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1.
Am J Hum Genet ; 102(4): 685-695, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576219

RESUMO

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.


Assuntos
Alelos , Perda Auditiva Neurossensorial/genética , Hipoglicemia/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/genética , Feminino , Fibroblastos/metabolismo , Perda Auditiva Neurossensorial/complicações , Humanos , Hipoglicemia/complicações , Lactente , Recém-Nascido , Masculino , Doenças Mitocondriais/complicações , Proteínas Mitocondriais/química , Fosforilação Oxidativa , Subunidades Proteicas/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/química
2.
J Inherit Metab Dis ; 43(6): 1382-1391, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32418222

RESUMO

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3 . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.


Assuntos
Cútis Laxa/genética , Antígenos de Histocompatibilidade Menor/genética , Mutação de Sentido Incorreto , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Pele/patologia , Sequência de Aminoácidos , Animais , Criança , Cútis Laxa/patologia , Feminino , Glicosilação , Homozigoto , Humanos , Camundongos , Camundongos Knockout , Linhagem , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência
3.
Respir Res ; 20(1): 128, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234847

RESUMO

BACKGROUND: Elastin breakdown and the resultant loss of lung elastic recoil is a hallmark of pulmonary emphysema in susceptible individuals as a consequence of tobacco smoke exposure. Systemic alterations to the synthesis and degradation of elastin may be important to our understanding of disease phenotypes in chronic obstructive pulmonary disease. We investigated the association of skin elasticity with pulmonary emphysema, obstructive lung disease, and blood biomarkers of inflammation and tissue protease activity in tobacco-exposed individuals. METHODS: Two hundred and thirty-six Caucasian individuals were recruited into a sub-study of the University of Pittsburgh Specialized Center for Clinically Orientated Research in chronic obstructive pulmonary disease, a prospective cohort study of current and former smokers. The skin viscoelastic modulus (VE), a determinant of skin elasticity, was recorded from the volar forearm and facial wrinkling severity was determined using the Daniell scoring system. RESULTS: In a multiple regression analysis, reduced VE was significantly associated with cross-sectional measurement of airflow obstruction (FEV1/FVC) and emphysema quantified from computed tomography (CT) images, ß = 0.26, p = 0.001 and ß = 0.24, p = 0.001 respectively. In emphysema-susceptible individuals, elasticity-determined skin age was increased (median 4.6 years) compared to the chronological age of subjects without emphysema. Plasma biomarkers of inflammation (TNFR1, TNFR2, CRP, PTX3, and SAA) and matrix metalloproteinase activity (MMP1, TIMP1, TIMP2, and TIMP4) were inversely associated with skin elasticity. CONCLUSIONS: We report that an objective non-invasive determinant of skin elasticity is independently associated with measures of lung function, pulmonary emphysema, and biomarkers of inflammation and tissue proteolysis in tobacco-exposed individuals. Loss of skin elasticity is a novel observation that may link the common pathological processes that drive tissue elastolysis in the extracellular matrix of the skin and lung in emphysema-susceptible individuals.


Assuntos
Mediadores da Inflamação/sangue , Metaloproteinases da Matriz/sangue , Enfisema Pulmonar/sangue , Envelhecimento da Pele/patologia , Fumantes , Fumar Tabaco/sangue , Idoso , Biomarcadores/sangue , Estudos de Coortes , Elasticidade/fisiologia , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , Estudos Prospectivos , Enfisema Pulmonar/diagnóstico , Método Simples-Cego , Fumar Tabaco/efeitos adversos
4.
Hum Mol Genet ; 24(14): 4024-36, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25882708

RESUMO

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFß) signaling. Despite elevated extracellular TGFß activity, downstream signaling molecules of the TGFß pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFß receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFß1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFß receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFß signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFß receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFß receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.


Assuntos
Cútis Laxa/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Estudos de Casos e Controles , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Endocitose/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Proteínas de Ligação a TGF-beta Latente/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo
5.
Hum Mol Genet ; 21(6): 1248-59, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22116938

RESUMO

Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-ß (TGFß) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFß-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFß receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFß reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFß inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFß signaling.


Assuntos
Anormalidades Cardiovasculares/etiologia , Proteínas Facilitadoras de Transporte de Glucose/fisiologia , Mitocôndrias/metabolismo , Notocorda/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Anormalidades Cardiovasculares/metabolismo , Anormalidades Cardiovasculares/patologia , Luciferases/metabolismo , Mitocôndrias/patologia , Dados de Sequência Molecular , Morfolinos/farmacologia , Mutação/genética , Notocorda/patologia , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcriptoma , Fator de Crescimento Transformador beta/antagonistas & inibidores , Peixe-Zebra/crescimento & desenvolvimento
6.
Pediatr Dermatol ; 31(3): 347-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24758204

RESUMO

We report a 3-year-old girl from Vietnam with severe congenital cutis laxa; no cardiovascular, pulmonary, neurologic, or visceral involvement; and no family history of cutis laxa. Mutational analysis of the elastin gene identified heterozygosity for a previously unreported de novo c.2184delT mutation in exon 30 not present in either parent.


Assuntos
Povo Asiático/genética , Cútis Laxa/genética , Cútis Laxa/patologia , Elastina/genética , Mutação Puntual , Pré-Escolar , Saúde da Família , Feminino , Heterozigoto , Humanos , Pais , Vietnã
7.
Hum Mutat ; 34(1): 111-21, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22829427

RESUMO

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFß) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFß activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.


Assuntos
Cútis Laxa/genética , Proteínas da Matriz Extracelular/genética , Proteínas de Ligação a TGF-beta Latente/genética , Mutação , Adolescente , Sequência de Bases , Western Blotting , Criança , Pré-Escolar , Consanguinidade , Cútis Laxa/complicações , Proteínas da Matriz Extracelular/metabolismo , Saúde da Família , Feminino , Expressão Gênica , Humanos , Lactente , Proteínas de Ligação a TGF-beta Latente/metabolismo , Masculino , Microscopia Eletrônica , Linhagem , Enfisema Pulmonar/complicações , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Pele/metabolismo , Pele/patologia , Pele/ultraestrutura , Adulto Jovem
8.
J Biol Chem ; 287(26): 22055-67, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22573328

RESUMO

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.


Assuntos
Processamento Alternativo , Cútis Laxa/genética , Elastina/biossíntese , Elastina/genética , Mutação , Animais , Aorta/metabolismo , Cromossomos Artificiais Bacterianos , Reagentes de Ligações Cruzadas/química , Elasticidade , Elastina/metabolismo , Éxons , Fibroblastos/citologia , Mutação da Fase de Leitura , Genes Dominantes , Humanos , Camundongos , Camundongos Transgênicos , Estrutura Terciária de Proteína , RNA/química , Transgenes
9.
Exp Dermatol ; 22(2): 88-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23088642

RESUMO

Elastic fibres are critical connective tissue components providing elasticity and resilience to skin and other tissues. These fibres are composed of elastin and a number of elastin-associated microfibrillar proteins that assemble in a complex fibre network in a multi-step process. Multiple cellular processes, including mitochondrial function, specific molecules in the secretory pathways and temporally and spatially ordered production of elastic fibre components, are required for the biogenesis of functional elastic fibres. Abnormalities in these processes can lead to loss of functional elastic fibres manifesting phenotypically as a skin disease. The paradigm of elastic fibre diseases affecting the skin is cutis laxa, a clinically and genetically heterogeneous group of disorders characterized by loose and sagging skin, frequently associated with extracutaneous manifestations in the lungs and the arterial blood vessels. The complexity of cutis laxa is emphasized by the fact that as many as 10 distinct genes can harbour mutations in this and related disorders. Understanding of the pathomechanistic pathways involved in perturbed elastic fibre assembly in cutis laxa provides information potentially helpful for the development of molecular strategies towards treatment of these, currently intractable, diseases.


Assuntos
Cútis Laxa/patologia , Tecido Elástico/patologia , Mutação , Pele/patologia , Artérias/patologia , Cromossomos Humanos X/genética , Cútis Laxa/diagnóstico , Elastina/genética , Genes Recessivos , Humanos , Pneumopatias/complicações , Pneumopatias/patologia , Microfibrilas/patologia , Mitocôndrias/metabolismo , Fenótipo
10.
Am J Hum Genet ; 85(5): 593-605, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19836010

RESUMO

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.


Assuntos
Derme/anormalidades , Intestinos/anormalidades , Proteínas de Ligação a TGF-beta Latente/genética , Pulmão/anormalidades , Mutação , Sistema Urinário/anormalidades , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Meios de Cultivo Condicionados/química , DNA/genética , DNA/isolamento & purificação , Derme/metabolismo , Derme/ultraestrutura , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Homozigoto , Humanos , Imuno-Histoquímica , Lactente , Mucosa Intestinal/metabolismo , Proteínas de Ligação a TGF-beta Latente/química , Pulmão/metabolismo , Masculino , Sistema Musculoesquelético , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Pele/citologia , Síndrome , Sistema Urinário/metabolismo
11.
J Am Acad Dermatol ; 66(5): 842.e1-17, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22387031

RESUMO

Cutis laxa is a rare disorder of elastic tissue resulting in loose, redundant, hypoelastic skin. Both acquired and inherited forms exist, some of which have significant systemic manifestations. Here, we review the various forms of cutis laxa, with focus on the inherited forms. Recent molecular studies have provided many new insights into the causes of cutis laxa and revealed greater genetic heterogeneity than previously appreciated.


Assuntos
Cútis Laxa/genética , Cútis Laxa/patologia , Proteínas da Matriz Extracelular/genética , Predisposição Genética para Doença/epidemiologia , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/epidemiologia , Anormalidades Múltiplas/genética , Biópsia por Agulha , Cútis Laxa/epidemiologia , Tecido Elástico/patologia , Feminino , Humanos , Imuno-Histoquímica , Incidência , Masculino , Prognóstico , Medição de Risco , Índice de Gravidade de Doença
12.
J Clin Invest ; 132(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34990407

RESUMO

Obstructive arterial diseases, including supravalvular aortic stenosis (SVAS), atherosclerosis, and restenosis, share 2 important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic, or deletion mutations in the elastin gene (ELN), and SVAS patients and elastin-mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking, as the underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models, and aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the NOTCH pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain, and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln-/- mutants. Eln-/- mice expressed higher levels of NOTCH ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln-/- mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.


Assuntos
Estenose Aórtica Supravalvular , Elastina , Proteína Jagged-1/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Aorta/metabolismo , Estenose Aórtica Supravalvular/genética , Estenose Aórtica Supravalvular/metabolismo , Estenose Aórtica Supravalvular/patologia , Constrição Patológica , Elastina/genética , Elastina/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , Receptor Notch3/genética
13.
Hum Mutat ; 32(4): 445-55, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21309044

RESUMO

Autosomal dominant cutis laxa (ADCL) is characterized by a typical facial appearance and generalized loose skin folds, occasionally associated with aortic root dilatation and emphysema. We sequenced exons 28-34 of the ELN gene in five probands with ADCL features and found five de novo heterozygous mutations: c.2296_2299dupGCAG (CL-1), c.2333delC (CL-2), c.2137delG (CL-3), c.2262delA (monozygotic twin CL-4 and CL-5), and c.2124del25 (CL-6). Four probands (CL-1,-2,-3,-6) presented with progressive aortic root dilatation. CL-2 and CL-3 also had bicuspid aortic valves. CL-2 presented with severe emphysema. Electron microscopy revealed elastic fiber fragmentation and diminished dermal elastin deposition. RT-PCR studies showed stable mutant mRNA in all patients. Exon 32 skipping explains a milder phenotype in patients with exon 32 mutations. Mutant protein expression in fibroblast cultures impaired deposition of tropoelastin onto microfibril-containing fibers, and enhanced tropoelastin coacervation and globule formation leading to lower amounts of mature, insoluble elastin. Mutation-specific effects also included endoplasmic reticulum stress and increased apoptosis. Increased pSMAD2 staining in ADCL fibroblasts indicated enhanced transforming growth factor beta (TGF-ß) signaling. We conclude that ADCL is a systemic disease with cardiovascular and pulmonary complications, associated with increased TGF-ß signaling and mutation-specific differences in endoplasmic reticulum stress and apoptosis.


Assuntos
Cútis Laxa/genética , Elastina/genética , Adolescente , Criança , Pré-Escolar , Transtornos Cromossômicos , Cútis Laxa/patologia , Tecido Elástico/metabolismo , Tecido Elástico/patologia , Feminino , Humanos , Masculino , Mutação , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Tropoelastina/genética , Tropoelastina/metabolismo
14.
Hum Mol Genet ; 18(12): 2149-65, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19321599

RESUMO

Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse-chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.


Assuntos
Cútis Laxa/metabolismo , Cútis Laxa/fisiopatologia , Vesículas Citoplasmáticas/metabolismo , Mutação , ATPases Translocadoras de Prótons/metabolismo , Tropoelastina/metabolismo , Sequência de Aminoácidos , Apoptose , Sobrevivência Celular , Células Cultivadas , Pré-Escolar , Estudos de Coortes , Cútis Laxa/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Transporte Proteico , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética
15.
J Clin Invest ; 118(5): 1606-15, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18452001

RESUMO

Williams-Beuren syndrome (WBS) is a microdeletion disorder caused by heterozygous loss of approximately 1.5-Mb pairs of DNA from chromosome 7. Patients with WBS have a characteristic constellation of medical and cognitive findings, with a hallmark feature of generalized arteriopathy presenting as stenoses of elastic arteries and hypertension. Human and mouse studies establish that defects in the elastin gene, leading to elastin haploinsufficiency, underlie the arteriopathy. In this review we describe potential links between elastin expression and arteriopathy, possible explanations for disease variability, and current treatment options and their limitations, and we propose several new directions for the development of nonsurgical preventative therapies based on insights from elastin biology.


Assuntos
Doenças Cardiovasculares/etiologia , Síndrome de Williams/complicações , Animais , Aorta/citologia , Aorta/patologia , Vasos Sanguíneos/anormalidades , Doenças Cardiovasculares/genética , Elastina/genética , Humanos , Síndrome de Williams/genética
16.
Genes (Basel) ; 12(6)2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071145

RESUMO

Latent transforming growth factor ß (TGFß)-binding protein (LTBP) 4, a member of the LTBP family, shows structural homology with fibrillins. Both these protein types are characterized by calcium-binding epidermal growth factor-like repeats interspersed with 8-cysteine domains. Based on its domain composition and distribution, LTBP4 is thought to adopt an extended structure, facilitating the linear deposition of tropoelastin onto microfibrils. In humans, mutations in LTBP4 result in autosomal recessive cutis laxa type 1C, characterized by redundant skin, pulmonary emphysema, and valvular heart disease. LTBP4 is an essential regulator of TGFß signaling and is related to development, immunity, injury repair, and diseases, playing a central role in regulating inflammation, fibrosis, and cancer progression. In this review, we focus on medical disorders or diseases that may be manipulated by LTBP4 in order to enhance the understanding of this protein.


Assuntos
Cútis Laxa/genética , Proteínas de Ligação a TGF-beta Latente/genética , Animais , Cútis Laxa/metabolismo , Cútis Laxa/patologia , Predisposição Genética para Doença , Humanos , Proteínas de Ligação a TGF-beta Latente/metabolismo , Distrofia Muscular de Duchenne/genética
17.
Cell Death Dis ; 12(10): 943, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645813

RESUMO

Transforming growth factor beta (TGFß) signalling regulates extracellular matrix accumulation known to be essential for the pathogenesis of renal fibrosis; latent transforming growth factor beta binding protein 4 (LTBP4) is an important regulator of TGFß activity. To date, the regulation of LTBP4 in renal fibrosis remains unknown. Herein, we report that LTBP4 is upregulated in patients with chronic kidney disease and fibrotic mice kidneys created by unilateral ureteral obstruction (UUO). Mice lacking the short LTBP4 isoform (Ltbp4S-/-) exhibited aggravated tubular interstitial fibrosis (TIF) after UUO, indicating that LTBP4 potentially protects against TIF. Transcriptomic analysis of human proximal tubule cells overexpressing LTBP4 revealed that LTBP4 influences angiogenic pathways; moreover, these cells preserved better mitochondrial respiratory functions and expressed higher vascular endothelial growth factor A (VEGFA) compared to wild-type cells under hypoxia. Results of the tube formation assay revealed that additional LTBP4 in human umbilical vein endothelial cell supernatant stimulates angiogenesis with upregulated vascular endothelial growth factor receptors (VEGFRs). In vivo, aberrant angiogenesis, abnormal mitochondrial morphology and enhanced oxidative stress were observed in Ltbp4S-/- mice after UUO. These results reveal novel molecular functions of LTBP4 stimulating angiogenesis and potentially impacting mitochondrial structure and function. Collectively, our findings indicate that LTBP4 protects against disease progression and may be of therapeutic use in renal fibrosis.


Assuntos
Rim/patologia , Proteínas de Ligação a TGF-beta Latente/metabolismo , Mitocôndrias/ultraestrutura , Neovascularização Fisiológica , Animais , Diferenciação Celular , Meios de Cultivo Condicionados/farmacologia , Fibrose , Perfilação da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunidade , Túbulos Renais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Biológicos , Fenótipo , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
18.
Hum Mutat ; 31(5): 594-601, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20437614

RESUMO

Mutations in the Notch pathway ligand Jagged1 (JAG1) cause Alagille syndrome (AGS), as well as cardiac defects in seemingly nonsyndromic individuals. To estimate the frequency of JAG1 mutations in cases with right-sided cardiac defects not otherwise diagnosed with AGS, we screened 94 cases with tetralogy of Fallot (TOF) and 50 with pulmonic stenosis/peripheral pulmonary stenosis (PS/PPS) or pulmonary valve atresia with intact ventricular septum (PA) for mutations. Sequence changes were identified in three TOF and three PS/PPS/PA patients, that were not present in 100 controls. We identified one frameshift and two missense mutations in the TOF cases, and one frameshift and two missense mutations in cases with PS/PPS/PA. The four missense mutations were assayed for their effect on protein localization, posttranslational modification, and ability to activate Notch signaling. The missense mutants displayed heterogeneous behavior in these assays, some with complete haploinsufficiency, suggesting that there are additional modifiers leading to organ specific features. We identified functionally significant mutations in 2% (2/94) of TOF patients and 4% (2/50) of PS/PPS/PA patients. Patients with right-sided cardiac defects should be carefully screened for features of AGS or a family history of cardiac defects that might suggest the presence of a JAG1 mutation.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estenose da Valva Pulmonar/genética , Tetralogia de Fallot/genética , Síndrome de Alagille/genética , Animais , Análise Mutacional de DNA , Feminino , Glicosilação , Humanos , Proteína Jagged-1 , Masculino , Camundongos , Mutação , Mutação de Sentido Incorreto , Células NIH 3T3 , Linhagem , Processamento de Proteína Pós-Traducional , Proteínas Serrate-Jagged , Transdução de Sinais/genética
19.
JMIR Res Protoc ; 9(7): e17329, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32706746

RESUMO

BACKGROUND: The prevalence of obesity and diabetes in Samoa, like many other Pacific Island nations, has reached epidemic proportions. Although the etiology of these conditions can be largely attributed to the rapidly changing economic and nutritional environment, a recently identified genetic variant, rs373863828 (CREB 3 regulatory factor, CREBRF: c.1370G>A p.[R457Q]) is associated with increased odds of obesity, but paradoxically, decreased odds of diabetes. OBJECTIVE: The overarching goal of the Soifua Manuia (Good Health) study was to precisely characterize the association of the CREBRF variant with metabolic (body composition and glucose homeostasis) and behavioral traits (dietary intake, physical activity, sleep, and weight control behaviors) that influence energy homeostasis in 500 adults. METHODS: A cohort of adult Samoans who participated in a genome-wide association study of adiposity in Samoa in 2010 was followed up, based on the presence or absence of the CREBRF variant, between August 2017 and March 2019. Over a period of 7-10 days, each participant completed the main study protocol, which consisted of anthropometric measurements (weight, height, circumferences, and skinfolds), body composition assessment (bioelectrical impedance and dual-energy x-ray absorptiometry), point-of-care glycated hemoglobin measurement, a fasting blood draw and oral glucose tolerance test, urine collection, blood pressure measurement, hand grip strength measurement, objective physical activity and sleep apnea monitoring, and questionnaire measures (eg, health interview, cigarette and alcohol use, food frequency questionnaire, socioeconomic position, stress, social support, food and water insecurity, sleep, body image, and dietary preferences). In January 2019, a subsample of the study participants (n=118) completed a buttock fat biopsy procedure to collect subcutaneous adipose tissue samples. RESULTS: Enrollment of 519 participants was completed in March 2019. Data analyses are ongoing, with results expected in 2020 and 2021. CONCLUSIONS: While the genetic variant rs373863828, in CREBRF, has the largest known effect size of any identified common obesity gene, very little is currently understood about the mechanisms by which it confers increased odds of obesity but paradoxically lowered odds of type 2 diabetes. The results of this study will provide insights into how the gene functions on a whole-body level, which could provide novel targets to prevent or treat obesity, diabetes, and associated metabolic disorders. This study represents the human arm of a comprehensive and integrated approach involving humans as well as preclinical models that will provide novel insights into metabolic disease. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/17329.

20.
J Mol Biol ; 369(3): 841-51, 2007 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-17459412

RESUMO

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.


Assuntos
Reagentes de Ligações Cruzadas/química , Tropoelastina/química , Animais , Western Blotting , Bovinos , Sobrevivência Celular , Desmosina/química , Ensaio de Imunoadsorção Enzimática , Éxons , Fibrilinas , Proteínas dos Microfilamentos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química
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