RESUMO
Compelling lines of evidence indicate that overexpression of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) in subjects with trisomy 21 (Down syndrome[DS]) contributes to the abnormal structure and function of the DS brain. In the present study, we used a novel, phospho-dependent antibody recognizing DYRK1A only with nonphosphorylated tyrosine 145 and 147 (DYRK1A Tyr-145/147P(-)), to investigate the expression pattern of this DYRK1A species in trisomic and disomic human and mouse brains. Immunoblotting and dephosphorylation experiments demonstrated higher levels of DYRK1A Tyr-145/147P(-) in postnatal trisomic brains in comparison with controls (by â¼40%) than those of the DYRK1A visualized by three other N- and C-terminally directed antibodies to DYRK1A. By immunofluorescence, the immunoreactivity to DYRK1A Tyr-145/147P(-) was the strongest in the nuclei of astroglial cells, which contrasted with the predominantly neuronal localization of DYRK1A visualized by the three other antibodies to DYRK1A we used. In addition, DYRK1A Tyr-145/147P(-) was enriched in the nuclei of neuronal progenitors and newly born neurons in the adult hippocampal proliferative zone and also occurred in some cholinergic axonal terminals. Our data show a distinctive expression pattern of DYRK1A forms nonphosphorylated at Tyr-145 and Tyr-147 in the brain tissue and suggest that DS subjects may exhibit not only upregulation of total DYRK1A, but also more subtle differences in phosphorylation levels of this kinase in comparison with control individuals.
Assuntos
Astrócitos/enzimologia , Síndrome de Down/enzimologia , Hipocampo/enzimologia , Células-Tronco Neurais/enzimologia , Fosfotirosina/metabolismo , Terminações Pré-Sinápticas/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Acetilcolina/metabolismo , Adolescente , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Western Blotting , Núcleo Celular/enzimologia , Criança , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Adulto Jovem , Quinases DyrkRESUMO
The classic late infantile form of neuronal ceroid lipofuscinosis (CLN2, cLINCL) is associated with mutations in the gene encoding tripeptidyl-peptidase I (TPP-I), a lysosomal aminopeptidase that cleaves off tripeptides from the free N-termini of oligopeptides. To date over 30 different mutations and 14 polymorphisms associated with CLN2 disease process have been identified. In the present study, we analysed the molecular basis of 15 different mutations of TPP-I by using immunocytochemistry, immunofluorescence, Western blotting, enzymatic assay and subcellular fractionation. In addition, we studied the expression of TPP-I in other lysosomal storage disorders such as CLN1, CLN3, muccopolysaccharidoses and GM1 and GM2 gangliosidoses. Our study shows that TPP-I is absent or appears in very small amounts not only in cLINCL subjects with mutations producing severely truncated protein, but also in individuals with missense point mutations, which correlates with loss of TPP-I activity. Of interest, small amounts of TPP-I were detected in lysosomal fraction from fibroblasts from cLINCL subject with protracted form. This observation suggests that the presence of small amounts of TPP-I in lysosomes is able to delay significantly CLN2 disease process. We also show that TPP-I immunoreactivity is increased in the brain tissue of CLN1 and CLN3 subjects, stronger in glial cells and macrophages than neurons. Less prominent increase of TPP-I staining was found in muccopolysaccharidoses and GM1 and GM2 gangliosidoses. These data suggest that TPP-I participates in lysosomal turnover of proteins in pathological conditions associated with cell/tissue injury.
Assuntos
Endopeptidases/genética , Lipofuscinoses Ceroides Neuronais/genética , Aminopeptidases , Anticorpos , Encéfalo/enzimologia , Células Cultivadas , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases/imunologia , Endopeptidases/metabolismo , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Lactente , Doenças por Armazenamento dos Lisossomos/enzimologia , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/enzimologia , Mutação , Lipofuscinoses Ceroides Neuronais/enzimologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/metabolismo , Polimorfismo Genético , Sítios de Splice de RNA/genética , Serina Proteases , Pele/citologia , Frações Subcelulares/enzimologia , Tripeptidil-Peptidase 1RESUMO
Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid-beta protein precursor and cathepsin D in vitro. The Leu101Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin.
Assuntos
Glicoproteínas de Membrana , Chaperonas Moleculares , Mutação de Sentido Incorreto , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteínas/genética , Proteínas/metabolismo , Elementos Antissenso (Genética) , Criança , Humanos , Técnicas In Vitro , Rim/citologia , Microscopia Confocal , Neuroblastoma , Neurônios/citologia , Neurônios/enzimologia , Proteínas/análise , Transfecção , Células Tumorais CultivadasRESUMO
The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders characterized by accumulation of ceroid lipopigment in lysosomes in various tissues and organs. The childhood forms of the NCLs represent the most common neurogenetic disorders of childhood and are inherited in an autosomal-recessive mode. The adult form of NCL is rare and shows either an autosomal-recessive or autosomal dominant mode of inheritance. Currently, five genes associated with various childhood forms of NCLs, designated CLN1, CLN2, CLN3, CLN5, and CLN8, have been isolated and characterized. Two of these genes, CLN1 and CLN2, encode lysosomal enzymes: palmitoyl protein thioesterase 1 (PPT1) and tripetidyl peptidase 1 (TPP1), respectively. CLN3, CLN5, and CLN8 encode proteins of predicted transmembrane topology, whose function has not been characterized yet. Two other genes, CLN6 and CLN7, have been assigned recently to small chromosomal regions. Gene(s) associated with the adult form of NCLs (CLN4) are at present unknown. This study summarizes the current classification and new diagnostic criteria of NCLs based on clinicopathological, biochemical, and molecular genetic data. Material includes 159 probands with NCL (37 CLNI, 72 classical CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR) as well as a comprehensive review of the literature. The results of our study indicate that although only biochemical and molecular genetic studies allow for definitive diagnosis, ultrastructural studies of the biopsy material are still very useful. Thus, although treatments for NCLs are not available at present, the diagnosis has become better defined.
Assuntos
Lipofuscinoses Ceroides Neuronais/classificação , Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/genética , Adolescente , Adulto , Criança , Pré-Escolar , Genótipo , Humanos , Lactente , Recém-Nascido , Mutação , Fenótipo , Tripeptidil-Peptidase 1RESUMO
Lysosomal accumulation of autofluorescent, ceroid lipopigment material in various tissues and organs is a common feature of the neuronal ceroid lipofuscinoses (NCLs). However, recent clinicopathologic and genetic studies have evidenced that NCLs encompass a group of highly heterogeneous disorders. In five of the eight NCL variants distinguished at present, genes associated with the disease process have been isolated and characterized (CLN1, CLN2, CLN3, CLN5, CLN8). Only products of two of these genes, CLN 1 and CLN2, have structural and functional properties of lysosomal enzymes. Nevertheless, according to the nature of the material accumulated in the lysosomes, NCLs in humans as well as natural animal models of these disorders can be divided into two major groups: those characterized by the prominent storage of saposins A and D, and those showing the predominance of subunit c of mitochondrial ATP synthase accumulation. Thus, taking into account the chemical character of the major component of the storage material, NCLs can be classified currently as proteinoses. Of importance, although lysosomal storage material accumulates in NCL subjects in various organs, only brain tissue shows severe dysfunction and cell death, another common feature of the NCL disease process. However, the relation between the genetic defects associated with the NCL forms, the accumulation of storage material, and tissue damage is still unknown. This chapter introduces the reader to the complex pathogenesis of NCLs and summarizes our current knowledge of the potential consequences of the genetic defects of NCL-associated proteins on the biology of the cell.
Assuntos
Lipofuscinoses Ceroides Neuronais/diagnóstico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/patologia , Adolescente , Adulto , Aminopeptidases , Animais , Antígenos CD , Criança , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases/deficiência , Humanos , Lactente , Recém-Nascido , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/deficiência , Serina Proteases , Tioléster Hidrolases/deficiência , Tripeptidil-Peptidase 1RESUMO
Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.
Assuntos
Encéfalo/patologia , Endopeptidases/análise , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminopeptidases , Encéfalo/enzimologia , Infarto Encefálico/enzimologia , Infarto Encefálico/patologia , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Criança , Dipeptidil Peptidases e Tripeptidil Peptidases , Encefalite/enzimologia , Encefalite/patologia , Humanos , Imuno-Histoquímica , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/enzimologia , Pessoa de Meia-Idade , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/patologia , Neurônios/enzimologia , Neurônios/patologia , Serina Proteases , Tripeptidil-Peptidase 1RESUMO
This study describes the phenotype/genotype analysis of 159 probands with neuronal ceroid lipofuscinosis (37 CLN1, 72 classic CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR). Phenotype/genotype comparison showed that mutations in the CLN1 gene were associated with different phenotypes: infantile, late infantile, and juvenile. Two common mutations (223A-->C and 451C-->T) were found in 26 of 37 CLN1 subjects (64% of alleles examined). A nonsense point mutation, 451C-->T, was the most common in CLN1 subjects with infantile onset at 0-2 years, accounting for 50% of alleles studied. A missense point mutation, 223A-->C, was the most common among CLN1 subjects with juvenile onset older than 4 years, accounting for 45% of alleles examined. Twenty-one other CLN1 mutations were identified in 4 of 37 subjects with infantile onset, 6 of 37 with late-infantile onset, and 6 of 37 with juvenile onset. All CLN1 probands were palmitoyl-protein thioesterase (PPT)-deficient and showed granular osmiophilic deposits (GROD) at the electron microscopic (EM) level. In the group of classic CLN2 (72 probands), two common mutations were found: an intronic 3556G-->C transversion in the invariant AG of 3' splice junction in 55% of probands, and a nonsense mutation 3670C-->T in 30% of probands. Classic late-infantile onset (2-4 years) was found in 68 of 72 (95%) cases, whereas juvenile onset (> 4 years) occurred only in 4 of 72 (5%) cases. All probands had deficiency of tripeptidyl-peptidase I (TPP1) activity and, at the EM level, curvilinear profiles. Ten probands with late-infantile onset did not show mutations in the CLN2 gene, had normal TPP1 activity, and at the EM level had mixed profiles. Further studies are in progress to identify genetic defect(s) in these subjects. The CLN3 group (40 probands) was divided into two categories: classic or typical presentation, and delayed classic or atypical presentation. All CLN3 patients had onset of symptoms after 4 years of age. In 40 probands, the 1.02-kb common deletion was found in one or two alleles of the CLN3 gene. Homozygotes for the common CLN3 deletion showed the classic phenotype. The phenotype in compound heterozygotes was either the classic or the delayed classic or atypical form. Thus, our study indicates that some mutations in the CLN1 and CLN2 genes may be associated with juvenile onset of the disease process and a more benign clinical course. Interfamilial and intrafamilial variations also were found, especially in the speed of becoming blind and neurologically disabled.
Assuntos
Glicoproteínas de Membrana , Chaperonas Moleculares , Lipofuscinoses Ceroides Neuronais/genética , Mutação Puntual , Aminopeptidases , Criança , Pré-Escolar , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases , Saúde da Família , Genótipo , Humanos , Lactente , Proteínas de Membrana/genética , Linhagem , Peptídeo Hidrolases/genética , Fenótipo , Proteínas/genética , Serina Proteases , Tioléster Hidrolases , Tripeptidil-Peptidase 1RESUMO
Immunocytochemical, biochemical, and molecular genetic studies indicate that apolipoprotein E (apoE) plays an important role in the process of amyloidogenesis-beta. However, there is still no clear translation of these data into the pathogenesis of amyloidosis-beta. Previous studies demonstrated sodium dodecyl sulfate (SDS)-resistant binding of apoE to the main component of Alzheimer's amyloid-A beta and modulation of A beta aggregation by apoE in vitro. To more closely characterize apoE-A beta interactions, we have studied the binding of thrombolytic fragments of apoE3 to A beta in vitro by using SDS-polyacrylamide gel electrophoresis and intrinsic fluorescence quenching. Here we demonstrate that SDS-resistant binding of A beta is mediated by the receptor-binding, N-terminal domain of apoE3. Under native conditions, both the N- and C-terminal domains of apoE3 bind A beta; however, the former does so with higher affinity. We propose that the modulation of A beta binding to the N-terminal domain of apoE is a potential therapeutic target for the treatment of amyloidosis-beta.
Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Apolipoproteína E3 , Apolipoproteínas E/efeitos dos fármacos , Sítios de Ligação , Humanos , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Dodecilsulfato de Sódio/farmacologia , Espectrometria de FluorescênciaRESUMO
Maintenance of the appropriate pH in the intracellular vacuolar compartments is essential for normal cell function. Here, we report that CLN3 protein, which is associated with the juvenile form of neuronal ceroid lipofuscinosis (JNCL), participates in lysosomal pH homeostasis in human cells. We show that CLN3 protein increases lysosomal pH in cultured human embryonal kidney cells, whereas inhibition of CLN3 protein synthesis by antisense approach acidifies lysosomal compartments. These changes in lysosomal pH are sufficient to exert a significant biological effect and modify intracellular processing of amyloid-beta protein precursor and cathepsin D, model proteins whose metabolism is influenced by the pH of acidic organelles. Mutant CLN3 protein (R334C) that is associated with the classical JNCL phenotype was devoid of biological activities of wild-type CLN3 protein. These data suggest that the pathogenesis of juvenile neuronal ceroid lipofuscinosis is associated with altered acidification of lysosomal compartments. Furthermore, our study indicates that CLN3 protein affects metabolism of proteins essential for cell functions, such as amyloid-beta protein precursor, implicated in Alzheimer's disease pathogenesis.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Catepsina D/metabolismo , Rim/metabolismo , Lisossomos/metabolismo , Glicoproteínas de Membrana , Chaperonas Moleculares , Proteínas/fisiologia , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Concentração de Íons de Hidrogênio , Rim/embriologia , Proteínas Luminescentes/biossíntese , Microscopia de Fluorescência , Mutação , Lipofuscinoses Ceroides Neuronais/metabolismo , Processamento de Proteína Pós-Traducional , Frações Subcelulares , TransfecçãoRESUMO
This study describes the phenotype/genotype analyses of 56 probands with a juvenile onset, some of which had atypical features of neuronal ceroid lipofuscinosis, collected at the New York State Institute for Basic Research (IBR). In this group, we found probands with abundant curvilinear profiles in lysosomal storage material, deficiency of pepstatin-insensitive peptidase, and mutations in the CLN2 gene, as well as patients with a predominance of granular osmiophilic deposits in the lysosomal storage material, deficiency of palmitoyl-protein thioesterase, and mutations in the CLN1 gene. We have divided the probands into two categories: typical (or classic) and atypical. Most of the typical and atypical probands had onset of symptoms about or after 4 years of age. Interfamiliar and intrafamiliar variations were found, especially in the speed of becoming practically blind. Thus, our study indicates that some mutations in the CLN1, CLN2, and CLN3 genes may be associated with late onset of the disease process, may have a more benign clinical course, and clinic overlap with other forms of neuronal ceroid lipofuscinosis.
Assuntos
Ciclinas , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/genética , Proteínas de Saccharomyces cerevisiae , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Lactente , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Linhagem , Fenótipo , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Tripeptidil-Peptidase 1RESUMO
The product of the CLN3 gene is a novel protein of unknown function. Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins. Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. In fact, the sequence of CLN3 protein contains several such signaling sequences, which are conserved among mammals. First, at the N-terminus, potential N-myristoylation motif is present. Second, the C-terminal part of CLN3 protein contains both the dileucine motif, which is a potential lysosomal targeting signal, and the prenylation motif. There is scanty evidence of lysosomal and/or mitochondrial localization of CLN3 protein. However, the question of where the functional site of the cln3 protein exists in vivo remains unanswered. From theoretical calculations, we hypothesized that CLN3 should be an integral part of the membranous micro-environment. First, to test this hypothesis, we initiated detergent-partitioning experiments, localizing CLN3 predominantly in a pool of membranous protein. Further studies have shown that CLN3 protein integrates spontaneously with cellular membranes. Second, based on the prenylation results of CLN3 protein in vitro, we discussed the possible topological consequences of C-terminal fragment of CLN3 protein.
Assuntos
Membrana Celular/metabolismo , Ciclinas , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae , Western Blotting , Sistema Livre de Células , Eletroforese em Gel de Poliacrilamida , Humanos , Octoxinol , Polietilenoglicóis/metabolismo , Prenilação de Proteína , Proteínas Recombinantes de FusãoRESUMO
CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern. By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments. Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae. Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments. However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein.
Assuntos
Ciclinas , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Antígenos CD/metabolismo , Células CHO , Cricetinae , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Técnicas In Vitro , Proteínas de Membrana Lisossomal , Microscopia Confocal , Microscopia de Fluorescência , Monensin/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão , Tunicamicina/metabolismoRESUMO
The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors.
Assuntos
Ciclinas , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Fosfatase Alcalina/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Fluorescência Verde , Immunoblotting , Proteínas Luminescentes/metabolismo , Manosefosfatos/metabolismo , Fosfoaminoácidos/isolamento & purificação , Fosfoaminoácidos/metabolismo , Fosforilação , Testes de Precipitina , Radioisótopos/metabolismo , Proteínas Recombinantes de Fusão , Serina/metabolismo , Treonina/metabolismoRESUMO
The gene for Batten disease, the CLN3 gene, encodes a novel, highly hydrophobic, multitransmembrane protein, predicted to consist of 438 amino acid residues. We have expressed a full-length CLN3 protein in fusion with green fluorescent protein in various cell lines to provide its initial biochemical characterization and subcellular localization. By using Western blotting, Percoll density gradient fractionation, and Triton X-114 extraction, we demonstrate that the product of the CLN3 gene, which we call battenin, in mammalian expression system studied is a highly glycosylated protein of lysosomal membrane. In addition our data suggest that CLN3 protein is processed proteolytically in acidic compartments of the cell. Thus, battenin represents the novel constituent of a growing family of lysosomal membrane proteins.
Assuntos
Ciclinas , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Western Blotting , Células CHO , Fracionamento Celular , Membrana Celular/metabolismo , Cricetinae , Endossomos/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Técnicas In Vitro , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Testes de Precipitina , Proteínas Recombinantes de Fusão , Sulfonas/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
We present a clinicopathological study and the first molecular genetic analysis of a family with 2 siblings affected by a rare, protracted form of juvenile neuronal ceroid lipofuscinosis (JNCL). Molecular genetic studies showed that both siblings, in addition to being heterozygous for the 1.02-kb CLN3 deletion, a common mutation in JNCL, also had a G-to-A missense mutation at nucleotide 1,020 of the CLN3 cDNA sequence on the non-1.02-kb deletion chromosomes. This point mutation resulted in a substitution of glutamic acid by lysine at position 295 of the CLN3 protein. Thus, a single point mutation at residue 295 of the CLN3 protein in protracted JNCL may underlie the phenotype in this form, which differs from that in classic JNCL.
Assuntos
Heterozigoto , Lipofuscinoses Ceroides Neuronais/genética , Adulto , Eletroencefalografia , Feminino , Genótipo , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Biologia Molecular , Mutação , Lipofuscinoses Ceroides Neuronais/patologia , Lipofuscinoses Ceroides Neuronais/fisiopatologia , Linhagem , Pele/patologiaRESUMO
Recently, the CLN3 gene associated with Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), a recessively inherited, progressive, neurodegenerative disorder of childhood, has been identified. The CLN3 gene encodes a novel protein (battenin) of a predicted 438 amino acids containing several potential posttranslational modifications. We have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein (GFP) to evaluate whether CLN3 protein is phosphorylated. By using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that the CLN3 protein is phosphorylated on both serine and threonine residues. We also demonstrate that CLN3 protein is not modified by mannose 6-phosphate. Furthermore, we show that phosphorylation of CLN3 protein is carried out by protein kinase A (cAMP-dependent protein kinase, PKA), protein kinase G (cGMP-dependent protein kinase, PKG), and casein kinase II and that it is enhanced by inhibition of protein phosphatase 1 (PP 1) or protein phosphatase 2A (PP 2A).
Assuntos
Glicoproteínas de Membrana , Chaperonas Moleculares , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteínas/metabolismo , Animais , Células CHO , Caseína Quinase II , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
Expression of the gene for Batten disease (CLN3) was studied in Escherichia coli and in a cell-free rabbit reticulocyte expression systems. A full-length recombinant fusion CLN3 protein was not produced in the bacterial systems used. However, both N-terminal fragment encompassing 246 amino acids and short C-terminal fragment containing 428-438 amino acids of the CLN3 protein were successfully overexpressed in bacteria. Further studies showed that the C-terminal sequence of the CLN3 protein corresponding to the 356-438 amino acid residues was responsible for inhibition of protein synthesis in bacteria. The full-length CLN3 gene product was readily synthesized in vitro in the cell-free rabbit reticulocyte expression system. The product obtained, corresponding to core CLN3 protein, showed an approximate molecular weight of 43 kDa. Immunoprecipitation of this product with pAb to 4-19 amino acids of the CLN3 protein allows us to suggest that CLN3 protein translation starts at Met-1.
Assuntos
Ciclinas , Lipofuscinoses Ceroides Neuronais/genética , Proteínas de Saccharomyces cerevisiae , Adolescente , Animais , Sistema Livre de Células , Criança , Pré-Escolar , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica/fisiologia , Humanos , Lactente , Glicoproteínas de Membrana , Chaperonas Moleculares , Lipofuscinoses Ceroides Neuronais/diagnóstico , Fragmentos de Peptídeos/genética , Biossíntese de Proteínas/genética , Coelhos , Proteínas Recombinantes de Fusão/genética , ReticulócitosRESUMO
We have collected 122 late-infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) and 191 juvenile NCL (JNCL, CLN3) cases, diagnosed on the basis of age-at-onset, clinical symptomatology, and pathological findings and representing the most common forms of NCL in the United States, and Europe. However, careful analysis of available data revealed that about 80% of cases show typical and 20% show atypical clinical course and/or pathological findings and thus, may represent variants of LINCL and JNCL, respectively. Recent progress in the biochemistry and molecular genetics of NCL inclined us to reevaluate these atypical NCL cases. The gene responsible for LINCL has not yet been identified, except for the Finnish variant. Accumulation of subunit c of mitochondrial ATP synthase, to curvilinear profiles, is found in LINCL cases. A novel variant of LINCL, with predominantly granular profiles in the lysosomal storage, as well as normal excretion of subunit c in urine samples, was found in five cases. When the palmitoyle-protein thioesterase (PPT) was studied in these five cases, it was found that the level was deficient, suggesting that they are not LINCL, but the infantile form of neuronal ceroid lipofuscinosis (INCL). Using molecular genetic techniques in the typical JNCL cases, common 1.02 kb deletion to CLN3 was found in 23/27 (homozygotes) and in one allele 4/27 (heterozygotes) in affected pedigrees. In atypical JNCL pedigrees, it was found in 5/16 heterozygotes, while in 1/5 pedigrees, a novel mutation of one atypical JNCL where a single amino acid substitution at 295 E-->K was found in one allele. None of the atypical JNCL cases was homozygote. In atypical JNCL cases where mutation in CLN3 has not been identified (11/16 probands), several possibilities may exist. The phenotype may be caused by a yet undefined mutation in CLN3 or may be due to phenotypically overlapping with other forms of NCL. Pheno/genotypic correlation and the diagnostic difficulties are discussed.
Assuntos
Lipofuscinoses Ceroides Neuronais/diagnóstico , Adolescente , Feminino , Genótipo , Humanos , Lactente , Lisossomos/ultraestrutura , Masculino , Lipofuscinoses Ceroides Neuronais/genética , Linhagem , Fenótipo , Tripeptidil-Peptidase 1RESUMO
Several apolipoproteins are known to be closely associated with amyloid fibrillogenesis. Serum amyloid A, apolipoprotein (apo) AII and apo A1 are each deposited as biochemically distinct forms of amyloid. Late-onset Alzheimer's disease is linked to one isotype of apo E, apo E4. Apo E and apo E4 in particular have been shown to modulate amyloid fibril formation by amyloid-beta peptides in vitro. Furthermore, the carboxy terminus of apo E has been shown to be a constituent of plaque amyloid. We show immunohistochemically and electron microscopically the presence of apo A1 in senile plaques. The intact apo A1 can itself form amyloid-like fibrils in vitro that are Congo Red positive. We propose that some proteins when misfolded can propagate this misfolding to identical units, either autocatalytically or to other proteins that are induced to fold into the same abnormal conformation. This conformational mimicry may initiate and/or augment fibrillogenesis in Alzheimer's disease.