glomerulosclerose Segmentar e Focal/enzimologia , Glomerulosclerose Segmentar e Focal/genética , Ubiquinona/análogos & derivados , Adolescente , Adulto , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/patologia , Estudos de Casos e Controles , Glomerulosclerose Segmentar e Focal/etnologia , Haplótipos , Humanos , Ativação Linfocitária/genética , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Ubiquinona/deficiência , Ubiquinona/metabolismo , Adulto JovemRESUMO
Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable (13)C(6)-isotope of pABA (p- amino[aromatic-(13)C(6)]benzoic acid ([(13)C(6)]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[(13)C(6)]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [(13)C(6)]pABA or [(13)C(6)]4HB generate both (13)C(6)-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product (13)C(6)-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ(6). This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ(6) quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.
Assuntos
Ácido 4-Aminobenzoico/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biossíntese , Biocatálise , Ácido Corísmico/metabolismo , Genes Fúngicos/genética , Metabolismo dos Lipídeos , Liases/metabolismo , Parabenos/metabolismo , Prenilação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ubiquinona/metabolismoRESUMO
Rhodoquinone (RQ) is an important cofactor used in the anaerobic energy metabolism of Rhodospirillum rubrum. RQ is structurally similar to ubiquinone (coenzyme Q or Q), a polyprenylated benzoquinone used in the aerobic respiratory chain. RQ is also found in several eukaryotic species that utilize a fumarate reductase pathway for anaerobic respiration, an important example being the parasitic helminths. RQ is not found in humans or other mammals, and therefore inhibition of its biosynthesis may provide a parasite-specific drug target. In this report, we describe several in vivo feeding experiments with R. rubrum used for the identification of RQ biosynthetic intermediates. Cultures of R. rubrum were grown in the presence of synthetic analogs of ubiquinone and the known Q biosynthetic precursors demethylubiquinone, demethoxyubiquinone, and demethyldemethoxyubiquinone, and assays were monitored for the formation of RQ(3). Data from time course experiments and S-adenosyl-l-methionine-dependent O-methyltransferase inhibition studies are discussed. Based on the results presented, we have demonstrated that Q is a required intermediate for the biosynthesis of RQ in R. rubrum.
Assuntos
Rhodospirillum rubrum/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Cromatografia Líquida , Espectrometria de Massas , Modelos Biológicos , Estrutura Molecular , Ubiquinona/biossíntese , Ubiquinona/químicaRESUMO
Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E(121)K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.
Assuntos
Proteínas Mitocondriais/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquinona/biossíntese , Sequência de Aminoácidos , Metiltransferases/metabolismo , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mutação Puntual , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Mucosal surfaces provide first-line defense against microbial invasion through their complex secretions. The antimicrobial activities of proteins in these secretions have been well delineated, but the contributions of lipids to mucosal defense have not been defined. We found that normal human nasal fluid contains all major lipid classes (in micrograms per milliliter), as well as lipoproteins and apolipoprotein A-I. The predominant less polar lipids were myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid, cholesterol, and cholesteryl palmitate, cholesteryl linoleate, and cholesteryl arachidonate. Normal human bronchioepithelial cell secretions exhibited a similar lipid composition. Removal of less-polar lipids significantly decreased the inherent antibacterial activity of nasal fluid against Pseudomonas aeruginosa, which was in part restored after replenishing the lipids. Furthermore, lipids extracted from nasal fluid exerted direct antibacterial activity in synergism with the antimicrobial human neutrophil peptide HNP-2 and liposomal formulations of cholesteryl linoleate and cholesteryl arachidonate were active against P. aeruginosa at physiological concentrations as found in nasal fluid and exerted inhibitory activity against other Gram-negative and Gram-positive bacteria. These data suggest that host-derived lipids contribute to mucosal defense. The emerging concept of host-derived antimicrobial lipids unveils novel roads to a better understanding of the immunology of infectious diseases.
Assuntos
Antibacterianos/imunologia , Ácidos Araquidônicos/fisiologia , Ésteres do Colesterol/metabolismo , Mucosa Nasal/imunologia , Mucosa Nasal/microbiologia , Antibacterianos/química , Antibacterianos/farmacologia , Ácidos Araquidônicos/química , Ácidos Araquidônicos/deficiência , Células Cultivadas , Ésteres do Colesterol/química , Ésteres do Colesterol/farmacologia , Enterobacter cloacae/crescimento & desenvolvimento , Enterobacter cloacae/imunologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/imunologia , Humanos , Imunidade Inata , Lipossomos/imunologia , Lipossomos/metabolismo , Mucosa Nasal/química , Mucosa Nasal/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/imunologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologiaRESUMO
The reversible redox chemistry of coenzyme Q serves a crucial function in respiratory electron transport. Biosynthesis of Q in Escherichia coli depends on the ubi genes. However, very little is known about UbiX, an enzyme thought to be involved in the decarboxylation step in Q biosynthesis in E. coli and Salmonella enterica. Here we characterize an E. coli ubiX gene deletion strain, LL1, to further elucidate E. coli ubiX function in Q biosynthesis. LLI produces very low levels of Q, grows slowly on succinate as the sole carbon source, accumulates 4-hydroxy-3-octaprenyl-benzoate, and has reduced UbiG O-methyltransferase activity. Expression of either E. coli ubiX or the Saccharomyces cerevisiae ortholog PAD1, rescues the deficient phenotypes of LL1, identifying PAD1 as an ortholog of ubiX. Our results suggest that both UbiX and UbiD are required for the decarboxylation of 4-hydroxy-3-octaprenyl-benzoate in E. coli coenzyme Q biosynthesis, especially during logarithmic growth.
Assuntos
Carboxiliases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Ubiquinona/biossíntese , Proliferação de Células , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologiaRESUMO
This paper describes for the first time the direct measurement of boric acid (B(OH)(3)) and borate (B(OH)(4) (-)) adduction to NAD(+) and NADH by electrospray ionization mass spectrometry (ESI-MS) and (11)B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD(+) forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD(+), and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD(+) were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD(+) and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 micro M for NAD(+) and boric acid (1 : 1) to detect NAD(+)-borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance.
Assuntos
Boratos/química , NAD/química , Esterificação , Guanosina Difosfato/análise , Guanosina Difosfato/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
UNLABELLED: Quinone compounds act as membrane resident carriers of electrons between components of the electron transport chain in the periplasmic space of prokaryotes and in the mitochondria of eukaryotes. Vitamin K is a quinone compound in the human body in a storage form as menaquinone (MK); distribution includes regulated amounts in mitochondrial membranes. The human brain, which has low amounts of typical vitamin K dependent function (e.g., gamma carboxylase) has relatively high levels of MK, and different regions of brain have different amounts. Coenzyme Q (Q), is a quinone synthesized de novo, and the levels of synthesis decline with age. The levels of MK are dependent on dietary intake and generally increase with age. MK has a characterized role in the transfer of electrons to fumarate in prokaryotes. A newly recognized fumarate cycle has been identified in brain astrocytes. The MK precursor menadione has been shown to donate electrons directly to mitochondrial complex III. HYPOTHESIS: Vitamin K compounds function in the electron transport chain of human brain astrocytes.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Transporte de Elétrons/fisiologia , Modelos Biológicos , Quinonas/metabolismo , Vitamina K 2/metabolismo , Encéfalo/citologia , Ácido Glutâmico/metabolismo , Humanos , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ubiquinona/metabolismo , Vitamina K 1/química , Vitamina K 1/metabolismo , Vitamina K 2/química , Vitamina K 3/química , Vitamina K 3/metabolismoRESUMO
The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.
Assuntos
Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintases/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Modelos Estatísticos , Transplante de Neoplasias , Transdução de Sinais , Estearoil-CoA Dessaturase/metabolismo , Esteróis/metabolismoRESUMO
Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.
Assuntos
Ácido Linoleico/farmacologia , Peroxidação de Lipídeos , Antioxidantes/química , Antioxidantes/metabolismo , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Cobre/farmacologia , Deutério/química , Deutério/metabolismo , Ácido Eicosapentaenoico/metabolismo , Ácido Eicosapentaenoico/farmacologia , Cinética , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Oxidantes/farmacologia , Oxirredução , Estresse Oxidativo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Ubiquinona/metabolismoRESUMO
The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D(2)-linoleic or 11,11,14,14-D(4)-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.
Assuntos
Citoproteção/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Antioxidantes/farmacologia , Citoproteção/genética , Deutério/química , Deutério/metabolismo , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Cromatografia Gasosa-Espectrometria de Massas , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Marcação por Isótopo , Organismos Geneticamente Modificados , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquinona/genética , Leveduras/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismoRESUMO
Therapy of mitochondrial respiratory chain diseases is complicated by limited understanding of cellular mechanisms that cause the widely variable clinical findings. Here, we show that focal segmental glomerulopathy-like kidney disease in Pdss2 mutant animals with primary coenzyme Q (CoQ) deficiency is significantly ameliorated by oral treatment with probucol (1% w/w). Preventative effects in missense mutant mice are similar whether fed probucol from weaning or for 3 weeks prior to typical nephritis onset. Furthermore, treating symptomatic animals for 2 weeks with probucol significantly reduces albuminuria. Probucol has a more pronounced health benefit than high-dose CoQ(10) supplementation and uniquely restores CoQ(9) content in mutant kidney. Probucol substantially mitigates transcriptional alterations across many intermediary metabolic domains, including peroxisome proliferator-activated receptor (PPAR) pathway signaling. Probucol's beneficial effects on the renal and metabolic manifestations of Pdss2 disease occur despite modest induction of oxidant stress and appear independent of its hypolipidemic effects. Rather, decreased CoQ(9) content and altered PPAR pathway signaling appear, respectively, to orchestrate the glomerular and global metabolic consequences of primary CoQ deficiency, which are both preventable and treatable with oral probucol therapy.
Assuntos
Alquil e Aril Transferases/genética , Metabolismo Energético/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Probucol/farmacologia , Ubiquinona/deficiência , Albuminúria/tratamento farmacológico , Alquil e Aril Transferases/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Feminino , Hiperglicemia/tratamento farmacológico , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Nefropatias/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Estresse Oxidativo , Probucol/uso terapêutico , Transdução de Sinais/fisiologiaRESUMO
Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.
Assuntos
Alquil e Aril Transferases/genética , Suplementos Nutricionais , Mutação , Nefrite/prevenção & controle , Ubiquinona/análogos & derivados , Albuminúria/urina , Alquil e Aril Transferases/metabolismo , Animais , Feminino , Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/prevenção & controle , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Camundongos Transgênicos , Mitocôndrias/metabolismo , Nefrite/genética , Nefrite/patologia , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transfecção , Ubiquinona/administração & dosagem , Ubiquinona/metabolismo , Ubiquinona/uso terapêuticoRESUMO
Coenzyme Q (Q) is a redox active lipid that is an essential component of the electron transport chain. Here, we show that steady state levels of Coq3, Coq4, Coq6, Coq7 and Coq9 polypeptides in yeast mitochondria are dependent on the expression of each of the other COQ genes. Submitochondrial localization studies indicate Coq9p is a peripheral membrane protein on the matrix side of the mitochondrial inner membrane. To investigate whether Coq9p is a component of a complex of Q-biosynthetic proteins, the native molecular mass of Coq9p was determined by Blue Native-PAGE. Coq9p was found to co-migrate with Coq3p and Coq4p at a molecular mass of approximately 1 MDa. A direct physical interaction was shown by the immunoprecipitation of HA-tagged Coq9 polypeptide with Coq4p, Coq5p, Coq6p and Coq7p. These findings, together with other work identifying Coq3p and Coq4p interactions, identify at least six Coq polypeptides in a multi-subunit Q biosynthetic complex.
Assuntos
Proteínas Mitocondriais/química , Complexos Multienzimáticos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquinona/biossíntese , Eletroforese em Gel de Poliacrilamida , Membranas Mitocondriais/química , Saccharomyces cerevisiae/genética , Ubiquinona/químicaRESUMO
Coenzyme Q (ubiquinone or Q) functions in the respiratory electron transport chain and serves as a lipophilic antioxidant. In the budding yeast Saccharomyces cerevisiae, Q biosynthesis requires nine Coq proteins (Coq1-Coq9). Previous work suggests both an enzymatic activity and a structural role for the yeast Coq7 protein. To define the functional roles of yeast Coq7p we test whether Escherichia coli ubiF can functionally substitute for yeast COQ7. The ubiF gene encodes a flavin-dependent monooxygenase that shares no homology to the Coq7 protein and is required for the final monooxygenase step of Q biosynthesis in E. coli. The ubiF gene expressed at low copy restores growth of a coq7 point mutant (E194K) on medium containing a non-fermentable carbon source, but fails to rescue a coq7 null mutant. However, expression of ubiF from a multicopy vector restores growth and Q synthesis for both mutants, although with a higher efficiency in the point mutant. We attribute the more efficient rescue of the coq7 point mutant to higher steady state levels of the Coq3, Coq4, and Coq6 proteins and to the presence of demethoxyubiquinone, the substrate of UbiF. Coq7p co-migrates with the Coq3 and Coq4 polypeptides as a high molecular mass complex. Here we show that addition of Q to the growth media also stabilizes the Coq3 and Coq4 polypeptides in the coq7 null mutant. The data suggest that Coq7p, and the lipid quinones (demethoxyubiquinone and Q) function to stabilize other Coq polypeptides.
Assuntos
Proteínas de Escherichia coli/genética , Oxigenases de Função Mista/genética , Mutação , Saccharomyces cerevisiae/genética , Ubiquinona/química , Sequência de Aminoácidos , Eletroquímica , Teste de Complementação Genética , Vetores Genéticos , Lipídeos , Mitocôndrias/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutação Puntual , Homologia de Sequência de AminoácidosRESUMO
Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.
Assuntos
Mutação , Consumo de Oxigênio , Saccharomyces cerevisiae/genética , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Coenzimas , Redutases do Citocromo/metabolismo , Primers do DNA/química , DNA Complementar/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Elétrons , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Genótipo , Histidina/química , Humanos , Lipídeos/química , Mitocôndrias/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , NAD/química , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fases de Leitura Aberta , Oxigênio/química , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Quinonas/química , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Succinatos/metabolismo , Ubiquinona/genética , Ubiquinona/fisiologiaRESUMO
Currently, eight genes are known to be involved in coenzyme Q6 biosynthesis in Saccharomyces cerevisiae. Here, we report a new gene designated COQ9 that is also required for the biosynthesis of this lipoid quinone. The respiratory-deficient pet mutant C92 was found to be deficient in coenzyme Q and to have low mitochondrial NADH-cytochrome c reductase activity, which could be restored by addition of coenzyme Q2. The mutant was used to clone COQ9, corresponding to reading frame YLR201c on chromosome XII. The respiratory defect of C92 is complemented by COQ9 and suppressed by COQ8/ABC1. The latter gene has been shown to be required for coenzyme Q biosynthesis in yeast and bacteria. Suppression by COQ8/ABC1 of C92, but not other coq9 mutants tested, has been related to an increase in the mitochondrial concentration of several enzymes of the pathway. Coq9p may either catalyze a reaction in the coenzyme Q biosynthetic pathway or have a regulatory role similar to that proposed for Coq8p.
Assuntos
Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Ubiquinona/biossíntese , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Citocromos/química , Mutação , NAD/metabolismo , Oxirredutases/metabolismo , Fenótipo , Espectrofotometria , Ubiquinona/genética , Ubiquinona/fisiologiaRESUMO
Coenzyme Q (Q) is a redox active lipid essential for aerobic respiration in eukaryotes. In Saccharomyces cerevisiae at least eight mitochondrial polypeptides, designated Coq1-Coq8, are required for Q biosynthesis. Here we present physical evidence for a coenzyme Q-biosynthetic polypeptide complex in isolated mitochondria. Separation of digitonin-solubilized mitochondrial extracts in one- and two-dimensional Blue Native PAGE analyses shows that Coq3 and Coq4 polypeptides co-migrate as high molecular mass complexes. Similarly, gel filtration chromatography shows that Coq1p, Coq3p, Coq4p, Coq5p, and Coq6p elute in fractions higher than expected for their respective subunit molecular masses. Coq3p, Coq4p, and Coq6p coelute with an apparent molecular mass exceeding 700 kDa. Coq3 O-methyltransferase activity, a surrogate for Q biosynthesis and complex activity, also elutes at this high molecular mass. We have determined the quinone content in lipid extracts of gel filtration fractions by liquid chromatography-tandem mass spectrometry and find that demethoxy-Q(6) is enriched in fractions with Coq3p. Co-precipitation of biotinylated-Coq3 and Coq4 polypeptide from digitonin-solubilized mitochondrial extracts shows their physical association. This study identifies Coq3p and Coq4p as defining members of a Q-biosynthetic Coq polypeptide complex.