RESUMO
Mitochondrial sirtuins, SIRT3-5, are NAD+-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.
Assuntos
Mitocôndrias/metabolismo , Mapas de Interação de Proteínas , Sirtuína 3/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras , Sirtuínas/classificação , Sirtuínas/metabolismoRESUMO
Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.
Assuntos
Mapas de Interação de Proteínas , Proteômica/métodos , Esclerose Lateral Amiotrófica/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.
Assuntos
Distonia/genética , Deficiência Intelectual/genética , Fenilcetonúrias/genética , Proteínas Repressoras/genética , Alelos , Sequência de Aminoácidos , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Estudos de Casos e Controles , Dopamina/deficiência , Dopamina/metabolismo , Éxons , Feminino , Fibroblastos/metabolismo , Deleção de Genes , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico HSP70/genética , Humanos , Masculino , Linhagem , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/genética , Serotonina/deficiência , Serotonina/metabolismo , Triptofano/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.
Assuntos
Células Endoteliais/enzimologia , Regulação da Expressão Gênica , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Sirtuína 1/genética , Sirtuína 1/metabolismo , Acetilação , Animais , Células Endoteliais/citologia , Técnicas de Inativação de Genes , Inativação Gênica , Células HEK293 , Humanos , Camundongos , Mutação , Receptor Notch1/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
JmjC domain-containing proteins play a crucial role in the control of gene expression by acting as protein hydroxylases or demethylases, thereby controlling histone methylation or splicing. Here, we demonstrate that silencing of Jumonji domain-containing protein 6 (Jmjd6) impairs angiogenic functions of endothelial cells by changing the gene expression and modulating the splicing of the VEGF-receptor 1 (Flt1). Reduction of Jmjd6 expression altered splicing of Flt1 and increased the levels of the soluble form of Flt1, which binds to VEGF and placental growth factor (PlGF) and thereby inhibits angiogenesis. Saturating VEGF or PlGF or neutralizing antibodies directed against soluble Flt1 rescued the angiogenic defects induced by Jmjd6 silencing. Jmjd6 interacts with the splicing factors U2AF65 that binds to Flt1 mRNA. In conclusion, Jmjd6 regulates the splicing of Flt1, thereby controlling angiogenic sprouting.
Assuntos
Endotélio Vascular/citologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Neovascularização Fisiológica/fisiologia , Splicing de RNA , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Células Cultivadas , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Neovascularização Fisiológica/genética , Fator de Crescimento Placentário , Proteínas da Gravidez , Processamento de Proteína Pós-Traducional , Fator A de Crescimento do Endotélio VascularAssuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Aminas Biogênicas/metabolismo , Fenilcetonúrias/diagnóstico , Agregação Patológica de Proteínas/genética , Proteínas Repressoras/deficiência , Diagnóstico Diferencial , Distonia/genética , Homozigoto , Humanos , Deficiência Intelectual/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transtornos Parkinsonianos/genética , Fenilcetonúrias/genética , Proteostase/genética , Proteínas Repressoras/genética , alfa-Sinucleína/metabolismoRESUMO
In embryogenesis, coronary blood vessels are formed by vasculogenesis from epicardium-derived progenitors. Subsequently, growing or regenerating myocardium increases its vasculature by angiogenesis, forming new vessels from the pre-existing ones. Recently, cell therapies for myocardium ischemia that used different protocols have given promising results, using either extra-cardiac blood vessel cell progenitors or stimulating the cardiac angiogenesis. We have questioned whether cardiomyocytes could sustain both vasculogenesis and angiogenesis. We used a 3D culture model of tissue-like spheroids in co-cultures of cardiomyocytes supplemented either with endothelial cells or with bone marrow-derived mesenchymal stroma cells. Murine foetal cardiomyocytes introduced into non-adherent U-wells formed 3D contractile structures. They were coupled by gap junctions. Cardiomyocytes segregated inside the 3D structure into clumps separated by connective tissue septa, rich in fibronectin. Three vascular endothelial growth factor isoforms were produced (VEGF 120, 164 and 188). When co-cultured with human umbilical cord endothelial cells, vascular structures were produced in fibronectin-rich external layer and in radial septa, followed by angiogenic sprouting into the cardiomyocyte microtissue. Presence of vascular structures led to the maintenance of long-term survival and contractile capacity of cardiac microtissues. Conversely, bone marrow mesenchymal cells formed isolated cell aggregates, which progressively expressed the endothelial markers von Willebrand's antigen and CD31. They proceeded to typical vasculogenesis forming new blood vessels organised in radial pattern. Our results indicate that the in vitro 3D model of cardiomyocyte spheroids provides the two basic elements for formation of new blood vessels: fibronectin and VEGF. Within the myocardial environment, endothelial and mesenchymal cells can proceed to formation of new blood vessels either through angiogenesis or vasculogenesis, respectively.
Assuntos
Vasos Coronários/fisiologia , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Miócitos Cardíacos/citologia , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Técnicas de Cocultura , Fibronectinas/biossíntese , Camundongos , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients' fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation.
Assuntos
Hepatopatias/genética , Hepatopatias/patologia , Proteínas de Membrana/deficiência , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/patologia , Proteínas Mitocondriais/deficiência , Mutação , Feminino , Humanos , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , IrmãosRESUMO
The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Animais , Linhagem Celular , Respiração Celular/fisiologia , Drosophila , Células HEK293 , Humanos , Mitocôndrias/fisiologia , Células Musculares/metabolismo , Neurônios/metabolismo , Ligação Proteica/fisiologia , Proteômica/métodosRESUMO
Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Complexo I de Transporte de Elétrons/genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Proteômica/métodosRESUMO
Blood vessels deliver nutrients and oxygen to cells and tissues in the body. When blood supply is insufficient new vessels form to meet the metabolic tissue requirements. Several studies have examined the cellular and molecular principles of blood vessel formation, yet little is known about how vessels sense and integrate environmental signals originating from nutrient- and oxygen-deprived tissues to achieve functional vascular patterning. The NAD(+)-dependent deacetylase SIRT1 mediates adaptation to environmental stresses by adjusting cellular responses to the energetic state of the cell and recent studies highlight important functions of SIRT1 in regulating vascular growth, shape, and function. Here, we review the emerging role of SIRT1 as a metabolic sensor coupling energy and oxygen homeostasis to the growth and function of the vasculature.