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1.
Hum Mol Genet ; 33(11): 1001-1014, 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38483348

RESUMO

The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.


Assuntos
Repetições Minissatélites , Pâncreas Exócrino , Humanos , Repetições Minissatélites/genética , Animais , Camundongos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/enzimologia , Células HEK293 , Mutagênese Insercional/genética , Alelos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/enzimologia , Frequência do Gene , Masculino , Feminino , Lipase/genética
2.
Diabetologia ; 66(12): 2226-2237, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37798422

RESUMO

AIMS/HYPOTHESIS: Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes. METHODS: We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers' phenotype and treatment response to sulfonylurea. RESULTS: In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic. CONCLUSIONS/INTERPRETATION: Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY.


Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Criança , Projetos Piloto , Diabetes Mellitus Tipo 2/metabolismo , Fenótipo , Autoanticorpos/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Noruega/epidemiologia , Compostos de Sulfonilureia , Mutação
3.
J Biol Chem ; 296: 100661, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862081

RESUMO

Variable number of tandem repeat (VNTR) sequences in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which is specifically expressed in pancreatic acinar cells and encodes the digestive enzyme carboxyl ester lipase. Rare single-base deletions (DELs) within the first (DEL1) or fourth (DEL4) VNTR segment of CEL cause maturity-onset diabetes of the young, type 8 (MODY8), an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Studies on the DEL1 variant have suggested that MODY8 is initiated by CEL protein misfolding and aggregation. However, it is unclear how the position of single-base deletions within the CEL VNTR affects pathogenic properties of the protein. Here, we investigated four naturally occurring CEL variants, arising from single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, and DEL13). When the four variants were expressed in human embryonic kidney 293 cells, only DEL1 and DEL4 led to significantly reduced secretion, increased intracellular aggregation, and increased endoplasmic reticulum stress compared with normal CEL protein. The level of O-glycosylation was affected in all DEL variants. Moreover, all variants had enzymatic activity comparable with that of normal CEL. We conclude that the longest aberrant protein tails, resulting from single-base deletions in the proximal VNTR segments, have highest pathogenic potential, explaining why DEL1 and DEL4 but not DEL9 and DEL13 have been observed in patients with MODY8. These findings further support the view that CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to endoplasmic reticulum stress and activation of the unfolded protein response.


Assuntos
Estresse do Retículo Endoplasmático , Lipase/genética , Lipase/metabolismo , Repetições Minissatélites , Mutação , Proteostase , Glicosilação , Células HEK293 , Humanos
4.
Pancreatology ; 22(8): 1099-1111, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36379850

RESUMO

BACKGROUND & AIMS: The CEL gene encodes the digestive enzyme carboxyl ester lipase. CEL-HYB1, a hybrid allele of CEL and its adjacent pseudogene CELP, is a genetic variant suggested to increase the risk of chronic pancreatitis (CP). Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms. METHODS: We established a knock-in mouse strain where the variable number of tandem repeat (VNTR) region of the endogenous mouse Cel gene was substituted with the mutated VNTR of the human CEL-HYB1 allele. Heterozygous and homozygous Cel-HYB1 mice and littermate wildtype controls were characterized with respect to pancreatic pathology and function. RESULTS: We successfully constructed a mouse model with pancreatic expression of a humanized CEL-HYB1 protein. The Cel-HYB1 mice spontaneously developed features of CP including inflammation, acinar atrophy and fatty replacement, and the phenotype became more pronounced as the animals aged. Moreover, Cel-HYB1 mice were normoglycemic at age 6 months, whereas at 12 months they exhibited impaired glucose tolerance. Immunostaining of pancreatic tissue indicated the formation of CEL protein aggregates, and electron microscopy showed dilated endoplasmic reticulum. Upregulation of the stress marker BiP/GRP78 was seen in pancreatic parenchyma obtained both from Cel-HYB1 animals and from a human CEL-HYB1 carrier. CONCLUSIONS: We have developed a new mouse model for CP that confirms the pathogenicity of the human CEL-HYB1 variant. Our findings place CEL-HYB1 in the group of genes that increase CP risk through protein misfolding-dependent pathways.


Assuntos
Lipase , Pancreatite Crônica , Humanos , Camundongos , Animais , Idoso , Lactente , Lipase/genética , Pancreatite Crônica/genética , Alelos , Repetições Minissatélites , Fatores de Risco
5.
Pancreatology ; 20(3): 377-384, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32007358

RESUMO

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.


Assuntos
Lipase/genética , Pancreatite Crônica/epidemiologia , Pancreatite Crônica/genética , Adulto , Idoso , Alelos , DNA/genética , Feminino , Duplicação Gênica , Frequência do Gene , Testes Genéticos , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/patologia , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/genética , Pancreatite Alcoólica/epidemiologia , Pancreatite Alcoólica/genética , Pancreatite Crônica/patologia , Risco
6.
J Biol Chem ; 293(50): 19476-19491, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30315106

RESUMO

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.


Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Carboxilesterase/química , Carboxilesterase/metabolismo , Pâncreas/enzimologia , Polissacarídeos/metabolismo , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Humanos , Domínios Proteicos
7.
Pancreatology ; 18(1): 12-19, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29233499

RESUMO

The enzyme carboxyl ester lipase (CEL), also known as bile salt-dependent or -stimulated lipase (BSDL, BSSL), hydrolyzes dietary fat, cholesteryl esters and fat-soluble vitamins in the duodenum. CEL is mainly expressed in pancreatic acinar cells and lactating mammary glands. The human CEL gene resides on chromosome 9q34.3 and contains a variable number of tandem repeats (VNTR) region that encodes a mucin-like protein tail. Although the number of normal repeats does not appear to significantly influence the risk for pancreatic disease, single-base pair deletions in the first VNTR repeat cause a syndrome of endocrine and exocrine dysfunction denoted MODY8. Hallmarks are low fecal elastase levels and pancreatic lipomatosis manifesting before the age of twenty, followed by development of diabetes and pancreatic cysts later in life. The mutant protein forms intracellular and extracellular aggregates, suggesting that MODY8 is a protein misfolding disease. Recently, a recombined allele between CEL and its pseudogene CELP was discovered. This allele (CEL-HYB) encodes a chimeric protein with impaired secretion increasing five-fold the risk for chronic pancreatitis. The CEL gene has proven to be exceptionally polymorphic due to copy number variants of the CEL-CELP locus and alterations involving the VNTR. Genome-wide association studies or deep sequencing cannot easily pick up this wealth of genetic variation. CEL is therefore an attractive candidate gene for further exploration of links to pancreatic disease.


Assuntos
Lipase/genética , Lipase/metabolismo , Pancreatopatias/enzimologia , Pancreatopatias/genética , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Mutação , Pancreatopatias/metabolismo
8.
Diabetologia ; 60(4): 625-635, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27913849

RESUMO

AIMS/HYPOTHESIS: MODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes. METHODS: Using next-generation sequencing, we screened the HNF1A, HNF4A, HNF1B, GCK and INS genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic). RESULTS: We identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3-5 (vs 2.4% in controls; p = 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls; p = 1.6 × 10-5). HNF1A showed the strongest enrichment of class 3-5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls; p = 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5. CONCLUSIONS/INTERPRETATION: This is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Adolescente , Anticorpos/metabolismo , Proteínas Reguladoras de Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Quinases do Centro Germinativo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Humanos , Lactente , Recém-Nascido , Lipase/genética , Masculino , Noruega , Fatores de Transcrição Box Pareados/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Receptores de Sulfonilureias/genética , Transativadores/genética , Quinases da Família src/genética
9.
Pancreatology ; 17(1): 83-88, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27773618

RESUMO

BACKGROUND/OBJECTIVES: We have recently described copy number variants (CNVs) of the human carboxyl-ester lipase (CEL) gene, including a recombined deletion allele (CEL-HYB) that is a genetic risk factor for chronic pancreatitis. Associations with pancreatic disease have also been reported for the variable number of tandem repeat (VNTR) region located in CEL exon 11. Here, we examined if CEL CNVs and VNTR length polymorphisms affect the risk for developing pancreatic cancer. METHODS: CEL CNVs and VNTR were genotyped in a German family with non-alcoholic chronic pancreatitis and pancreatic cancer, in 265 German and 197 Norwegian patients diagnosed with pancreatic adenocarcinoma, and in 882 controls. CNV screening was performed using PCR assays followed by agarose gel electrophoresis whereas VNTR lengths were determined by DNA fragment analysis. RESULTS: The investigated family was CEL-HYB-positive. However, an association of CEL-HYB or a duplication CEL allele with pancreatic cancer was not seen in our two patient cohorts. The frequency of the 23-repeat VNTR allele was borderline significant in Norwegian cases compared to controls (1.2% vs. 0.3%; P = 0.05). For all other VNTR lengths, no statistically significant difference in frequency was observed. Moreover, no association with pancreatic cancer was detected when CEL VNTR lengths were pooled into groups of short, normal or long alleles. CONCLUSIONS: We could not demonstrate an association between CEL CNVs and pancreatic cancer. An association is also unlikely for CEL VNTR lengths, although analyses in larger materials are necessary to completely exclude an effect of rare VNTR alleles.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Lipase/genética , Repetições Minissatélites , Neoplasias Pancreáticas/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Fatores de Risco
10.
Am J Hum Genet ; 93(1): 150-7, 2013 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-23810379

RESUMO

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Transtornos do Crescimento/enzimologia , Hipercalcemia/enzimologia , Doenças Metabólicas/enzimologia , Nefrocalcinose/enzimologia , Transdução de Sinais , Adipócitos/metabolismo , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Índice de Massa Corporal , Diferenciação Celular , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Análise Mutacional de DNA , Ativação Enzimática , Exoma , Feminino , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Genética Populacional/métodos , Transtornos do Crescimento/patologia , Humanos , Hipercalcemia/patologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Doenças Metabólicas/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Nefrocalcinose/patologia , Linhagem , Adulto Jovem , Domínios de Homologia de src
11.
J Biol Chem ; 289(42): 29097-111, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25160620

RESUMO

Maturity-onset diabetes of the young, type 8 (MODY8) is characterized by a syndrome of autosomal dominantly inherited diabetes and exocrine pancreatic dysfunction. It is caused by deletion mutations in the last exon of the carboxyl ester lipase (CEL) gene, resulting in a CEL protein with increased tendency to aggregate. In this study we investigated the intracellular distribution of the wild type (WT) and mutant (MUT) CEL proteins in cellular models. We found that both CEL-WT and CEL-MUT were secreted via the endoplasmic reticulum and Golgi compartments. However, their subcellular distributions differed, as only CEL-MUT was observed as an aggregate at the cell surface and inside large cytoplasmic vacuoles. Many of the vacuoles were identified as components of the endosomal system, and after its secretion, the mutant CEL protein was re-internalized, transported to the lysosomes, and degraded. Internalization of CEL-MUT also led to reduced viability of pancreatic acinar and beta cells. These findings may have implications for the understanding of how the acinar-specific CEL-MUT protein causes both exocrine and endocrine pancreatic disease.


Assuntos
Carboxilesterase/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Endocitose , Lipase/metabolismo , Pâncreas Exócrino/metabolismo , Animais , Apoptose , Membrana Celular/enzimologia , Sobrevivência Celular , Meios de Cultivo Condicionados/química , Cicloeximida/química , Células HEK293 , Células HeLa , Humanos , Mutação , Ligação Proteica , Ratos
12.
J Biol Chem ; 288(8): 5353-6, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23306198

RESUMO

Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetes is not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODY-hiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs.


Assuntos
Técnicas de Cultura de Células/métodos , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Adolescente , Adulto , Biópsia/métodos , Criança , Feminino , Fibroblastos/citologia , Humanos , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Cariotipagem , Fator 4 Semelhante a Kruppel , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Pâncreas/metabolismo , Linhagem , Pele/metabolismo , Fatores de Transcrição/metabolismo
13.
J Biol Chem ; 288(8): 5951-62, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23297408

RESUMO

Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic ß-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 ß-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic ß-cells.


Assuntos
Regulação Enzimológica da Expressão Gênica , Glucoquinase/química , Pâncreas/enzimologia , Sumoilação , Animais , Carboidratos/química , Catálise , Eletroforese em Gel Bidimensional/métodos , Células Secretoras de Insulina/citologia , Cinética , Fígado/enzimologia , Espectrometria de Massas/métodos , Camundongos , Mutação , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química
14.
J Mol Diagn ; 26(6): 530-541, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38575066

RESUMO

Precision medicine relies on accurate and consistent classification of sequence variants. A correct diagnosis of hepatocyte nuclear factor (HNF) 1B maturity-onset diabetes of the young, caused by pathogenic variants in the HNF1B gene, is important for optimal disease management and prognosis, and it has implications for genetic counseling and follow-up of at-risk family members. We hypothesized that the functional characterization could provide valuable information to assist the interpretation of pathogenicity of HNF1B variants. Using different in vitro functional assays, variants identified among 313 individuals, suspected to have monogenic diabetes with or without kidney disease, were characterized. The data from the functional assays were subsequently conjugated with obtained clinical, biochemical, and in silico data. Two variants (p.A167P, p.H336Pfs∗22) showed severe loss of function due to impaired transactivation, reduced DNA binding (p.A167P), and mRNA instability (p.A167P). Although both these variant carriers were diagnosed with diabetes, the p.H336Pfs∗22 carrier also had congenital absence of a kidney, which is a characteristic trait for HNF1B maturity-onset diabetes of the young. Functional analysis of the p.A167P variant revealed damaging effects on HNF-1B protein function, which may warrant imaging of the kidneys and/or pancreas. In addition, the current study has generated important data, including evidence supporting the benign functional impact of five variants (p.D82N, p.T88A, p.N394D, p.V458G, and p.T544A), and piloting new approaches that will prove critical for the growth of HNF1B-diabetes diagnosis.


Assuntos
Diabetes Mellitus Tipo 2 , Fator 1-beta Nuclear de Hepatócito , Humanos , Fator 1-beta Nuclear de Hepatócito/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/diagnóstico , Feminino , Masculino , Adulto , Medicina de Precisão/métodos , Mutação , Adolescente , Pessoa de Meia-Idade , Adulto Jovem
15.
Biochim Biophys Acta ; 1822(11): 1705-15, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22820548

RESUMO

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 ß-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.


Assuntos
Diabetes Mellitus Tipo 2/genética , Glucoquinase , Proteínas Mutantes , Deficiências na Proteostase , Diabetes Mellitus Tipo 2/metabolismo , Glucoquinase/química , Glucoquinase/genética , Glucoquinase/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Octoxinol , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Proteólise , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Reticulócitos/metabolismo
16.
J Biol Chem ; 286(40): 34593-605, 2011 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-21784842

RESUMO

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.


Assuntos
Carboxilesterase/genética , Diabetes Mellitus Tipo 2/genética , Mutação , Pâncreas Exócrino/metabolismo , Sequência de Aminoácidos , Animais , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Pâncreas Exócrino/fisiopatologia , Polilisina/química , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
17.
Exp Cell Res ; 317(3): 293-306, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20974127

RESUMO

p120 Catenin (p120(ctn)) regulates cadherin stability, and thus facilitates strong cell-cell adhesion. Previously, we demonstrated that Gα(12) interacts with p120(ctn). In the present study, we have delineated a region of p120(ctn) that binds to Gα(12). We report that the N-terminal region of p120(ctn) (amino acids 1-346) is necessary and sufficient for the interaction. While the coiled-coiled domain and a charged region, comprising a.a 102-120, were found to be dispensable, amino acids 121-323 were required for p120(ctn) binding to Gα(12). This region harbors the phosphorylation domain of p120(ctn) and has been postulated as important for RhoA regulation. Downregulation of Src family kinase-induced tyrosine phosphorylation of p120(ctn) was observed in the presence of activated Gα(12). This down-regulation was triggered by three different Gα(12) mutants uncoupled from RhoA signalling. Furthermore, a dominant active form of RhoA did not reduce Src-induced phosphoryaltion of p120(ctn). In summary, our results suggest that Gα(12) binds to p120(ctn) and modulates its phosphorylation status through a Rho-independent mechanism. Gα(12) emerges as an important regulator of p120(ctn) function, and possibly of cadherin-mediated adhesion and/or cell motility.


Assuntos
Cateninas/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Humanos , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Tirosina/metabolismo , delta Catenina
18.
J Clin Endocrinol Metab ; 107(4): e1455-e1466, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-34850019

RESUMO

CONTEXT: Maturity onset diabetes of the young, type 8 (MODY8) is associated with mutations in the CEL gene, which encodes the digestive enzyme carboxyl ester lipase. Several diabetes cases and families have in recent years been attributed to mutations in CEL without any functional or clinical evidence provided. OBJECTIVE: To facilitate correct MODY8 diagnostics, we screened 2 cohorts of diabetes patients and delineated the phenotype. METHODS: Young, lean Swedish and Finnish patients with a diagnosis of type 2 diabetes (352 cases, 406 controls) were screened for mutations in the CEL gene. We also screened 58 Czech MODY cases who had tested negative for common MODY genes. For CEL mutation-positive subjects, family history was recorded, and clinical investigations and pancreatic imaging performed. RESULTS: Two cases (1 Swedish and 1 Czech) with germline mutation in CEL were identified. Clinical and radiological investigations of these 2 probands and their families revealed dominantly inherited insulin-dependent diabetes, pancreatic exocrine dysfunction, and atrophic pancreas with lipomatosis and cysts. Notably, hereditary pancreatitis was the predominant phenotype in 1 pedigree. Both families carried single-base pair deletions in the proximal part of the CEL variable number of tandem repeat (VNTR) region in exon 11. The mutations are predicted to lead to aberrant protein tails that make the CEL protein susceptible to aggregation. CONCLUSION: The diagnosis of MODY8 requires a pancreatic exocrine phenotype and a deletion in the CEL VNTR in addition to dominantly inherited diabetes. CEL screening may be warranted also in families with hereditary pancreatitis of unknown genetic etiology.


Assuntos
Diabetes Mellitus Tipo 2 , Lipase/genética , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Humanos , Mutação , Pancreatite Crônica
19.
Nat Metab ; 4(1): 76-89, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-35058633

RESUMO

MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to ß-cells as a form of cross-talk between exocrine and endocrine cells. Human ß-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to ß-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant ß-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of ß-cells to cause diabetes.


Assuntos
Comunicação Celular , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas Exócrino/metabolismo , Células Acinares/metabolismo , Animais , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Lipase/química , Lipase/genética , Lipase/metabolismo , Camundongos , Mutação , Pâncreas/metabolismo , Pâncreas/patologia , Transporte Proteico , Solubilidade
20.
Nat Metab ; 4(3): 344-358, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35315439

RESUMO

Early childhood obesity is a growing global concern; however, the role of common genetic variation on infant and child weight development is unclear. Here, we identify 46 loci associated with early childhood body mass index at specific ages, matching different child growth phases, and representing four major trajectory patterns. We perform genome-wide association studies across 12 time points from birth to 8 years in 28,681 children and their parents (27,088 mothers and 26,239 fathers) in the Norwegian Mother, Father and Child Cohort Study. Monogenic obesity genes are overrepresented near identified loci, and several complex association signals near LEPR, GLP1R, PCSK1 and KLF14 point towards a major influence for common variation affecting the leptin-melanocortin system in early life, providing a link to putative treatment strategies. We also demonstrate how different polygenic risk scores transition from birth to adult profiles through early child growth. In conclusion, our results offer a fine-grained characterization of a changing genetic landscape sustaining early childhood growth.


Assuntos
Obesidade Infantil , Adulto , Índice de Massa Corporal , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Humanos , Lactente , Mães
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