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1.
Mol Cell ; 79(3): 406-415.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692975

RESUMO

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.


Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
2.
Traffic ; 17(2): 117-30, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26572236

RESUMO

The long-standing paradigm that all peroxisomal proteins are imported post-translationally into pre-existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co-translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N-terminal transmembrane segment (TMS) of ribosome-bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3-containing ribosome•nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP-dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.


Assuntos
Retículo Endoplasmático/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Peroxinas , Peroxissomos/metabolismo , Transporte Proteico/fisiologia , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo
3.
Biochim Biophys Acta ; 1863(5): 870-80, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26392202

RESUMO

The importance of peroxisomes is highlighted by severe inherited human disorders linked to impaired peroxisomal biogenesis. Besides the simple architecture of these ubiquitous and dynamic organelles, their biogenesis is surprisingly complex and involves specialized proteins, termed peroxins, which mediate targeting and insertion of peroxisomal membrane proteins (PMPs) into the peroxisomal bilayer, and the import of soluble proteins into the protein-dense matrix of the organelle. The long-standing paradigm that all peroxisomal proteins are imported directly into preexisting peroxisomes has been challenged by the detection of PMPs inside the endoplasmic reticulum (ER). New models propose that the ER originates peroxisomal biogenesis by mediating PMP trafficking to the peroxisomes via budding vesicles. However, the relative contribution of this ER-derived pathway to the total peroxisome population in vivo, and the detailed mechanisms of ER entry and exit of PMPs are controversially discussed. This review aims to summarize present knowledge about how PMPs are targeted to the ER, instead of being inserted directly into preexisting peroxisomes. Moreover, molecular mechanisms that facilitate bilayer insertion of PMPs among different species are discussed.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Retículo Endoplasmático/química , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peroxinas , Peroxissomos/química , Plantas/química , Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
4.
PLoS Pathog ; 10(12): e1004554, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25503639

RESUMO

Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antígenos Virais/genética , Vírus da Varíola Bovina/genética , Mutação da Fase de Leitura/genética , Genoma Viral/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , Animais , Antígenos Virais/imunologia , Antígenos Virais/fisiologia , Retículo Endoplasmático , Retroalimentação Fisiológica/fisiologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Dados de Sequência Molecular , Células Sf9
5.
J Biol Chem ; 286(48): 41402-41412, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21984826

RESUMO

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas da Matriz Viral/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apresentação de Antígeno/genética , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Infecções por Vírus Epstein-Barr/genética , Células HeLa , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Spodoptera , Proteínas da Matriz Viral/genética
6.
Viruses ; 14(2)2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35215846

RESUMO

Flaviviruses such as dengue virus (DENV) and Zika virus (ZIKV) have evolved sophisticated mechanisms to suppress the host immune system. For instance, flavivirus infections were found to sabotage peroxisomes, organelles with an important role in innate immunity. The current model suggests that the capsid (C) proteins of DENV and ZIKV downregulate peroxisomes, ultimately resulting in reduced production of interferons by interacting with the host protein PEX19, a crucial chaperone in peroxisomal biogenesis. Here, we aimed to explore the importance of peroxisomes and the role of C interaction with PEX19 in the flavivirus life cycle. By infecting cells lacking peroxisomes we show that this organelle is required for optimal DENV replication. Moreover, we demonstrate that DENV and ZIKV C bind PEX19 through a conserved PEX19-binding motif, which is also commonly found in cellular peroxisomal membrane proteins (PMPs). However, in contrast to PMPs, this interaction does not result in the targeting of C to peroxisomes. Furthermore, we show that the presence of C results in peroxisome loss due to impaired peroxisomal biogenesis, which appears to occur by a PEX19-independent mechanism. Hence, these findings challenge the current model of how flavivirus C might downregulate peroxisomal abundance and suggest a yet unknown role of peroxisomes in flavivirus biology.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Vírus da Dengue/fisiologia , Proteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Zika virus/fisiologia , Animais , Linhagem Celular , Vírus da Dengue/química , Humanos , Biogênese de Organelas , Peroxissomos/fisiologia , Replicação Viral , Zika virus/química
7.
Biochem Biophys Res Commun ; 377(1): 176-80, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18834860

RESUMO

Strikingly variable clinical phenotypes can be found in X-linked adrenoleukodystrophy (X-ALD) even with the same ABCD1 mutation. ABCD2 is the closest homolog to ABCD1. Since ABCD2 overexpression complements the loss of ABCD1 in vivo and in vitro, we have investigated the possible role of the ABCD2 gene locus as determinant of X-ALD phenotypes. Sequence and segregation analysis of the ABCD2 gene, in a large X-ALD family with different phenotypes disclosed that the identical ABCD2 alleles were inherited in brothers affected by mild (noncerebral) versus severe (childhood cerebral) X-ALD phenotypes. Moreover, two independent association studies of ABCD2 polymorphisms and clinical phenotypes showed an even allele distribution in different X-ALD phenotypes and controls. Based on these findings ABCD2 can be excluded as a major modifier locus for clinical diversity in X-ALD. These findings are of particular importance for the attempt of pharmacological induction of ABCD2 as a possible therapeutic approach in X-ALD.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/patologia , Polimorfismo de Nucleotídeo Único , Subfamília D de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Adolescente , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Mutação , Linhagem , Fenótipo
8.
Hum Mutat ; 27(8): 748-59, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16835865

RESUMO

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions. We elaborated the rationale for decision making in 3-methylcrotonyl-coenzyme A (CoA) carboxylase deficiency (MCCD), which afflicts leucine catabolism, with reported outcomes ranging from asymptomatic to death. In Bavaria, we screened 677,852 neonates for 25 conditions, including MCCD, based on elevated concentrations of 3-hydroxyisovalerylcarnitine (3-HIVA-C). Genotypes of MCCA (MCCC1) and MCCB (MCCC2) were assessed in identified newborns, their relatives, and in individuals (n = 17) from other regions, and correlated to biochemical and clinical phenotypes. NBS revealed eight newborns and six relatives with MCCD, suggesting a higher frequency than previously assumed (1:84,700). We found a strikingly heterogeneous spectrum of 22 novel and eight reported mutations. Allelic variants were neither related to biochemical nor anamnestic data of our probands showing all asymptomatic or benign phenotypes. Comparative analysis of case reports with NBS data implied that only few individuals (< 10%) develop symptoms. In addition, none of the symptoms reported so far can clearly be attributed to MCCD. MCCD is a genetic condition with low clinical expressivity and penetrance. It largely represents as nondisease. So far, there are no genetic or biochemical markers that would identify the few individuals potentially at risk for harmful clinical expression. The low ratio of benefit to harm was pivotal to the decision to exclude MCCD from NBS in Germany. MCCD may be regarded as exemplary of the ongoing controversy arising from the inclusion of potentially asymptomatic conditions, which generates a psychological burden for afflicted families and a financial burden for health care systems.


Assuntos
Carbono-Carbono Ligases/deficiência , Heterogeneidade Genética , Mutação , Triagem Neonatal/legislação & jurisprudência , Alelos , Carbono-Carbono Ligases/genética , Estudos de Coortes , Deficiências Nutricionais/diagnóstico , Deficiências Nutricionais/genética , Feminino , Genótipo , Alemanha , Humanos , Recém-Nascido , Masculino , Penetrância , Medição de Risco
9.
J Mol Biol ; 427(5): 1102-18, 2015 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-25224907

RESUMO

Protein homeostasis results in a steady supply of peptides, which are further degraded to fuel protein synthesis or metabolic needs of the cell. In higher vertebrates, a small fraction of the resulting peptidome, however, is translocated into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Antigenic peptides are guided to major histocompatibility complex class I (MHC I) molecules and are finally displayed on the cell surface, where they mount an adaptive immune response against viral infected or malignantly transformed cells. Here, we review the structural organization and the molecular mechanism of this specialized antigen translocon. We discuss how the ATP-binding cassette (ABC) transporter TAP communicates and cooperates within the multi-component peptide loading machinery, mediating the proper assembly and editing of kinetically stable peptide/MHC I complexes. In light of its important role within the MHC I antigen processing pathway, TAP is a prime target for viral immune evasion strategies, and we summarize how this antigen translocation machinery is sabotaged by viral factors. Finally, we compare TAP with other ABC systems that facilitate peptide translocation.


Assuntos
Imunidade Adaptativa/imunologia , Apresentação de Antígeno/imunologia , Antígenos Virais/imunologia , Evasão da Resposta Imune/imunologia , Vírus/imunologia , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Transporte Proteico/imunologia
10.
Eur J Cell Biol ; 82(8): 401-10, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14533738

RESUMO

In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein. This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood. Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting. However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself. Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency. Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome. The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1. Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting. The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency. The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria. Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Peroxissomos/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adrenoleucodistrofia/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
12.
J Biol Chem ; 284(15): 10232-42, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19211561

RESUMO

After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.


Assuntos
Retículo Endoplasmático/metabolismo , Microssomos/metabolismo , Ricina/química , Membrana Celular/metabolismo , Citosol/metabolismo , Conformação Molecular , Fosfolipídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Espectrometria de Fluorescência/métodos , Propriedades de Superfície , Temperatura
13.
Biochem Biophys Res Commun ; 334(3): 939-46, 2005 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-16023992

RESUMO

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog. The amino-termini containing 39 (MCCalpha) or 20 amino acids (MCCbeta) were both necessary and sufficient for targeting. Structural requirements for mitochondrial import were defined by site-directed mutagenesis. Our studies provide the prerequisite to understand the impact of specific mutations on the clinical phenotype of MCC deficiency.


Assuntos
Carbono-Carbono Ligases/metabolismo , Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Carbono-Carbono Ligases/química , Carbono-Carbono Ligases/deficiência , Carbono-Carbono Ligases/genética , Proteínas de Transporte/metabolismo , Humanos , Rim/química , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo
14.
Biochem Biophys Res Commun ; 291(5): 1180-6, 2002 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11883941

RESUMO

PEX19 has been shown to play a central role in the early steps of peroxisomal membrane synthesis. Computational database analysis of the PEX19 sequence revealed three different conserved domains: D1 (aa 1--87), D2 (aa 88--272), and D3 (aa 273--299). However, these domains have not yet been linked to specific biological functions. We elected to functionally characterize the proteins derived from two naturally occurring PEX19 splice variants: PEX19DeltaE2 lacking the N-terminal domain D1 and PEX19DeltaE8 lacking the domain D3. Both interact with peroxisomal ABC transporters (ALDP, ALDRP, PMP70) and with full-length PEX3 as shown by in vitro protein interaction studies. PEX19DeltaE8 also interacts with a PEX3 protein lacking the peroxisomal targeting region located at the N-terminus (Delta66aaPEX3), whereas PEX19DeltaE2 does not. Functional complementation studies in PEX19-deficient human fibroblasts revealed that transfection of PEX19DeltaE8-cDNA leads to restoration of both peroxisomal membranes and of functional peroxisomes, whereas transfection of PEX19DeltaE2-cDNA does not restore peroxisomal biogenesis. Human PEX19 is partly farnesylated in vitro and in vivo. The farnesylation consensus motif CLIM is located in the PEX19 domain D3. The finding that the protein derived from the splice variant lacking D3 is able to interact with several peroxisomal membrane proteins and to restore peroxisomal biogenesis challenges the previous assumption that farnesylation of PEX19 is essential for its biological functionality. The data presented demonstrate a considerable functional diversity of the proteins encoded by two PEX19 splice variants and thereby provide first experimental evidence for specific biological functions of the different predicted domains of the PEX19 protein.


Assuntos
Proteínas de Membrana/fisiologia , Peroxissomos/fisiologia , Processamento Alternativo , Animais , Células COS , Chlorocebus aethiops , Teste de Complementação Genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína
15.
Am J Hum Genet ; 70(1): 20-5, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11719902

RESUMO

Enteropeptidase (enterokinase [E.C.3.4.21.9]) is a serine protease of the intestinal brush border in the proximal small intestine. It activates the pancreatic proenzyme trypsinogen, which, in turn, releases active digestive enzymes from their inactive pancreatic precursors. Congenital enteropeptidase deficiency is a rare recessively inherited disorder leading, in affected infants, to severe failure to thrive. The genomic structure of the proenteropeptidase gene (25 exons, total gene size 88 kb) was characterized in order to perform DNA sequencing in three clinically and biochemically proved patients with congenital enteropeptidase deficiency who were from two families. We found compound heterozygosity for nonsense mutations (S712X/R857X) in two affected siblings and found compound heterozygosity for a nonsense mutation (Q261X) and a frameshift mutation (FsQ902) in the third patient. In accordance with the biochemical findings, all four defective alleles identified are predicted null alleles leading to a gene product not containing the active site of the enzyme. These data provide first evidence that proenteropeptidase-gene mutations are the primary cause of congenital enteropeptidase deficiency.


Assuntos
Enteropeptidase/deficiência , Precursores Enzimáticos/deficiência , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Mutação/genética , Alelos , Sequência de Bases , Sítios de Ligação/genética , Domínio Catalítico/genética , Códon sem Sentido/genética , Análise Mutacional de DNA , Enteropeptidase/química , Enteropeptidase/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Éxons/genética , Insuficiência de Crescimento/enzimologia , Insuficiência de Crescimento/genética , Feminino , Mutação da Fase de Leitura/genética , Heterozigoto , Humanos , Íntrons , Masculino , Linhagem , Polimorfismo Genético/genética , Polimorfismo de Nucleotídeo Único/genética
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