RESUMO
BACKGROUND: Propionic acidemia is an inborn error of metabolism caused by a deficiency in the mitochondrial enzyme propionyl-CoA carboxylase that converts the propionyl CoA to methyl malonyl CoA. This leads to profound changes in distinct metabolic pathways, including the urea cycle, with consequences in ammonia detoxification. The implication of the tricarboxylic acid cycle is less well known, but its repercussions could explain both some of the acute and long-term symptoms of this disease. MATERIALS AND METHODS: The present observational study investigates the amino acid profiles of patients with propionic acidemia being monitored at the Hospital Ramón y Cajal (Madrid, Spain), between January 2015 and September 2017, comparing periods of metabolic stability with those of decompensation with ketosis and/or hyperammonemia. RESULTS: The concentrations of 19 amino acids were determined in 188 samples provided by 10 patients. We identified 40 metabolic decompensation episodes (22 only with ketosis and 18 with hyperammonemia). Plasma glutamine and alanine levels were reduced during these metabolic crises, probably indicating deficiency of anaplerosis (p < 0.001 for both alanine and glutamine). Hypocitrulllinemia and hypoprolinemia were also detected during hyperammonemia (p < 0.001 and 0.03, respectively). CONCLUSIONS: The amino acid profile detected during decompensation episodes suggests deficient anaplerosis from propionyl-CoA and its precursors, with implications in other metabolic pathways like synthesis of urea cycle amino acids and ammonia detoxification.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Hiperamonemia , Cetose , Acidemia Propiônica , Alanina , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Aminoácidos , Amônia , Glutamina , Humanos , Acidemia Propiônica/metabolismo , Acidemia Propiônica/patologia , UreiaRESUMO
Mutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2, are causal of gray platelet syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a, and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively. Proximity ligation assays show that these 2 proteins are physically proximal to Nbeal2 in human megakaryocytes. In addition, we demonstrate that Nbeal2 is primarily localized in the cytoplasm and Dock7 on the membrane of or in α-granules. Interestingly, platelets from GPS cases and Nbeal2-/- mice are almost devoid of Dock7, resulting in a profound dysregulation of its signaling pathway, leading to defective actin polymerization, platelet activation, and shape change. This study shows for the first time proteins interacting with Nbeal2 and points to the dysregulation of the canonical signaling pathway of Dock7 as a possible cause of the aberrant formation of platelets in GPS cases and Nbeal2-deficient mice.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Megacariócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Plaquetas/citologia , Proteínas Sanguíneas/genética , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Megacariócitos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Ligação Proteica , Proteínas de Transporte Vesicular/genéticaRESUMO
The identification of bona fide protein-protein interactions and the mapping of proteomes was greatly enhanced by protein tagging for generic affinity purification methods and analysis by mass spectrometry (AP-MS). The high quality of AP-MS data permitted the development of proteomic navigation by sequential tagging of identified interactions. However AP-MS is laborious and limited to relatively high affinity protein-protein interactions. Proximity labeling, first with the biotin ligase BirA, termed BioID, and then with ascorbate peroxidase, termed APEX, permits a greater reach into the proteome than AP-MS enabling both the identification of a wider field and weaker protein-protein interactions. This additional reach comes with the need for stringent controls. Proximity labeling also permits experiments in living cells allowing spatiotemporal investigations of the proteome. Here we discuss proximity labeling with accompanying methodological descriptions for E. coli and mammalian cells.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Coloração e Rotulagem/métodos , Animais , Ascorbato Peroxidases/metabolismo , Biotina/química , Biotina/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/instrumentação , Proteínas Repressoras/metabolismo , Análise Espaço-TemporalRESUMO
The congenital disorders of glycosylation (CDG) are defects in glycoprotein and glycolipid glycan synthesis and attachment. They affect multiple organ/systems, but non-specific symptoms render the diagnosis of the different CDG very challenging. Phosphomannomutase 2 (PMM2)-CDG is the most common CDG, but advances in genetic analysis have shown others to occur more commonly than previously thought. The present work reports the clinical and mutational spectrum of 25 non-PMM2 CDG patients. The most common clinical symptoms were hypotonia (80%), motor or psychomotor disability (80%) and craniofacial dysmorphism (76%). Based on their serum transferrin isoform profile, 18 were classified as CDG-I and 7 as CDG-II. Pathogenic variations were found in 16 genes (ALG1, ALG6, ATP6V0A2, B4GALT1, CCDC115, COG7, DOLK, DPAGT1, DPM1, GFPT1, MPI, PGM1, RFT1, SLC35A2, SRD5A3, and SSR4). Overall, 27 variants were identified, 12 of which are novel. The results highlight the importance of combining genetic and biochemical analyses for the early diagnosis of this heterogeneous group of disorders.
Assuntos
Defeitos Congênitos da Glicosilação/diagnóstico , Defeitos Congênitos da Glicosilação/genética , Fosfotransferases (Fosfomutases)/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , EspanhaRESUMO
A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.
Assuntos
Proteína C9orf72/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Proteína C9orf72/genética , Expansão das Repetições de DNA , Dipeptídeos/metabolismo , Drosophila , Demência Frontotemporal/genética , HumanosRESUMO
Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.
Assuntos
Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/isolamento & purificação , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/metabolismo , Animais , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas/métodos , Camundongos , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in derepression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway.
Assuntos
Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Proteínas Metiltransferases/fisiologia , Animais , Diferenciação Celular/genética , Citoplasma/química , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Metilação , Camundongos , Proteína-Arginina N-MetiltransferasesRESUMO
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Quinases Relacionadas a NIMA , Prometáfase/fisiologia , Ligação Proteica , Multimerização Proteica , Fatores de Tempo , Células Tumorais Cultivadas , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
MYSM1 is a chromatin-binding transcriptional cofactor that deubiquitinates histone H2A. Studies of Mysm1-deficient mice have shown that it is essential for hematopoietic stem cell (HSC) function and lymphopoiesis. Human carriers of a rare MYSM1-inactivating mutation display similar lymphopoietic deficiencies. However, the mechanism by which MYSM1 regulates hematopoietic homeostasis remains unclear. Here, we show that Mysm1-deficiency results in p53 protein elevation in many hematopoietic cell types. p53 is a central regulator of cellular stress responses and HSC homeostasis. We thus generated double-knockout mice to assess a potential genetic interaction between Mysm1 and p53 in hematopoiesis. Mysm1(-/-)p53(-/-) mouse characterization showed a full rescue of Mysm1(-/-) developmental and hematopoietic defects. This included restoration of lymphopoiesis, and HSC numbers and functions. These results establish p53 activation as the driving mechanism for hematopoietic abnormalities in Mysm1 deficiency. Our findings may advance the understanding of p53 regulation in hematopoiesis and implicate MYSM1 as a potential p53 cofactor.
Assuntos
Endopeptidases/genética , Células-Tronco Hematopoéticas/patologia , Linfopenia/genética , Linfopoese , Proteína Supressora de Tumor p53/metabolismo , Animais , Deleção de Genes , Células-Tronco Hematopoéticas/metabolismo , Linfopenia/metabolismo , Linfopenia/patologia , Camundongos , Camundongos Knockout , Transativadores , Proteína Supressora de Tumor p53/genética , Proteases Específicas de UbiquitinaRESUMO
Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.
Assuntos
Homeostase/fisiologia , Intestinos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Células-Tronco/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas de Transporte Vesicular/fisiologia , Animais , Proteínas de Transporte/análise , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/análise , Feminino , Deleção de Genes , Humanos , Intestinos/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Células-Tronco/citologia , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologia , Proteínas de Transporte Vesicular/genéticaRESUMO
OBJECTIVES: The aim of the study was to evaluate indications, results, and clinical and neurological evolution in children who have undergone liver transplantation for classical maple syrup urine disease (MSUD). METHODS: Descriptive study of liver transplantation for MSUD between 1991 and 2012. Eight patients were transplanted. RESULTS: Indications for transplant were poor metabolic control expressed as significant psychomotor disabilities (4 had psychomotor delays, 5 had spasticity, and 5 had epilepsy) and poor quality of life (mean number of acute metabolic decompensations and mean number of total hospitalizations before transplantation 5 and 12, respectively). Four required nasogastric tube, with a maximum 4 g/day protein-restricted diet in all of them. Seven sustained significant alterations in brain magnetic resonance imaging. Mean leucine and alloisoleucine levels were 608 (standard deviation [SD] 516) and 218 µmol/L (SD 216), respectively. All of the patients received transplants with deceased-donor livers, with ages between 1.5 and 2.5 years (mean 1.78 years). Mean posttransplantation follow-up period was 12.2 years (range 5-21 years). Final patient and graft survival was 87.5% and 75%, respectively. Following transplantation, none required hospitalization in the last 3 years nor did any have new acute metabolic decompensations following a normal diet. Five followed normal schooling, 2 had motor disabilities, and 2 had convulsive crises. Brain magnetic resonance imaging was taken in 4 patients, showing neuroimage improvement in 3 of them. Mean leucine levels were <350 µmol/L from the immediate posttransplantation period (mean 225 µmol/L, SD 78), with a maximum alloisoleucine level of 20 µmol/L. CONCLUSIONS: Liver transplantation is an effective treatment for classical MSUD that arrests brain damage, although it does not reverse the process.
Assuntos
Encéfalo/patologia , Sobrevivência de Enxerto , Transplante de Fígado , Doença da Urina de Xarope de Bordo/cirurgia , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Isoleucina/sangue , Leucina/sangue , Transplante de Fígado/mortalidade , Masculino , Doença da Urina de Xarope de Bordo/sangue , Qualidade de Vida , Sobreviventes/estatística & dados numéricos , Resultado do TratamentoRESUMO
This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others.
Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/genética , Doença da Urina de Xarope de Bordo/genética , Fosfoproteínas Fosfatases/genética , Apoptose , Western Blotting , Sobrevivência Celular , Análise Mutacional de DNA , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Frequência do Gene , Genótipo , Humanos , Lactente , Isoleucina/sangue , Leucina/sangue , Doença da Urina de Xarope de Bordo/diagnóstico , Microscopia de Fluorescência , Mutação , Fenótipo , Proteína Fosfatase 2C , Complexo Piruvato Desidrogenase/genética , Espécies Reativas de Oxigênio , Análise de Sequência de DNA , Pele/citologia , Pele/metabolismoRESUMO
Recent years have seen great advances in our knowledge of congenital disorders of glycosylation (CDG), a clinically and biochemically heterogeneous group of genetic diseases caused by defects in the synthesis (CDG-I) or processing (CDG-II) of glycans that form glycoconjugates. This paper reports a new subtype of non-neurological CDG involving the impaired cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. A patient presented with muscle fatigue, elevated creatine kinase, growth hormone deficiency, and first branchial arch syndrome. These findings, together with the abnormal type II plasma transferrin isoform profile detected, was compatible with a CDG. Functional testing and clinical analyses suggested a deficiency in the interconversion of glucose-1-phosphate and glucose-6-phosphate catalyzed by phosphoglucomutase (PGM1), a defect previously described as glycogenosis type XIV (GSDXIV, MIM 612934). PGM1 activity in patient-derived fibroblasts was significantly reduced, as was the quantity of immunoreactive PGM1 protein (Western blot assays). Mutation analysis of PGM1 and subsequent functional analysis investigating transient expression of PGM1 in immortalized patient fibroblasts, followed by ex vivo splicing assays using minigenes, allowed the characterization of two novel pathogenic mutations: c.871G>A (p.Gly291Arg) and c.1144 + 3A>T. The latter represents a severe splicing mutation leading to the out-of-frame skipping of exon 7 and the formation of a truncated protein (p.Arg343fs). MALDI mass spectra of permethylated protein N-glycans from the patient's serum suggested a marked hypoglycosylation defect. The present findings confirm that, in addition to a rare muscular glycolytic defect, PGM1 deficiency causes a non-neurological disorder of glycosylation.
Assuntos
Sistema Nervoso Central/metabolismo , Defeitos Congênitos da Glicosilação/classificação , Defeitos Congênitos da Glicosilação/genética , Mutação de Sentido Incorreto , Fosfoglucomutase/genética , Adolescente , Metabolismo dos Carboidratos/genética , Sequência de Carboidratos , Sistema Nervoso Central/fisiopatologia , Defeitos Congênitos da Glicosilação/complicações , Análise Mutacional de DNA , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/fisiologia , Polimorfismo de Nucleotídeo Único/fisiologiaRESUMO
BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive disease that belongs to a group of disorders resulting from inborn errors of protein metabolism. It was the first disease included in neonatal screening. Neonatal screening has allowed an early diagnosis and treatment of the disease. As a result, an increasing number of women diagnosed with phenylketonuria have reached the reproductive phase of life in good health, and management of pregnancy in women with PKU is becoming more frequent. CASE PRESENTATION: In this study, we report the case of a 28-year-old Caucasian patient being followed up for phenylketonuria at Ramón y Cajal Hospital's Metabolic Diseases Unit. We describe the patient's gestation, impacted by her and her partner's diagnosis of PKU, classic and mild phenotypes, respectively, resulting in the fetus affectation. CONCLUSIONS: The description of PKU management-diagnosis, follow-up, and treatment-for both that of patient and that of the gestation with fetus affectation covers a wide sample scenario that shows the effectiveness of pregnancy planning and monitoring of females with PKU and questions the need to carry out a genetic study of gene PKU in the study of fertility.
Assuntos
Fenilcetonúrias , Gravidez , Recém-Nascido , Humanos , Feminino , Adulto , Fenilcetonúrias/diagnóstico , Triagem Neonatal/métodosRESUMO
Malaria transmission to mosquitoes requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei, the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of ten mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in the determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain as well as the protein interactors of a female-determining zinc-finger protein indicate that germ-granule-like ribonucleoprotein complexes complement transcriptional processes in the regulation of both male and female development of a malaria parasite.
Assuntos
Culicidae , Malária , Parasitos , Animais , Feminino , Masculino , Parasitos/metabolismo , Malária/parasitologia , Plasmodium berghei/genética , Desenvolvimento Sexual/genética , Culicidae/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The combination of affinity purification with mass spectrometry analysis has become the method of choice for protein complex characterization. With the improved performance of mass spectrometry technology, the sensitivity of the analyses is increasing, probing deeper into molecular interactions and yielding longer lists of proteins. These identify not only core complex subunits but also the more inaccessible proteins that interact weakly or transiently. Alongside them, contaminant proteins, which are often abundant proteins in the cell, tend to be recovered in affinity experiments because they bind nonspecifically and with low affinity to matrix, tag, and/or antibody. The challenge now lies in discriminating nonspecific binders from true interactors, particularly at the low level and in a larger scale. This review aims to summarize the variety of methods that have been used to distinguish contaminants from specific interactions in the past few years, ranging from manual elimination using heuristic rules to more sophisticated probabilistic scoring approaches. We aim to give awareness on the processing that takes place before an interaction list is reported and on the different types of list curation approaches suited to the different experiments.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Animais , Cromatografia de Afinidade/métodos , Humanos , Marcação por Isótopo , Espectrometria de Massas/métodos , Modelos Estatísticos , Ligação Proteica , Proteoma/química , Proteoma/isolamento & purificação , Coloração e Rotulagem/métodosRESUMO
Candida albicans is the principal causative agent of lethal fungal infections, predominantly in immunocompromised hosts. Extracellular vesicles (EVs) have been described as crucial in the interaction of microorganisms with their host. Since the yeast-to-hypha transition is an important virulence trait with great impact in invasive candidiasis (IC), we have addressed the characterization of EVs secreted by hyphal cells (HEVs) from C. albicans, comparing them to yeast EVs (YEVs). YEVs comprised a larger population of bigger EVs with mainly cell wall proteins, while HEVs were smaller, in general, and had a much higher protein diversity. YEVs were able to rescue the sensitivity of a cell wall mutant against calcofluor white, presumably due to the larger amount of cell wall proteins they contained. On the other hand, HEVs also contained many cytoplasmic proteins related to protein metabolism and intracellular protein transport and the endosomal sorting complexes required for transport (ESCRT) pathway related to exosome biogenesis, pointing to an intracellular origin of HEVs. Interestingly, an active 20S proteasome complex was secreted exclusively in HEVs. Moreover, HEVs contained a greater number of virulence-related proteins. As for their immunogenic role, both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis; however, under our conditions, only HEVs showed a cytotoxic effect on human macrophages and could elicit the release of tumor necrosis factor alpha (TNF-α) by these macrophages. IMPORTANCE This first analysis of HEVs of C. albicans has shown clear differences between them and the YEVs of C. albicans, showing their relevance and possible use in the discovery of new diagnostic markers and treatment targets against C. albicans infections. The data obtained point to different mechanisms of biogenesis of YEVs and HEVs, as well as different involvements in cell biology and host interaction. YEVs played a more relevant role in cell wall maintenance, while HEVs were more closely related to virulence, as they had greater effects on human immune cells. Importantly, an active 20S proteosome complex was described as a fungal-EV cargo. A deeper study of its role and those of many other proteins exclusively detected in HEVs and involved in different relevant biological processes of this fungus could open up interesting new areas of research in the battle against C. albicans.
Assuntos
Candidíase Invasiva , Vesículas Extracelulares , Candida albicans/metabolismo , Candidíase , Candidíase Invasiva/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Hifas/metabolismo , Imunidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Aneuploidy results in decreased cellular fitness in many species and model systems. However, aneuploidy is commonly found in cancer cells and often correlates with aggressive growth, suggesting that the impact of aneuploidy on cellular fitness is context dependent. The BRG1 (SMARCA4) subunit of the SWI/SNF chromatin remodelling complex is frequently lost in cancer. Here, we use a chromosomally stable cell line to test the effect of BRG1 loss on the evolution of aneuploidy. BRG1 deletion leads to an initial loss of fitness in this cell line that improves over time. Notably, we find increased tolerance to aneuploidy immediately upon loss of BRG1, and the fitness recovery over time correlates with chromosome gain. These data show that BRG1 loss creates an environment where karyotype changes can be explored without a fitness penalty. At least in some genetic backgrounds, therefore, BRG1 loss can affect the progression of tumourigenesis through tolerance of aneuploidy.
Assuntos
Aneuploidia , Montagem e Desmontagem da Cromatina , Linhagem Celular , Aberrações Cromossômicas , Cromossomos , DNA Helicases/genética , Humanos , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Propionic acidaemia (PA) is an innate error of metabolism involving a deficiency in the enzyme propionyl-CoA carboxylase. Better control of acute decompensation episodes together with better treatment and monitoring have improved the prognosis of patients with this problem. However, long-term complications can arise in those in whom good metabolic control is achieved, the result of mitochondrial dysfunction caused by deficient anaplerosis, increased oxidative stress, and reduced antioxidative capacity. Coenzyme Q10 (CoQ10) is a nutritional supplement that has a notable antioxidative effect and has been shown to improve mitochondrial function. The present prospective, interventional study examines the plasma concentration of CoQ10 in patients with PA, their tolerance of such supplementation with ubiquinol, and its benefits. Seven patients with PA (aged 2.5 to 20 years, 4 males) received supplements of CoQ10 in the form of ubiquinol (10 mg/kg/day for 6 months). A total of 6/7 patients showed reduced plasma CoQ10 concentrations that normalized after supplementation with ubiquinol (p-value < 0.001), which was well tolerated. Urinary citrate levels markedly increased during the study (p-value: 0.001), together with elevation of citrate/methlycitrate ratio (p-value: 0.03). No other significant changes were seen in plasma or urine biomarkers of PA. PA patients showed a deficiency of plasma CoQ10, which supplementation with ubiquinol corrected. The urinary excretion of Krebs cycle intermediate citrate and the citrate/methylcitrate ratio significantly increased compared to the baseline, suggesting improvement in anaplerosis. This treatment was well tolerated and should be further investigated as a means of preventing the chronic complications associated with likely multifactorial mitochondrial dysfunction in PA.
RESUMO
Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.