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OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.
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Human aging is linked to bone loss, resulting in bone fragility and an increased risk of fractures. This is primarily due to an age-related decline in the function of bone-forming osteoblastic cells and accelerated cellular senescence within the bone microenvironment. Here, we provide a detailed discussion of the hypothesis that age-related defective bone formation is caused by senescence of skeletal stem cells, as they are the main source of bone forming osteoblastic cells and influence the composition of bone microenvironment. Furthermore, this review discusses potential strategies to target cellular senescence as an emerging approach to treat age-related bone loss.
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OBJECTIVE: Drugs targeting the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) are emerging as treatments for type-2 diabetes and obesity. GIP acutely decreases serum markers of bone resorption and transiently increases bone formation markers in short-term clinical investigations. However, it is unknown whether GIP acts directly on bone cells to mediate these effects. Using a GIPR-specific antagonist, we aimed to assess whether GIP acts directly on primary human osteoclasts and osteoblasts. METHODS: Osteoclasts were differentiated from human CD14+ monocytes and osteoblasts from human bone. GIPR expression was determined using RNA-seq in primary human osteoclasts and in situ hybridization in human femoral bone. Osteoclastic resorptive activity was assessed using microscopy. GIPR signaling pathways in osteoclasts and osteoblasts were assessed using LANCE cAMP and AlphaLISA phosphorylation assays, intracellular calcium imaging and confocal microscopy. The bioenergetic profile of osteoclasts was evaluated using Seahorse XF-96. RESULTS: GIPR is robustly expressed in mature human osteoclasts. GIP inhibits osteoclastogenesis, delays bone resorption, and increases osteoclast apoptosis by acting upon multiple signaling pathways (Src, cAMP, Akt, p38, Akt, NFκB) to impair nuclear translocation of nuclear factor of activated T cells-1 (NFATc1) and nuclear factor-κB (NFκB). Osteoblasts also expressed GIPR, and GIP improved osteoblast survival. Decreased bone resorption and improved osteoblast survival were also observed after GIP treatment of osteoclast-osteoblast co-cultures. Antagonizing GIPR with GIP(3-30)NH2 abolished the effects of GIP on osteoclasts and osteoblasts. CONCLUSIONS: GIP inhibits bone resorption and improves survival of human osteoblasts, indicating that drugs targeting GIPR may impair bone resorption, whilst preserving bone formation.
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Reabsorção Óssea , Osteoclastos , Humanos , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Diferenciação CelularRESUMO
Bromodomain containing 1 (BRD1) encodes an epigenetic regulator that controls the expression of genetic networks linked to mental illness. BRD1 is essential for normal brain development and its role in psychopathology has been demonstrated in genetic and preclinical studies. However, the neurobiology that bridges its molecular and neuropathological effects remains poorly explored. Here, using publicly available datasets, we find that BRD1 targets nuclear genes encoding mitochondrial proteins in cell lines and that modulation of BRD1 expression, irrespective of whether it is downregulation or upregulation of one or the other existing BRD1 isoforms (BRD1-L and BRD1-S), leads to distinct shifts in the expression profile of these genes. We further show that the expression of nuclear genes encoding mitochondrial proteins is negatively correlated with the expression of BRD1 mRNA during human brain development. In accordance, we identify the key gate-keeper of mitochondrial metabolism, Peroxisome proliferator-activated receptor (PPAR) among BRD1's co-transcription factors and provide evidence that BRD1 acts as a co-repressor of PPAR-mediated transcription. Lastly, when using quantitative PCR, mitochondria-targeted fluorescent probes, and the Seahorse XFe96 Analyzer, we demonstrate that modulation of BRD1 expression in cell lines alters mitochondrial physiology (mtDNA content and mitochondrial mass), metabolism (reducing power), and bioenergetics (among others, basal, maximal, and spare respiration) in an expression level- and isoform-dependent manner. Collectively, our data suggest that BRD1 is a transcriptional regulator of nuclear-encoded mitochondrial proteins and that disruption of BRD1's genomic actions alters mitochondrial functions. This may be the mechanism underlying the cellular and atrophic changes of neurons previously associated with BRD1 deficiency and suggests that mitochondrial dysfunction may be a possible link between genetic variation in BRD1 and psychopathology in humans.
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Histona Acetiltransferases , Esquizofrenia , Metabolismo Energético , Histona Acetiltransferases/fisiologia , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/metabolismo , Esquizofrenia/genéticaRESUMO
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous, debilitating, and complex disease. Along with disabling fatigue, ME/CFS presents an array of other core symptoms, including autonomic nervous system (ANS) dysfunction, sustained inflammation, altered energy metabolism, and mitochondrial dysfunction. Here, we evaluated patients' symptomatology and the mitochondrial metabolic parameters in peripheral blood mononuclear cells (PBMCs) and plasma from a clinically well-characterised cohort of six ME/CFS patients compared to age- and gender-matched controls. We performed a comprehensive cellular assessment using bioenergetics (extracellular flux analysis) and protein profiles (quantitative mass spectrometry-based proteomics) together with self-reported symptom measures of fatigue, ANS dysfunction, and overall physical and mental well-being. This ME/CFS cohort presented with severe fatigue, which correlated with the severity of ANS dysfunction and overall physical well-being. PBMCs from ME/CFS patients showed significantly lower mitochondrial coupling efficiency. They exhibited proteome alterations, including altered mitochondrial metabolism, centred on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels. Overall, these results indicate that PBMCs from ME/CFS patients have a decreased ability to fulfill their cellular energy demands.
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Síndrome de Fadiga Crônica/sangue , Síndrome de Fadiga Crônica/imunologia , Síndrome de Fadiga Crônica/fisiopatologia , Adulto , Células Sanguíneas/citologia , Estudos de Coortes , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Leucócitos Mononucleares/citologia , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Projetos Piloto , Proteoma/metabolismo , Proteômica/métodosRESUMO
Standardization of the use of next-generation sequencing for the diagnosis of rare neurological disorders has made it possible to detect potential disease-causing genetic variations, including de novo variants. However, the lack of a clear pathogenic relevance of gene variants poses a critical limitation for translating this genetic information into clinical practice, increasing the necessity to perform functional assays. Genetic screening is currently recommended in the guidelines for diagnosis of hypomyelinating leukodystrophies (HLDs). HLDs represent a group of rare heterogeneous disorders that interfere with the myelination of the neurons in the central nervous system. One of the HLD-related genes is HSPD1, encoding the mitochondrial chaperone heat shock protein 60 (HSP60), which functions as folding machinery for the mitochondrial proteins imported into the mitochondrial matrix space. Disease-causing HSPD1 variants have been associated with an autosomal recessive form of fatal hypomyelinating leukodystrophy (HLD4, MitCHAP60 disease; MIM #612233) and an autosomal dominant form of spastic paraplegia, type 13 (SPG13; MIM #605280). In 2018, a de novo HSPD1 variant was reported in a patient with HLD. Here, we present another case carrying the same heterozygous de novo variation in the HSPD1 gene (c.139T > G, p.Leu47Val) associated with an HLD phenotype. Our molecular studies show that the variant HSP60 protein is stably present in the patient's fibroblasts, and functional assays demonstrate that the variant protein lacks in vivo function, thus confirming its disease association. We conclude that de novo variations of the HSPD1 gene should be considered as potentially disease-causing in the diagnosis and pathogenesis of the HLDs.
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Chaperonina 60/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Adulto , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Chaperonina 10/genética , Chaperonina 60/química , Criança , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Proteínas Mitocondriais/química , Modelos Moleculares , Mutação , Proteínas da Gravidez/genética , Conformação Proteica , Recidiva , Relação Estrutura-Atividade , Fatores Supressores Imunológicos/genéticaRESUMO
BACKGROUND: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is the most frequent fatty acid oxidation (FAO) defect in humans. MCAD-deficient fibroblasts are more resistant to oxidative stress-induced cell death than other FAO defects and healthy controls. METHODS: Herein we investigate the antioxidant response and mitochondrial function in fibroblasts from MCAD-deficient patients (c.985 A>G/c.985 A>G) and healthy controls. RESULTS: MCAD-deficient fibroblasts showed increased level of mitochondrial superoxide, while lipids were less oxidatively damaged, and higher amount of manganese superoxide dismutase were detected compared to healthy controls, showing forceful antioxidant system in MCADD. We showed increased maximal respiration and reserve capacity in MCAD-deficient fibroblasts compared to controls, indicating more capacity through the tricarboxylic acid (TCA) cycle and subsequently respiratory chain. This led us to study the pyruvate dehydrogenase complex (PDC), the key enzyme in the glycolysis releasing acetyl-CoA to the TCA cycle. MCAD-deficient fibroblasts displayed not only significantly increased PDC but also increased lipoylated PDC protein levels compared to healthy controls. CONCLUSIONS: Based on these findings, we raise the interesting hypothesis that increased PDC-bound lipoic acid, synthesized from accumulated octanoic acid in MCADD, may affect the cellular antioxidant pool in MCADD.
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Acil-CoA Desidrogenase/deficiência , Acil-CoA Desidrogenase/genética , Antioxidantes/farmacologia , Erros Inatos do Metabolismo Lipídico/metabolismo , Ácido Tióctico/química , Acil-CoA Desidrogenase/metabolismo , Antioxidantes/metabolismo , Caprilatos/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Morte Celular , Fibroblastos/metabolismo , Genótipo , Glicólise , Humanos , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Estresse Oxidativo , Fenótipo , Superóxidos/metabolismoRESUMO
In this review, we discuss the myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), which is characterised by extreme mental and physical fatigue with associated symptoms of pain, disturbed sleep, cognitive and autonomic dysfunction, as well as post-exertional malaise. This con-dition is often preceded by an infection, severe physiological and/or psychological strain. Over the last decades, research has demonstrated mitochondrial, neuroendocrine, immuno-logical, and metabolic perturbations in patients with ME/CFS, giving hope for the development of new biomarkers and new treatment modalities.
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Síndrome de Fadiga Crônica , Biomarcadores , Síndrome de Fadiga Crônica/diagnóstico , Síndrome de Fadiga Crônica/genética , Síndrome de Fadiga Crônica/imunologia , Humanos , Mitocôndrias , DorRESUMO
Besides providing the majority of ATP production in cells, mitochondria are also involved in many other cellular functions and are central for cellular stress signaling. Mitochondrial dysfunction induces not only inherited mitochondrial disorders but also contributes to neurodegenerative diseases, cancer, diabetes, and metabolic syndrome. The HSP60/HSP10 molecular chaperone complex facilitates folding of mitochondrial proteins and is thus an important factor for many mitochondrial functions. To model different degrees of oxidative stress and mitochondrial dysfunction we here describe a HEK293 derived Flp-In cell system with stable insertion and tunable expression of HSP60 cDNA carrying a dominant negative mutation. When expressed the dominant negative HSP60 mutant is incorporated into endogenously encoded HSP60/HSP10 complexes and impairs chaperone activity of the HSP60/HSP10 complex in a dose dependent manner. Using this system, different levels of oxidative stress and mitochondrial dysfunction challenges can be generated depending on the induction level of the mutant HSP60 cDNA insert. Here we describe our system and pertinent analysis methodology for use in studies of mitochondrial chaperone deficiency and resulting effects of increased production of reactive oxygen species and mitochondrial dysfunction.
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Chaperonina 60/deficiência , Suscetibilidade a Doenças , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Citometria de Fluxo , Humanos , Espectrometria de Massas , Potencial da Membrana MitocondrialRESUMO
The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.
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Ciclo Celular/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteoma/metabolismo , Sulfetos/metabolismo , Encefalopatias Metabólicas Congênitas , Adesão Celular , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Glutationa/metabolismo , Glicólise , Glicoproteínas/metabolismo , Humanos , Proteínas com Domínio LIM/metabolismo , Ácido Láctico/metabolismo , Masculino , Peroxirredoxinas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteômica , Púrpura , Proteínas RibossômicasRESUMO
AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.
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Tecido Adiposo Marrom/efeitos dos fármacos , Hipoglicemiantes/farmacocinética , Metformina/farmacocinética , Consumo de Oxigênio/efeitos dos fármacos , Animais , Cimetidina/administração & dosagem , Relação Dose-Resposta a Droga , Humanos , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , TranscriptomaRESUMO
Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) requires remodeling of mitochondria and a metabolic shift from an oxidative state to a more glycolytic state. In this study, we evaluated the nuclear reprogramming efficiency in relation to mitochondrial bioenergetic parameters of fibroblasts from seven different human individuals. Using the Seahorse extracellular energy flux analyzer, we measured oxygen consumption rate (OCR) profiles of the cells, along with their nuclear reprogramming efficiency into iPSCs. Our results showed that fibroblasts with the lowest mitochondrial spare respiratory capacity (SRC) had the highest nuclear reprogramming efficiency, opposed to fibroblasts with the highest mitochondrial SRC, which showed lowest reprogramming efficiency. Furthermore, we found that targeted fluorescent tagging of endogenous genes (MYH6 and COL2A1) by CRISPR/Cas9-mediated homologous recombination was accompanied by an increase in the SRC level of the modified fibroblasts and impaired reprogramming efficiency. Our findings indicate a negative correlation between high mitochondrial SRC in somatic cells and low reprogramming efficiencies. This type of analysis potentially allows screening and predicting reprogramming efficiency before reprogramming, and further suggests that nuclear reprogramming might be improved by approaches that modulate the SRC.
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Reprogramação Celular , Mitocôndrias/metabolismo , Adolescente , Respiração Celular , Criança , Pré-Escolar , Derme/citologia , Metabolismo Energético , Feminino , Fibroblastos/metabolismo , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at 3 months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature. To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although, we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.
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Heat shock protein 60 (HSP60) forms together with heat shock protein 10 (HSP10) double-barrel chaperonin complexes that are essential for folding to the native state of proteins in the mitochondrial matrix space. Two extremely rare monogenic disorders have been described that are caused by missense mutations in the HSPD1 gene that encodes the HSP60 subunit of the HSP60/HSP10 chaperonin complex. Investigations of the molecular mechanisms underlying these disorders have revealed that different degrees of reduced HSP60 function produce distinct neurological phenotypes. While mutations with deleterious or strong dominant negative effects are not compatible with life, HSPD1 gene variations found in the human population impair HSP60 function and depending on the mechanism and degree of HSP60 dys- and mal-function cause different phenotypes. We here summarize the knowledge on the effects of disturbances of the function of the HSP60/HSP10 chaperonin complex by disease-associated mutations.
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Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.
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Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1ß (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1ß proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1ß monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.
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Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1ß, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.