RESUMO
Valosin-containing protein (VCP) is an AAA+ ATPase that plays critical roles in multiple ubiquitin-dependent cellular processes. Dominant pathogenic variants in VCP are associated with adult-onset multisystem proteinopathy (MSP), which manifests as myopathy, bone disease, dementia, and/or motor neuron disease. Through GeneMatcher, we identified 13 unrelated individuals who harbor heterozygous VCP variants (12 de novo and 1 inherited) associated with a childhood-onset disorder characterized by developmental delay, intellectual disability, hypotonia, and macrocephaly. Trio exome sequencing or a multigene panel identified nine missense variants, two in-frame deletions, one frameshift, and one splicing variant. We performed in vitro functional studies and in silico modeling to investigate the impact of these variants on protein function. In contrast to MSP variants, most missense variants had decreased ATPase activity, and one caused hyperactivation. Other variants were predicted to cause haploinsufficiency, suggesting a loss-of-function mechanism. This cohort expands the spectrum of VCP-related disease to include neurodevelopmental disease presenting in childhood.
Assuntos
Doenças Musculares , Transtornos do Neurodesenvolvimento , Adulto , Humanos , Proteína com Valosina/genética , Hipotonia Muscular , Mutação de Sentido Incorreto/genéticaRESUMO
The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.
Assuntos
Encefalopatias/genética , Epilepsia/genética , Rim Fundido/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Osteocondrodisplasias/genética , Adolescente , Sequência de Aminoácidos , Animais , Encefalopatias/etiologia , Criança , Pré-Escolar , Epilepsia/complicações , Evolução Molecular , Feminino , Frequência do Gene , Humanos , Lactente , Masculino , Camundongos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Fenótipo , Estabilidade Proteica , Síndrome , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Adulto Jovem , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Hydrops fetalis, a pathological fluid accumulation in two or more body compartments, is aetiologically heterogeneous. We investigated a consanguineous family with recurrent pregnancy loss due to severe early-onset non-immune hydrops fetalis. METHODS AND RESULTS: Whole exome sequencing in four fetuses with hydrops fetalis revealed that they were homozygous for the angiopoietin-2 (ANGPT2) variant Chr8 (GRCh37/Hg19): 6385085T>C, NM_001147.2:c.557A>G. The substitution introduces a cryptic, exonic splice site predicted to result in loss of 10 nucleotides with subsequent shift in reading frame, leading to a premature stop codon. RNA analysis in the heterozygous parents demonstrated loss of detectable mutant allele, indicative of loss-of-function via nonsense-mediated mRNA decay. Serum ANGPT2 levels were reduced in the parents. In a pregnancy with a healthy, heterozygous child, transiently increased fetal nuchal translucency was noted. CONCLUSION: Pathogenic heterozygous ANGPT2 missense variants were recently shown to cause autosomal dominant primary lymphoedema. ANGPT2 is a ligand of the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) pathway. It is critical to the formation and remodelling of blood and lymphatic vessels and is involved in vessel maintenance. ANGPT2 knockout mice die from generalised lymphatic dysfunction. We show here that a homozygous pathogenic variant causes loss-of-function and results in severe early-onset hydrops fetalis. This is the first report of an autosomal recessive ANGPT2-related disorder in humans.
Assuntos
Angiopoietina-2 , Hidropisia Fetal , Animais , Feminino , Humanos , Camundongos , Gravidez , Angiopoietina-2/genética , Códon sem Sentido/genética , Heterozigoto , Hidropisia Fetal/genética , Hidropisia Fetal/metabolismo , Mutação de Sentido Incorreto , Recém-NascidoRESUMO
De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Exoma/genética , Mutação , Transtornos do Neurodesenvolvimento/etiologia , Proteínas Serina-Treonina Quinases/genética , Animais , Criança , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Heterozigoto , Humanos , Masculino , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Sequenciamento do ExomaRESUMO
PURPOSE: GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT. METHODS: All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records. RESULTS: Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively. CONCLUSION: Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described.
Assuntos
Deficiência de GATA2 , Transplante de Células-Tronco Hematopoéticas , Medula Óssea , Deficiência de GATA2/diagnóstico , Deficiência de GATA2/genética , Deficiência de GATA2/terapia , Fator de Transcrição GATA2/genética , Humanos , Noruega/epidemiologiaRESUMO
By clinical whole exome sequencing, we identified 12 individuals with ages 3 to 37 years, including three individuals from the same family, with a consistent phenotype of intellectual disability (ID), macrocephaly, and overgrowth of adenoid tissue. All 12 individuals harbored a rare heterozygous variant in ZBTB7A which encodes the transcription factor Zinc finger and BTB-domain containing protein 7A, known to play a role in lympho- and hematopoiesis. ID was generally mild. Fetal hemoglobin (HbF) fraction was elevated 2.2%-11.2% (reference value <2% in individuals > 6 months) in four of the five individuals for whom results were available. Ten of twelve individuals had undergone surgery at least once for lymphoid hypertrophy limited to the pharynx. In the most severely affected individual (individual 1), airway obstruction resulted in 17 surgical procedures before the age of 13 years. Sleep apnea was present in 8 of 10 individuals. In the nine unrelated individuals, ZBTB7A variants were novel and de novo. The six frameshift/nonsense and four missense variants were spread throughout the gene. This is the first report of a cohort of individuals with this novel syndromic neurodevelopmental disorder.
Assuntos
Deficiência Intelectual , Megalencefalia , Transtornos do Neurodesenvolvimento , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Hemoglobina Fetal , Humanos , Deficiência Intelectual/genética , Tecido Linfoide , Megalencefalia/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genéticaRESUMO
AIMS: A prolonged corrected QT interval (QTc) ≥500 ms is associated with high all-cause mortality in hospitalized patients. We aimed to explore any difference in short- and long-term mortality in patients with QTc ≥500 ms compared with patients with QTc <500 ms after adjustment for comorbidity and main diagnosis. METHODS AND RESULTS: Patients with QTc ≥500 ms who were hospitalized at Telemark Hospital Trust, Norway between January 2007 and April 2014 were identified. Thirty-day and 3-year all-cause mortality in 980 patients with QTc ≥500 ms were compared with 980 patients with QTc <500 ms, matched for age and sex and adjusting for Charlson comorbidity index (CCI), previous admissions, and main diagnoses. QTc ≥500 ms was associated with increased 30-day all-cause mortality [hazard ratio (HR) 1.90, 95% confidence interval (CI) 1.38-2.62; P < 0.001]. There was no significant difference in mortality between patients with QTc ≥500 ms and patients with QTc <500 ms who died between 30 days and 3 years; 32% vs. 29%, P = 0.20. Graded CCI was associated with increased 3-year all-cause mortality (CCI 1-2: HR 1.62, 95% CI 1.34-1.96; P < 0.001; CCI 3-4: HR 2.50, 95% CI 1.95-3.21; P < 0.001; CCI ≥5: HR 3.76, 95% CI 2.85-4.96; P < 0.001) but was not associated with 30-day all-cause mortality. CONCLUSION: QTc ≥500 ms is a powerful predictor of short-term mortality overruling comorbidities. QTc ≥500 ms also predicted long-term mortality, but this effect was mainly caused by the increased short-term mortality. For long-term mortality, comorbidity was more important.
Assuntos
Cardiopatias , Hospitalização/estatística & dados numéricos , Pacientes Internados/estatística & dados numéricos , Síndrome do QT Longo/diagnóstico , Neoplasias , Acidente Vascular Cerebral , Causas de Morte , Comorbidade , Eletrocardiografia/métodos , Feminino , Cardiopatias/diagnóstico , Cardiopatias/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Noruega/epidemiologia , Prognóstico , Medição de Risco , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , TempoAssuntos
Alelos , Endrin/análogos & derivados , Éxons/genética , Frequência do Gene , Humanos , MutaçãoRESUMO
BACKGROUND: Rare sequence variants in at least five genes are known to cause monogenic obesity. In this study we aimed to investigate the prevalence of, and characterize, rare coding and splice site variants in LEP, LEPR, MC4R, PCSK1 and POMC in patients with morbid obesity and normal weight controls. METHOD: Targeted next-generation sequencing of all exons in LEP, LEPR, MC4R, PCSK1 and POMC was performed in 485 patients with morbid obesity and 327 normal weight population-based controls from Norway. RESULTS: In total 151 variants were detected. Twenty-eight (18.5%) of these were rare, coding or splice variants and five (3.3%) were novel. All individuals, except one control, were heterozygous for the 28 variants, and the distribution of the rare variants showed a significantly higher carrier frequency among cases than controls (9.9% vs. 4.9%, p=0.011). Four variants in MC4R were classified as pathogenic or likely pathogenic. CONCLUSION: Four cases (0.8%) of monogenic obesity were detected, all due to MC4R variants previously linked to monogenic obesity. Significant differences in carrier frequencies among patients with morbid obesity and normal weight controls suggest an association between heterozygous rare coding variants in these five genes and morbid obesity. However, additional studies in larger cohorts and functional testing of the novel variants identified are required to confirm the findings.
Assuntos
Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Obesidade Mórbida/genética , Análise de Sequência de DNA/métodos , Adolescente , Adulto , Distribuição por Idade , Estudos de Casos e Controles , Criança , Feminino , Predisposição Genética para Doença , Humanos , Leptina/genética , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Noruega , Pró-Opiomelanocortina/genética , Pró-Proteína Convertase 1/genética , Receptor Tipo 4 de Melanocortina/genética , Receptores para Leptina/genética , Adulto JovemRESUMO
BACKGROUND: New DNA-sequencing technology is revolutionising medical diagnostics. Through the use of exome sequencing, it is now possible to sequence all human genes in parallel. This technology has been widely used in research over the last few years and is now also being applied to diagnostics. The aim of this study was to systematically examine initial experiences with diagnostic exome sequencing in Norway. MATERIAL AND METHOD: This is a retrospective observational study of the results of all exome sequencing performed by the Section of Medical Genetics at Telemark Hospital between December 2012 and October 2014, and includes 125 persons in 46 families. The majority of these families were being investigated for a syndrome (n = 35, 76%) or neurological disease (n = 9, 20%). RESULTS: Exome sequencing detected pathogenic sequence variants in 15 of 46 probands, and variants of unknown significance in 12 probands. Of the 100 patients who stated their wishes regarding feedback of any incidental findings, six indicated that they did not wish to receive such information. There were no incidental findings in this study, but neither were such sequence variants actively looked for. INTERPRETATION: Exome sequencing can enable more patients with syndromes or neurological diseases to receive a causal diagnosis, and to receive this diagnosis at an earlier stage. However, the patients in this study were quite highly selected, and the results must therefore be interpreted with caution.
Assuntos
Exoma , Doenças Genéticas Inatas/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Doenças do Sistema Nervoso , Análise de Sequência de DNA , Humanos , Consentimento Livre e Esclarecido , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Noruega , Estudos Retrospectivos , SíndromeRESUMO
BACKGROUND: Next-generation sequencing (NGS) is a genetic technique used to determine the order of nucleotides in DNA. The technique has proved to be more efficient than the traditional method, Sanger sequencing, for sequencing multiple genes. NGS is now being used to diagnose disorders in which multiple genes are involved. This study has examined whether next-generation sequencing produces a greater number of positive diagnoses than its traditional counterpart in patients with suspected hereditary peripheral neuropathy. MATERIAL AND METHOD: This study is a retrospective review of samples from 103 patients investigated for hereditary peripheral neuropathy, received by Telemark Hospital in the period 2012-14. After exclusion of duplication/deletion of PMP22, 96 samples were analysed by NGS with physical enrichment of 52 hereditary peripheral neuropathy genes. RESULTS: A genetic cause was identified in 35 patients (34%) with peripheral neuropathy, of which 28 (27%) were point mutations identified by NGS. INTERPRETATION: Of the pathogenic point mutations identified in this study, 12 were in genes that would previously have been analysed by Sanger sequencing in our department, whereas 16 were in genes that would not previously have been tested.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Doenças do Sistema Nervoso Periférico , Análise de Sequência de DNA , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Neuropatias Hereditárias Sensoriais e Autônomas/diagnóstico , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Neuropatia Hereditária Motora e Sensorial/diagnóstico , Neuropatia Hereditária Motora e Sensorial/genética , Humanos , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/genética , Mutação Puntual , Estudos RetrospectivosRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor homology domain repeat A of the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts recycling of the internalized LDLR. As a consequence, the LDLR is rerouted to the lysosomes for degradation. Although PCSK9 may bind to an LDLR lacking the ligand-binding domain, at least three ligand-binding repeats of the ligand-binding domain are required for PCSK9 to reroute the LDLR to the lysosomes. In this study, we have studied the binding of PCSK9 to an LDLR with or without the ligand-binding domain at increasingly acidic conditions in order to mimic the milieu of the LDLR:PCSK9 complex as it translocates from the cell membrane to the sorting endosomes. These studies have shown that PCSK9 is rapidly released from an LDLR lacking the ligand-binding domain at pH in the range of 6.9-6.1. A similar pattern of release at acidic pH was also observed for the binding to the normal LDLR of mutant PCSK9 lacking the C-terminal domain. Together these data indicate that an interaction between the negatively charged ligand-binding domain of the LDLR and the positively charged C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDLR during the early phase of endosomal acidification as the LDLR translocates from the cell membrane to the sorting endosome.
Assuntos
Ácidos/metabolismo , Endossomos/metabolismo , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Animais , Western Blotting , Células CHO , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Humanos , Concentração de Íons de Hidrogênio , Rim/citologia , Rim/metabolismo , Lisossomos/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de LDL/genética , Serina Endopeptidases/genética , Transdução de SinaisRESUMO
A 55-year-old woman with a history of bowel dysmotility presented with abdominal distension and peritonitis. Family history included premature deaths with intestinal symptomatology, suggesting autosomal dominant inheritance. Computed tomography showed a distended small bowel. Symptoms were alleviated by enterocutaneous stomas. Initial ileal biopsy suggested neuropathy; however, exome sequencing revealed an Arg148Ser mutation in the enteric smooth muscle actin gamma 2 (ACTG2) gene. Histological reassessment showed abnormal muscularis propria and smooth muscle actin, with the same findings in sibling, confirming familial visceral myopathy. Thus, noninvasive genomic analysis can provide early and specific diagnosis of familial visceral myopathy, which may help to avoid inappropriate surgery.
Assuntos
Actinas/genética , Duodeno/anormalidades , Pseudo-Obstrução Intestinal/genética , Pseudo-Obstrução Intestinal/patologia , Bexiga Urinária/anormalidades , Análise Mutacional de DNA , Duodeno/patologia , Exoma , Feminino , Humanos , Pessoa de Meia-Idade , Linhagem , Bexiga Urinária/patologiaRESUMO
BACKGROUND: Serine residues in the protein backbone of heavily glycosylated proteoglycans are bound to glycosaminoglycans through a tetrasaccharide linker. UXS1 encodes UDP-glucuronate decarboxylase 1, which catalyzes synthesis of UDP-xylose, the donor of the first building block in the linker. Defects in other enzymes involved in formation of the tetrasaccharide linker cause so-called linkeropathies, characterized by short stature, radio-ulnar synostosis, decreased bone density, congenital contractures, dislocations, and more. METHODS: Whole exome sequencing was performed in a father and son who presented with a mild skeletal dysplasia, as well as the father's unaffected parents. Wild-type and mutant UXS1 were recombinantly expressed in Escherichia coli and purified. Enzyme activity was evaluated by LC-MS/MS. In vivo effects were studied using HeparinRed assay and metabolomics. RESULTS: The son had short long bones, normal epiphysis, and subtle metaphyseal changes especially in his legs. The likely pathogenic heterozygous variant NM_001253875.1(UXS1):c.557T>A p.(Ile186Asn) detected in the son was de novo in the father. Purified Ile186Asn-UXS1, in contrast to the wild-type, was not able to convert UDP-glucuronic acid to UDP-xylose. Plasma glycosaminoglycan levels were decreased in both son and father. CONCLUSION: This is the first report linking UXS1 to short-limbed short stature in humans.
Assuntos
Nanismo , Humanos , Masculino , Nanismo/genética , Nanismo/metabolismo , Nanismo/patologia , Carboxiliases/genética , Carboxiliases/metabolismo , Alelos , Fenótipo , Mutação , Adulto , LinhagemRESUMO
Genetic repeat expansions cause neuronal degeneration in amyotrophic lateral sclerosis as well as other neurodegenerative disorders such as spinocerebellar ataxia, Huntington's disease and Kennedy's disease. Repeat expansions in the same gene can cause multiple clinical phenotypes. We aimed to characterize repeat expansions in a Norwegian amyotrophic lateral sclerosis cohort. Norwegian amyotrophic lateral sclerosis patients (n = 414) and neurologically healthy controls adjusted for age and gender (n = 713) were investigated for repeat expansions in AR, ATXN1, ATXN2 and HTT using short read exome sequencing and the ExpansionHunter software. Five amyotrophic lateral sclerosis patients (1.2%) and two controls (0.3%) carried ≥36 repeats in HTT (P = 0.032), and seven amyotrophic lateral sclerosis patients (1.7%) and three controls (0.4%) carried ≥29 repeats in ATXN2 (P = 0.038). One male diagnosed with amyotrophic lateral sclerosis carried a pathogenic repeat expansion in AR, and his diagnosis was revised to Kennedy's disease. In ATXN1, 50 amyotrophic lateral sclerosis patients (12.1%) and 96 controls (13.5%) carried ≥33 repeats (P = 0.753). None of the patients with repeat expansions in ATXN2 or HTT had signs of Huntington's disease or spinocerebellar ataxia type 2, based on a re-evaluation of medical records. The diagnosis of amyotrophic lateral sclerosis was confirmed in all patients, with the exception of one patient who had primary lateral sclerosis. Our findings indicate that repeat expansions in HTT and ATXN2 are associated with increased likelihood of developing amyotrophic lateral sclerosis. Further studies are required to investigate the potential relationship between HTT repeat expansions and amyotrophic lateral sclerosis.
RESUMO
In this study we have characterized a naturally occurring truncated form of the low density lipoprotein receptor (LDLR). Western blot analysis of transfected cells indicated that the truncated form (∆N-LDLR) is a degradation product of the full-length LDLR generated by cleavage in the linker region between ligand-binding repeats 4 and 5 of the ligand-binding domain. The cleavage of the linker was not caused by components of the culture media, as heat inactivation of the media did not prevent cleavage. Rather, it is assumed that cleavage was caused by an enzyme secreted from the cells. Biotinylation experiments showed that ∆N-LDLR is located on the cell surface and is detectable approximately 5 h after synthesis of the full-length LDLR. Flow cytometric analysis showed that ∆N-LDLR was not able to bind and internalize low density lipoprotein (LDL). ∆N-LDLR appeared to be equally stable as the full-length LDLR. Thus, generation of ∆N-LDLR does not appear to be the first signal for degradation of the LDLR. The existence of two functionally different populations of LDLRs on the cell surface, of which ∆N-LDLR constitutes 28%, must be taken into account when interpreting results of experiments to study LDLRs on the cell surface. Furthermore, if the cleavage of the linker between ligand-binding repeats 4 and 5 could be prevented by an enzyme inhibitor, this could represent a novel therapeutic strategy to increase the number of functioning LDLRs and thereby decrease the levels of plasma LDL cholesterol.
Assuntos
Proteólise , Receptores de LDL/metabolismo , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Meios de Cultura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Inibidores de Proteases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Receptores de LDL/antagonistas & inibidores , Receptores de LDL/química , Deleção de SequênciaRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.
Assuntos
Endossomos/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Endossomos/química , Células Hep G2 , Histidina/química , Histidina/metabolismo , Humanos , Dados de Sequência Molecular , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ligação Proteica , Receptores de LDL/química , Células Tumorais CultivadasRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31-53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32-40 and 48-50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs. Deletion of the acidic residues of the longest negatively charged segment increased PCSK9's ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9's activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9's activity by 36%. These findings indicate that the inhibitory effect of residues 31-53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9's activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9.
Assuntos
Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Sequência Conservada , Humanos , Dados de Sequência Molecular , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Estrutura Terciária de Proteína , Deleção de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
The low density lipoprotein receptor (LDLR) binds and internalizes low density lipoprotein (LDL). At the mildly acidic pH of the sorting endosomes, LDL is released from the receptor and the receptor recycles back to the cell membrane. Mutations in the LDLR gene may disrupt the normal function of the LDLR in different ways. Class 5 mutations result in receptors that are able to bind and internalize LDL, but they fail to release LDL in the sorting endosomes and fail to recycle. Instead they are rerouted to the lysosomes for degradation. However, the underlying mechanism remains to be determined. To study the role of the cytoplasmic domain of the LDLR for rerouting Class 5 mutants to the lysosomes, we have performed studies to determine whether Class 5 mutants caused by mutations E387K or V408M are degraded when the cytoplasmic domain has been altered or deleted. As determined by confocal laser-scanning microscopy, these mutant LDLR were inserted into the cell membrane and were able to internalize LDL. As determined by Western blot analysis, Class 5 mutants without a cytoplasmic domain still were degraded after binding LDL. Thus, the cytoplasmic domain does not play a role in rerouting Class 5 mutant LDLR to the lysosomes. Rather, one may speculate that sterical hindrance may prevent Class 5 mutants with bound LDL from entering the narrow recycling tubules of the sorting endosome.
Assuntos
Citoplasma/metabolismo , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Mutação , Estrutura Terciária de Proteína , Receptores de LDL/genéticaRESUMO
Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE(814) and/or ExD(837) had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.