RESUMO
Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ß is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit ß and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Ácidos Cetoglutáricos/farmacologia , Longevidade/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células Jurkat , Longevidade/efeitos dos fármacos , Longevidade/genética , Camundongos , ATPases Mitocondriais Próton-Translocadoras/genética , Ligação ProteicaRESUMO
HJV (haemojuvelin) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile haemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as RGMc (repulsive guidance molecule c), is a member of the three-protein RGM family. RGMs are GPI (glycosylphosphatidylinositol)-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteine residues in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. In the present study we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential MS steps composed of ETD (electron transfer dissociation) and CID (collision-induced dissociation), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. The results of the present study identified an HJV/RGMc molecular species containing four disulfide linkages. We predict using ab initio modelling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modelling to define unknown structural features in proteins.
Assuntos
Proteínas Reguladoras de Ferro/química , Proteínas de Membrana/química , Modelos Moleculares , Proteômica/métodos , Sequência de Aminoácidos , Animais , Dissulfetos/química , Proteínas Ligadas por GPI , Células HEK293 , Proteína da Hemocromatose , Humanos , Proteínas Reguladoras de Ferro/genética , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genéticaRESUMO
The six high-affinity insulin-like growth factor-binding proteins (IGFBPs) comprise a conserved family of secreted molecules that modulate IGF actions by regulating their half-life and access to signaling receptors, and also exert biological effects that are independent of IGF binding. IGFBPs are composed of cysteine-rich amino- (N-) and carboxyl- (C-) terminal domains, along with a cysteine-poor central linker segment. IGFBP-5 is the most conserved IGFBP, and contains 18 cysteines, but only 2 of 9 putative disulfide bonds have been mapped to date. Using a mass spectrometry (MS)-based strategy combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) steps, in which ETD fragmentation preferentially induces cleavage of disulfide bonds, and CID provides exact disulfide linkage assignments between liberated peptides, we now have definitively mapped 5 disulfide bonds in IGFBP-5. In addition, in conjunction with ab initio molecular modeling we are able to assign the other 4 disulfide linkages to within a GCGCCXXC motif that is conserved in five IGFBPs. Because of the nature of ETD fragmentation MS experiments were performed without chemical reduction of IGFBP-5. Our results not only establish a disulfide bond map of IGFBP-5 but also define a general approach that takes advantage of the specificity of ETD and the scalability of tandem MS, and the predictive power of ab initio molecular modeling to characterize unknown disulfide linkages in proteins.
Assuntos
Dissulfetos/química , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Modelos Moleculares , Mapeamento de Peptídeos/métodos , Motivos de Aminoácidos , Animais , Linhagem Celular , Cisteína , Humanos , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Espectrometria de Massas , CamundongosRESUMO
Inactivating mutations in hemojuvelin/repulsive guidance molecule c (HJV/RGMc) cause juvenile hemochromatosis (JH), a rapidly progressive iron overload disorder in which expression of hepcidin, a key liver-derived iron-regulatory hormone, is severely diminished. Several growth factors in the bone morphogenetic protein (BMP) family, including BMP2 and BMP6, can stimulate production of hepcidin, a biological effect that may be modified by RGMc. Here we demonstrate that soluble RGMc proteins are potent BMP inhibitors. We find that 50- and 40-kDa RGMc isoforms, when added to cells as highly purified IgG Fc fusion proteins, are able to block the acute effects of both BMP2 and BMP6 at the levels of Smad induction and gene activation, and thus represent a potentially unique class of broad-spectrum BMP antagonists. Whole transcript microarray analysis revealed that BMP2 and BMP6 each stimulated expression of a nearly identical cohort of approximately 40 mRNAs in Hep3B cells and demonstrated that 40-kDa RGMc was an effective inhibitor of both growth factors, although its potency was less than that of the known BMP2-selective antagonist, Noggin. We additionally show that JH-linked RGMc mutant proteins that retain the ability to bind BMPs are also able to function as BMP inhibitors, and like the wild type soluble RGMc species, can block BMP-activated hepcidin gene expression. The latter results raise the question of whether disease severity in JH will vary depending on the ability of a given mutant RGMc protein to interact with BMPs.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular , Proteínas Ligadas por GPI , Perfilação da Expressão Gênica , Proteína da Hemocromatose , Hepcidinas , Humanos , Sobrecarga de Ferro , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes de Fusão/química , Transdução de Sinais , Distribuição TecidualRESUMO
BACKGROUND: Repulsive guidance molecule c (RGMc or hemojuvelin), a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH), a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. RESULTS: We now show that pro-protein convertases (PC) are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. CONCLUSION: Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.
Assuntos
Proteínas Reguladoras de Ferro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Pró-Proteína Convertases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Furina/metabolismo , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Peso Molecular , Proteínas Musculares/genética , Mutação , Isoformas de Proteínas/metabolismoRESUMO
Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA). The transcriptional profile of PKO cells was similar to rho0 mtDNA deleted cells, with perturbations in cholesterol (FDR = 6.35 x 10-13), lipid (FDR = 3.21 x 10-11), and secondary alcohol (FDR = 1.04x10-12) metabolic pathway gene expression compared to wild type parental (TM6) MEFs. Transcriptome analysis indicates processes related to axonogenesis (FDR = 4.49 x 10-3), axon development (FDR = 4.74 x 10-3), and axonal guidance (FDR = 4.74 x 10-3) were overrepresented in PKO cells, consistent with previous studies detailing causative PNPase mutations in delayed myelination, hearing loss, encephalomyopathy, and chorioretinal defects in humans. Overrepresentation analysis revealed alterations in metabolic pathways in both PKO and rho0 cells. Therefore, we assessed the correlation of genes implicated in cell cycle progression and total metabolism and observed a strong positive correlation between PKO cells and rho0 MEFs compared to TM6 MEFs. We quantified the normalized biomass accumulation rate of PKO clones at 1.7% (SD ± 2.0%) and 2.4% (SD ± 1.6%) per hour, which was lower than TM6 cells at 3.3% (SD ± 3.5%) per hour. Furthermore, PKO in mouse inner ear hair cells caused progressive hearing loss that parallels human familial hearing loss previously linked to mutations in PNPase. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , DNA Mitocondrial/metabolismo , Regulação da Expressão Gênica , Perda Auditiva/fisiopatologia , Mitocôndrias/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Animais , Ciclo Celular , DNA Mitocondrial/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mutação , Polirribonucleotídeo Nucleotidiltransferase/genéticaRESUMO
The import of a modest number of nucleus-encoded RNAs into mitochondria has been reported in species ranging from yeast to human. With the advent of high-throughput RNA sequencing, additional nucleus-encoded mitochondrial RNAs are being identified. Confirming the mitochondrial localization of candidate RNAs of interest (e.g., small noncoding RNAs, miRNAs, tRNAs, and possibly lncRNAs and viral RNAs) and understanding their function within the mitochondrion is assisted by in vitro and in vivo import assay systems. Here we describe these two systems for studying mitochondrial RNA import, processing, and functions.
Assuntos
Transporte de RNA , RNA/genética , Sistema Livre de Células , Técnicas In Vitro , RNA/isolamento & purificação , RNA/metabolismo , RNA Mitocondrial , Transcrição GênicaRESUMO
Juvenile hemochromatosis is a severe and rapidly progressing hereditary disorder of iron overload, and it is caused primarily by defects in the gene encoding repulsive guidance molecule c/hemojuvelin (RGMc/HJV), a recently identified protein that undergoes a complicated biosynthetic pathway in muscle and liver, leading to cell membrane-linked single-chain and heterodimeric species, and two secreted single-chain isoforms. RGMc modulates expression of the hepatic iron regulatory factor, hepcidin, potentially through effects on signaling by the bone morphogenetic protein (BMP) family of soluble growth factors. To date, little is known about specific pathogenic defects in disease-causing RGMc/HJV proteins. Here we identify functional abnormalities in three juvenile hemochromatosis-linked mutants. Using a combination of approaches, we first show that BMP-2 could interact in biochemical assays with single-chain RGMc species, and also could bind to cell-associated RGMc. Two mouse RGMc amino acid substitution mutants, D165E and G313V (corresponding to human D172E and G320V), also could bind BMP-2, but less effectively than wild-type RGMc, while G92V (human G99V) could not. In contrast, the membrane-spanning protein, neogenin, a receptor for the related molecule, RGMa, preferentially bound membrane-associated heterodimeric RGMc and was able to interact on cells only with wild-type RGMc and G92V. Our results show that different isoforms of RGMc/HJV may play unique physiological roles through defined interactions with distinct signaling proteins and demonstrate that, in some disease-linked RGMc mutants, these interactions are defective.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Morfogenética Óssea 2 , Linhagem Celular , Membrana Celular/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica/fisiologia , Hemocromatose/genética , Proteína da Hemocromatose , Humanos , Proteínas de Membrana/genética , Mutação , Ligação Proteica , Isoformas de ProteínasRESUMO
The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of alpha-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10 degrees C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10 degrees C, respectively, indicating destabilization of the N-domain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and C-domain fold with a cooperative free energy of 1.0-1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca2+ binding sites of thin filament in two settings: Ca2+-dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca2+ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca2+ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca2+ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca2+ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.
Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Cardiomiopatias/genética , Mutação , Tropomiosina/genética , Adenosina Trifosfatases/metabolismo , Animais , Bovinos , Cinética , Desnaturação Proteica , Estrutura Terciária de Proteína , Coelhos , Ratos , Temperatura , Termodinâmica , Tropomiosina/químicaRESUMO
The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5'-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.
Assuntos
Actinas/química , Modelos Moleculares , Proteínas Motores Moleculares/química , Contração Muscular/fisiologia , Miosinas/química , Actinas/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/fisiologia , Animais , Galinhas , Simulação por Computador , Transferência de Energia , Hidrólise , Substâncias Macromoleculares , Proteínas Motores Moleculares/fisiologia , Movimento (Física) , Movimento/fisiologia , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Subfragmentos de Miosina/química , Subfragmentos de Miosina/fisiologia , Miosinas/fisiologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Coelhos , Estereoisomerismo , Estresse Mecânico , Trinitrobenzenos/químicaRESUMO
There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long- and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-epsilon or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergent-skinned mouse cardiac fiber bundles to measure changes in Ca(2+)-dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca(2+), and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca(2+) sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca(2+). Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.