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1.
Mol Cell ; 82(15): 2885-2899.e8, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35841888

RESUMO

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.


Assuntos
Regulação da Expressão Gênica , Ribossomos , Genoma Humano/genética , Humanos , Fases de Leitura Aberta/genética , Biossíntese de Proteínas , Proteínas/metabolismo , RNA/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(8): e2209177120, 2023 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-36787364

RESUMO

Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic protein aggregates in Alzheimer's disease (AD). Recent evidence indicates that a switch in energy production from mitochondrial respiration to glycolysis disrupts this important protective microglial function and may provide therapeutic targets for AD. Here, we demonstrate that the translocator protein (TSPO) and a member of its mitochondrial complex, hexokinase-2 (HK), play critical roles in microglial respiratory-glycolytic metabolism and phagocytosis. Pharmacological and genetic loss-of-function experiments showed that TSPO is critical for microglial respiratory metabolism and energy supply for phagocytosis, and its expression is enriched in phagocytic microglia of AD mice. Meanwhile, HK controlled glycolytic metabolism and phagocytosis via mitochondrial binding or displacement. In cultured microglia, TSPO deletion impaired mitochondrial respiration and increased mitochondrial recruitment of HK, inducing a switch to glycolysis and reducing phagocytosis. To determine the functional significance of mitochondrial HK recruitment, we developed an optogenetic tool for reversible control of HK localization. Displacement of mitochondrial HK inhibited glycolysis and improved phagocytosis in TSPO-knockout microglia. Mitochondrial HK recruitment also coordinated the inflammatory switch to glycolysis that occurs in response to lipopolysaccharide in normal microglia. Interestingly, cytosolic HK increased phagocytosis independent of its metabolic activity, indicating an immune signaling function. Alzheimer's beta amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in AD. Thus, targeting mitochondrial HK may offer an immunotherapeutic approach to promote phagocytic microglial function in AD.


Assuntos
Doença de Alzheimer , Animais , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Microglia/metabolismo , Fagocitose , Mitocôndrias/metabolismo
3.
Development ; 147(21)2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33033118

RESUMO

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.


Assuntos
Diferenciação Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Endoderma/embriologia , Doenças da Vesícula Biliar/genética , Doenças da Vesícula Biliar/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Atresia Intestinal/genética , Atresia Intestinal/patologia , Mutação/genética , Pâncreas/embriologia , Fatores de Transcrição de Fator Regulador X/genética , Alelos , Sequência de Bases , Diferenciação Celular/genética , Cromatina/metabolismo , Consanguinidade , Diabetes Mellitus/diagnóstico por imagem , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Família , Feminino , Doenças da Vesícula Biliar/diagnóstico por imagem , Genoma Humano , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Atresia Intestinal/diagnóstico por imagem , Masculino , Linhagem , Transcrição Gênica , Transcriptoma/genética , Microtomografia por Raio-X
4.
Proc Natl Acad Sci U S A ; 116(19): 9622-9627, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31015293

RESUMO

White matter abnormalities are a nearly universal pathological feature of neurodegenerative disorders including Huntington disease (HD). A long-held assumption is that this white matter pathology is simply a secondary outcome of the progressive neuronal loss that manifests with advancing disease. Using a mouse model of HD, here we show that white matter and myelination abnormalities are an early disease feature appearing before the manifestation of any behavioral abnormalities or neuronal loss. We further show that selective inactivation of mutant huntingtin (mHTT) in the NG2+ oligodendrocyte progenitor cell population prevented myelin abnormalities and certain behavioral deficits in HD mice. Strikingly, the improvements in behavioral outcomes were seen despite the continued expression of mHTT in nonoligodendroglial cells including neurons, astrocytes, and microglia. Using RNA-seq and ChIP-seq analyses, we implicate a pathogenic mechanism that involves enhancement of polycomb repressive complex 2 (PRC2) activity by mHTT in the intrinsic oligodendroglial dysfunction and myelination deficits observed in HD. Our findings challenge the long-held dogma regarding the etiology of white matter pathology in HD and highlight the contribution of epigenetic mechanisms to the observed intrinsic oligodendroglial dysfunction. Our results further suggest that ameliorating white matter pathology and oligodendroglial dysfunction may be beneficial for HD.


Assuntos
Comportamento Animal , Doenças Desmielinizantes , Proteína Huntingtina , Doença de Huntington , Mutação , Oligodendroglia , Animais , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Camundongos Mutantes , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Substância Branca/metabolismo , Substância Branca/patologia
5.
Circulation ; 140(11): 937-951, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31284728

RESUMO

BACKGROUND: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. METHODS: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart. RESULTS: We generated nucleotide-resolution translatome data during the transforming growth factor ß1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor ß1. CONCLUSIONS: We reveal widespread translational effects of transforming growth factor ß1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.


Assuntos
Cardiopatias/genética , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Perfilação da Expressão Gênica/métodos , Cardiopatias/patologia , Humanos , Análise de Sequência de RNA/métodos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
6.
Genome Res ; 27(3): 440-450, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28250018

RESUMO

The recoding of genetic information through RNA editing contributes to proteomic diversity, but the extent and significance of RNA editing in disease is poorly understood. In particular, few studies have investigated the relationship between RNA editing and disease at a genome-wide level. Here, we developed a framework for the genome-wide detection of RNA sites that are differentially edited in disease. Using RNA-sequencing data from 100 hippocampi from mice with epilepsy (pilocarpine-temporal lobe epilepsy model) and 100 healthy control hippocampi, we identified 256 RNA sites (overlapping with 87 genes) that were significantly differentially edited between epileptic cases and controls. The degree of differential RNA editing in epileptic mice correlated with frequency of seizures, and the set of genes differentially RNA-edited between case and control mice were enriched for functional terms highly relevant to epilepsy, including "neuron projection" and "seizures." Genes with differential RNA editing were preferentially enriched for genes with a genetic association to epilepsy. Indeed, we found that they are significantly enriched for genes that harbor nonsynonymous de novo mutations in patients with epileptic encephalopathy and for common susceptibility variants associated with generalized epilepsy. These analyses reveal a functional convergence between genes that are differentially RNA-edited in acquired symptomatic epilepsy and those that contribute risk for genetic epilepsy. Taken together, our results suggest a potential role for RNA editing in the epileptic hippocampus in the occurrence and severity of epileptic seizures.


Assuntos
Epilepsia/genética , Edição de RNA , Animais , Estudo de Associação Genômica Ampla , Hipocampo/metabolismo , Masculino , Camundongos , Transcriptoma
7.
Circ Res ; 118(3): 420-432, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26646931

RESUMO

RATIONALE: Platelets shed microRNAs (miRNAs). Plasma miRNAs change on platelet inhibition. It is unclear whether plasma miRNA levels correlate with platelet function. OBJECTIVE: To link small RNAs to platelet reactivity. METHODS AND RESULTS: Next-generation sequencing of small RNAs in plasma revealed 2 peaks at 22 to 23 and 32 to 33 nucleotides corresponding to miRNAs and YRNAs, respectively. Among YRNAs, predominantly, fragments of RNY4 and RNY5 were detected. Plasma miRNAs and YRNAs were measured in 125 patients with a history of acute coronary syndrome who had undergone detailed assessment of platelet function 30 days after the acute event. Using quantitative real-time polymerase chain reactions, 92 miRNAs were assessed in patients with acute coronary syndrome on different antiplatelet therapies. Key platelet-related miRNAs and YRNAs were correlated with platelet function tests. MiR-223 (rp=0.28; n=121; P=0.002), miR-126 (rp=0.22; n=121; P=0.016), and other abundant platelet miRNAs and YRNAs showed significant positive correlations with the vasodilator-stimulated phosphoprotein phosphorylation assay. YRNAs, miR-126, and miR-223 were also among the small RNAs showing the greatest dependency on platelets and strongly correlated with plasma levels of P-selectin, platelet factor 4, and platelet basic protein in the population-based Bruneck study (n=669). A single-nucleotide polymorphism that facilitates processing of pri-miR-126 to mature miR-126 accounted for a rise in circulating platelet activation markers. Inhibition of miR-126 in mice reduced platelet aggregation. MiR-126 directly and indirectly affects ADAM9 and P2Y12 receptor expression. CONCLUSIONS: Levels of platelet-related plasma miRNAs and YRNAs correlate with platelet function tests in patients with acute coronary syndrome and platelet activation markers in the general population. Alterations in miR-126 affect platelet reactivity.


Assuntos
Síndrome Coronariana Aguda/sangue , Plaquetas/metabolismo , MicroRNAs/sangue , Ativação Plaquetária , Síndrome Coronariana Aguda/tratamento farmacológico , Síndrome Coronariana Aguda/genética , Animais , Plaquetas/efeitos dos fármacos , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
8.
PLoS Genet ; 10(12): e1004813, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25474312

RESUMO

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.


Assuntos
Doenças Cardiovasculares/genética , Metilação de DNA , Genoma , Miocárdio/metabolismo , Animais , Sequência de Bases , Doenças Cardiovasculares/patologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Polimorfismo de Nucleotídeo Único , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos SHR , Análise de Sequência de DNA/métodos
9.
J Mol Cell Cardiol ; 92: 105-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26827899

RESUMO

The adult human myocardium is incapable of regeneration; yet, the zebrafish (Danio rerio) can regenerate damaged myocardium. Similar to the zebrafish heart, hearts of neonatal, but not adult mice are capable of myocardial regeneration. We performed a proteomics analysis of adult zebrafish hearts and compared their protein expression profile to hearts from neonatal and adult mice. Using difference in-gel electrophoresis (DIGE), there was little overlap between the proteome from adult mouse (>8weeks old) and adult zebrafish (18months old) hearts. Similarly, there was a significant degree of mismatch between the protein expression in neonatal and adult mouse hearts. Enrichment analysis of the selected proteins revealed over-expression of DNA synthesis-related proteins in the cardiac proteome of the adult zebrafish heart similar to neonatal and 4days old mice, whereas in hearts of adult mice there was a mitochondria-related predominance in protein expression. Importantly, we noted pronounced differences in the myofilament composition: the adult zebrafish heart lacks many of the myofilament proteins of differentiated adult cardiomyocytes such as the ventricular isoforms of myosin light chains and nebulette. Instead, troponin I and myozenin 1 were expressed as skeletal isoforms rather than cardiac isoforms. The relative immaturity of the adult zebrafish heart was further supported by cardiac microRNA data. Our assessment of zebrafish and mammalian hearts challenges the assertions on the translational potential of cardiac regeneration in the zebrafish model. The immature myofilament composition of the fish heart may explain why adult mouse and human cardiomyocytes lack this endogenous repair mechanism.


Assuntos
Coração/crescimento & desenvolvimento , Proteoma/biossíntese , Proteômica , Regeneração/genética , Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Humanos , Camundongos , MicroRNAs/biossíntese , Proteínas dos Microfilamentos/biossíntese , Proteínas Musculares/biossíntese , Miócitos Cardíacos/metabolismo , Proteoma/genética , Transcriptoma , Troponina I/biossíntese , Peixe-Zebra/crescimento & desenvolvimento
10.
Circ Res ; 115(10): 857-66, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25201911

RESUMO

RATIONALE: Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. OBJECTIVE: Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. METHODS AND RESULTS: Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E-deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E-deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. CONCLUSIONS: We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.


Assuntos
Aneurisma da Aorta Abdominal/sangue , MicroRNAs/fisiologia , Idoso , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Biomarcadores/sangue , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/sangue , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia
11.
Nature ; 467(7314): 460-4, 2010 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-20827270

RESUMO

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.


Assuntos
Diabetes Mellitus Tipo 1/genética , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Imunidade Inata/genética , Vírus/imunologia , Animais , Sequência de Bases , Cromossomos Humanos Par 13/genética , Cromossomos de Mamíferos/genética , Diabetes Mellitus Tipo 1/imunologia , Redes Reguladoras de Genes/genética , Estudo de Associação Genômica Ampla , Humanos , Inflamação/genética , Inflamação/imunologia , Fator Regulador 7 de Interferon/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
12.
Mol Cell Proteomics ; 13(10): 2545-57, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24958171

RESUMO

In an endotoxaemic mouse model of sepsis, a tissue-based proteomics approach for biomarker discovery identified long pentraxin 3 (PTX3) as the lead candidate for inflamed myocardium. When the redox-sensitive oligomerization state of PTX3 was further investigated, PTX3 accumulated as an octamer as a result of disulfide-bond formation in heart, kidney, and lung-common organ dysfunctions seen in patients with sepsis. Oligomeric moieties of PTX3 were also detectable in circulation. The oligomerization state of PTX3 was quantified over the first 11 days in critically ill adult patients with sepsis. On admission day, there was no difference in the oligomerization state of PTX3 between survivors and non-survivors. From day 2 onward, the conversion of octameric to monomeric PTX3 was consistently associated with a greater survival after 28 days of follow-up. For example, by day 2 post-admission, octameric PTX3 was barely detectable in survivors, but it still constituted more than half of the total PTX3 in non-survivors (p < 0.001). Monomeric PTX3 was inversely associated with cardiac damage markers NT-proBNP and high-sensitivity troponin I and T. Relative to the conventional measurements of total PTX3 or NT-proBNP, the oligomerization of PTX3 was a superior predictor of disease outcome.


Assuntos
Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Proteômica/métodos , Sepse/metabolismo , Sepse/mortalidade , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/metabolismo , Idoso , Animais , Biomarcadores/química , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Oxirredução , Prognóstico , Multimerização Proteica , Sepse/patologia
13.
Circulation ; 129(18): 1821-31, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24622385

RESUMO

BACKGROUND: The bulk of cardiovascular disease risk is not explained by traditional risk factors. Recent advances in mass spectrometry allow the identification and quantification of hundreds of lipid species. Molecular lipid profiling by mass spectrometry may improve cardiovascular risk prediction. METHODS AND RESULTS: Lipids were extracted from 685 plasma samples of the prospective population-based Bruneck Study (baseline evaluation in 2000). One hundred thirty-five lipid species from 8 different lipid classes were profiled by shotgun lipidomics with the use of a triple-quadrupole mass spectrometer. Levels of individual species of cholesterol esters (CEs), lysophosphatidylcholines, phosphatidylcholines, phosphatidylethanolamines (PEs), sphingomyelins, and triacylglycerols (TAGs) were associated with cardiovascular disease over a 10-year observation period (2000-2010, 90 incident events). Among the lipid species with the strongest predictive value were TAGs and CEs with a low carbon number and double-bond content, including TAG(54:2) and CE(16:1), as well as PE(36:5) (P=5.1 × 10⁻7, 2.2 × 10⁻4, and 2.5 × 10⁻³, respectively). Consideration of these 3 lipid species on top of traditional risk factors resulted in improved risk discrimination and classification for cardiovascular disease (cross-validated ΔC index, 0.0210 [95% confidence interval, 0.0010-0.0422]; integrated discrimination improvement, 0.0212 [95% confidence interval, 0.0031-0.0406]; and continuous net reclassification index, 0.398 [95% confidence interval, 0.175-0.619]). A similar shift in the plasma fatty acid composition was associated with cardiovascular disease in the UK Twin Registry (n=1453, 45 cases). CONCLUSIONS: This study applied mass spectrometry-based lipidomics profiling to population-based cohorts and identified molecular lipid signatures for cardiovascular disease. Molecular lipid species constitute promising new biomarkers that outperform the conventional biochemical measurements of lipid classes currently used in clinics.


Assuntos
Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/metabolismo , Lipidoses/epidemiologia , Lipidoses/metabolismo , Metabolômica , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Ésteres do Colesterol/metabolismo , Feminino , Humanos , Lisofosfatidilcolinas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Análise Multivariada , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Valor Preditivo dos Testes , Estudos Prospectivos , Sistema de Registros , Fatores de Risco , Esfingomielinas/metabolismo , Triglicerídeos/metabolismo , Reino Unido/epidemiologia
14.
Ann Rheum Dis ; 74(3): e18, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24515954

RESUMO

BACKGROUND: Osteoarthritis is the most common form of arthritis and a major socioeconomic burden. Our study is the first to explore the association between serum microRNA levels and the development of severe osteoarthritis of the knee and hip joint in the general population. METHODS: We followed 816 Caucasian individuals from 1995 to 2010 and assessed joint arthroplasty as a definitive outcome of severe osteoarthritis of the knee and hip. After a microarray screen, we validated 12 microRNAs by real-time PCR in the entire cohort at baseline. RESULTS: In Cox regression analysis, three microRNAs were associated with severe knee and hip osteoarthritis. let-7e was a negative predictor for total joint arthroplasty with an adjusted HR of 0.75 (95% CI 0.58 to 0.96; p=0.021) when normalised to U6, and 0.76 (95% CI 0.6 to 0.97; p=0.026) after normalisation to the Ct average. miRNA-454 was inversely correlated with severe knee or hip osteoarthritis with an adjusted HR of 0.77 (95% CI 0.61 to 0.97; p=0.028) when normalised to U6. This correlation was lost when data were normalised to Ct average (p=0.118). Finally, miRNA-885-5p showed a trend towards a positive relationship with arthroplasty when normalised to U6 (HR 1.24; 95% CI 0.95 to 1.62; p=0.107) or to Ct average (HR 1.30; 95% CI 0.99 to 1.70; p=0.056). CONCLUSIONS: Our study is the first to identify differentially expressed circulating microRNAs in osteoarthritis patients necessitating arthroplasty in a large, population-based cohort. Among these microRNAs, let-7e emerged as potential predictor for severe knee or hip osteoarthritis.


Assuntos
MicroRNAs/sangue , Osteoartrite do Quadril/genética , Osteoartrite do Joelho/genética , Idoso , Artroplastia de Quadril , Artroplastia do Joelho , Estudos de Coortes , Feminino , Humanos , Estudos Longitudinais , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Osteoartrite do Quadril/sangue , Osteoartrite do Quadril/cirurgia , Osteoartrite do Joelho/sangue , Osteoartrite do Joelho/cirurgia , Modelos de Riscos Proporcionais , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de Doença
15.
Circ Res ; 113(10): 1138-47, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24006456

RESUMO

RATIONALE: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. OBJECTIVE: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. METHODS AND RESULTS: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-ß. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre-miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre-miR-29 conditioned medium, whereas pre-miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. CONCLUSIONS: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , MicroRNAs/fisiologia , Miocárdio/metabolismo , Proteômica , Animais , Proteína C-Reativa/metabolismo , Células Cultivadas , Colágeno/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibrose , Fator de Crescimento Insulin-Like I/metabolismo , Fator Inibidor de Leucemia/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Miocárdio/patologia , Componente Amiloide P Sérico/metabolismo , Fator de Crescimento Transformador beta/farmacologia
16.
Circ Res ; 112(4): 595-600, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23283721

RESUMO

RATIONALE: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. OBJECTIVE: To analyze changes in plasma miRNAs in response to antiplatelet therapy. METHODS AND RESULTS: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. CONCLUSIONS: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.


Assuntos
Plaquetas/metabolismo , MicroRNAs/sangue , Plasma/metabolismo , Ativação Plaquetária , Plasma Rico em Plaquetas/metabolismo , Soro/metabolismo , Adulto , Aspirina/administração & dosagem , Aspirina/farmacologia , Aspirina/uso terapêutico , Biomarcadores , Plaquetas/efeitos dos fármacos , Coleta de Amostras Sanguíneas/métodos , Doenças das Artérias Carótidas/tratamento farmacológico , Estudos de Casos e Controles , Ensaios Clínicos como Assunto , Fatores de Confusão Epidemiológicos , Diabetes Mellitus Tipo 2/sangue , Relação Dose-Resposta a Droga , Monitoramento de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Perfilação da Expressão Gênica , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Ativação Plaquetária/genética , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Cloridrato de Prasugrel , Reação em Cadeia da Polimerase em Tempo Real , Tiofenos/administração & dosagem , Tiofenos/farmacologia , Adulto Jovem
17.
J Mol Cell Cardiol ; 55: 27-30, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23277191

RESUMO

Perhexiline is a potent anti-anginal drug used for treatment of refractory angina and other forms of heart disease. It provides an oxygen sparing effect in the myocardium by creating a switch from fatty acid to glucose metabolism through partial inhibition of carnitine palmitoyltransferase 1 and 2. However, the precise molecular mechanisms underlying the cardioprotective effects elicited by perhexiline are not fully understood. The present study employed a combined proteomics, metabolomics and computational approach to characterise changes in murine hearts upon treatment with perhexiline. According to results based on difference in-gel electrophoresis, the most profound change in the cardiac proteome related to the activation of the pyruvate dehydrogenase complex. Metabolomic analysis by high-resolution nuclear magnetic resonance spectroscopy showed lower levels of total creatine and taurine in hearts of perhexiline-treated mice. Creatine and taurine levels were also significantly correlated in a cross-correlation analysis of all metabolites. Computational modelling suggested that far from inducing a simple shift from fatty acid to glucose oxidation, perhexiline may cause complex rebalancing of carbon and nucleotide phosphate fluxes, fuelled by increased lactate and amino acid uptake, to increase metabolic flexibility and to maintain cardiac output. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".


Assuntos
Fármacos Cardiovasculares/farmacologia , Coração/efeitos dos fármacos , Metaboloma , Miocárdio/metabolismo , Perexilina/farmacologia , Proteoma , Animais , Análise por Conglomerados , Simulação por Computador , Masculino , Metabolômica , Camundongos , Modelos Biológicos , Proteômica
18.
Neuromolecular Med ; 25(4): 644-649, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37684514

RESUMO

Transcriptional and proteomics analyses in human fragile X syndrome (FXS) neurons identified markedly reduced expression of COMT, a key enzyme involved in the metabolism of catecholamines, including dopamine, epinephrine and norepinephrine. FXS is the most common genetic cause of intellectual disability and autism spectrum disorders. COMT encodes for catechol-o-methyltransferase and its association with neuropsychiatric disorders and cognitive function has been extensively studied. We observed a significantly reduced level of COMT in in FXS human neural progenitors and neurons, as well as hippocampal neurons from Fmr1 null mice. We show that deficits in COMT were associated with an altered response in an assay of dopaminergic activity in Fmr1 null mice. These findings demonstrate that loss of FMRP downregulates COMT expression and affects dopamine signaling in FXS, and supports the notion that targeting catecholamine metabolism may be useful in regulating certain neuropsychiatric aspects of FXS.


Assuntos
Catecol O-Metiltransferase , Síndrome do Cromossomo X Frágil , Animais , Humanos , Camundongos , Catecol O-Metiltransferase/genética , Dopamina/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Camundongos Knockout , Neurônios/metabolismo
19.
EBioMedicine ; 94: 104720, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37481821

RESUMO

BACKGROUND: In Huntington's disease (HD), a CAG repeat expansion mutation in the Huntingtin (HTT) gene drives a gain-of-function toxicity that disrupts mRNA processing. Although dysregulation of gene splicing has been shown in human HD post-mortem brain tissue, post-mortem analyses are likely confounded by cell type composition changes in late-stage HD, limiting the ability to identify dysregulation related to early pathogenesis. METHODS: To investigate gene splicing changes in early HD, we performed alternative splicing analyses coupled with a proteogenomics approach to identify early CAG length-associated splicing changes in an established isogenic HD cell model. FINDINGS: We report widespread neuronal differentiation stage- and CAG length-dependent splicing changes, and find an enrichment of RNA processing, neuronal function, and epigenetic modification-related genes with mutant HTT-associated splicing. When integrated with a proteomics dataset, we identified several of these differential splicing events at the protein level. By comparing with human post-mortem and mouse model data, we identified common patterns of altered splicing from embryonic stem cells through to post-mortem striatal tissue. INTERPRETATION: We show that widespread splicing dysregulation in HD occurs in an early cell model of neuronal development. Importantly, we observe HD-associated splicing changes in our HD cell model that were also identified in human HD striatum and mouse model HD striatum, suggesting that splicing-associated pathogenesis possibly occurs early in neuronal development and persists to later stages of disease. Together, our results highlight splicing dysregulation in HD which may lead to disrupted neuronal function and neuropathology. FUNDING: This research is supported by the Lee Kong Chian School of Medicine, Nanyang Technological University Singapore Nanyang Assistant Professorship Start-Up Grant, the Singapore Ministry of Education under its Singapore Ministry of Education Academic Research Fund Tier 1 (RG23/22), the BC Children's Hospital Research Institute Investigator Grant Award (IGAP), and a Scholar Award from the Michael Smith Health Research BC.


Assuntos
Doença de Huntington , Camundongos , Animais , Criança , Humanos , Doença de Huntington/metabolismo , Splicing de RNA/genética , Processamento Alternativo , Mutação , RNA Mensageiro/metabolismo , Proteína Huntingtina/genética
20.
J Mol Cell Cardiol Plus ; 6: 100056, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38143961

RESUMO

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ∼1: 200-500 and predisposes individuals to heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac fibroblasts. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

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