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1.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34344826

RESUMO

Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge ("trans") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arginina/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Arginina/farmacologia , Citosol/metabolismo , Feminino , Células HEK293 , Humanos , Cinética , Lisina/metabolismo , Lisina/farmacologia , Lisossomos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Xenopus , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
2.
J Med Chem ; 63(15): 8231-8249, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32608236

RESUMO

Sialin, encoded by the SLC17A5 gene, is a lysosomal sialic acid transporter defective in Salla disease, a rare inherited leukodystrophy. It also enables metabolic incorporation of exogenous sialic acids, leading to autoantibodies against N-glycolylneuraminic acid in humans. Here, we identified a novel class of human sialin ligands by virtual screening and structure-activity relationship studies. The ligand scaffold is characterized by an amino acid backbone with a free carboxylate, an N-linked aromatic or heteroaromatic substituent, and a hydrophobic side chain. The most potent compound, 45 (LSP12-3129), inhibited N-acetylneuraminic acid 1 (Neu5Ac) transport in a non-competitive manner with IC50 ≈ 2.5 µM, a value 400-fold lower than the KM for Neu5Ac. In vitro and molecular docking studies attributed the non-competitive character to selective inhibitor binding to the Neu5Ac site in a cytosol-facing conformation. Moreover, compound 45 rescued the trafficking defect of the pathogenic mutant (R39C) causing Salla disease. This new class of cell-permeant inhibitors provides tools to investigate the physiological roles of sialin and help develop pharmacological chaperones for Salla disease.


Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Lisossomos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Células HeLa , Humanos , Ligantes , Simulação de Acoplamento Molecular/métodos , Estrutura Secundária de Proteína , Ratos
3.
J Biol Chem ; 292(28): 11980-11991, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28572511

RESUMO

Vitamin B12 (cobalamin (Cbl)), in the cofactor forms methyl-Cbl and adenosyl-Cbl, is required for the function of the essential enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. Cbl enters mammalian cells by receptor-mediated endocytosis of protein-bound Cbl followed by lysosomal export of free Cbl to the cytosol and further processing to these cofactor forms. The integral membrane proteins LMBD1 and ABCD4 are required for lysosomal release of Cbl, and mutations in the genes LMBRD1 and ABCD4 result in the cobalamin metabolism disorders cblF and cblJ. We report a new (fifth) patient with the cblJ disorder who presented at 7 days of age with poor feeding, hypotonia, methylmalonic aciduria, and elevated plasma homocysteine and harbored the mutations c.1667_1668delAG [p.Glu556Glyfs*27] and c.1295G>A [p.Arg432Gln] in the ABCD4 gene. Cbl cofactor forms are decreased in fibroblasts from this patient but could be rescued by overexpression of either ABCD4 or, unexpectedly, LMBD1. Using a sensitive live-cell FRET assay, we demonstrated selective interaction between ABCD4 and LMBD1 and decreased interaction when ABCD4 harbored the patient mutations p.Arg432Gln or p.Asn141Lys or when artificial mutations disrupted the ATPase domain. Finally, we showed that ABCD4 lysosomal targeting depends on co-expression of, and interaction with, LMBD1. These data broaden the patient and mutation spectrum of cblJ deficiency, establish a sensitive live-cell assay to detect the LMBD1-ABCD4 interaction, and confirm the importance of this interaction for proper intracellular targeting of ABCD4 and cobalamin cofactor synthesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Lisossomos/metabolismo , Erros Inatos do Metabolismo/genética , Modelos Moleculares , Mutação , Proteínas de Transporte Nucleocitoplasmático/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Substituição de Aminoácidos , Domínio Catalítico , Linhagem Celular Transformada , Células Cultivadas , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/enzimologia , Lisossomos/patologia , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Simulação de Acoplamento Molecular , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/deficiência , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína , Vitamina B 12/metabolismo
4.
Proc Natl Acad Sci U S A ; 114(18): E3602-E3611, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28416685

RESUMO

Lysosomes degrade cellular components sequestered by autophagy or extracellular material internalized by endocytosis and phagocytosis. The macromolecule building blocks released by lysosomal hydrolysis are then exported to the cytosol by lysosomal transporters, which remain undercharacterized. In this study, we designed an in situ assay of lysosomal amino acid export based on the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis that detects lysosomal storage. This assay was used to screen candidate lysosomal transporters, leading to the identification of sodium-coupled neutral amino acid transporter 7 (SNAT7), encoded by the SLC38A7 gene, as a lysosomal transporter highly selective for glutamine and asparagine. Cell fractionation confirmed the lysosomal localization of SNAT7, and flux measurements confirmed its substrate selectivity and showed a strong activation by the lysosomal pH gradient. Interestingly, gene silencing or editing experiments revealed that SNAT7 is the primary permeation pathway for glutamine across the lysosomal membrane and it is required for growth of cancer cells in a low free-glutamine environment, when macropinocytosis and lysosomal degradation of extracellular proteins are used as an alternative source of amino acids. SNAT7 may, thus, represent a novel target for glutamine-related anticancer therapies.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Glutamina/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Glutamina/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/patologia , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral
5.
Proc Natl Acad Sci U S A ; 109(50): E3434-43, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23169667

RESUMO

Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Cisteamina/uso terapêutico , Cistinose/tratamento farmacológico , Cistinose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/genética , Canavanina/metabolismo , RNA Helicases DEAD-box , DNA Complementar/genética , Proteínas de Drosophila , Fenômenos Eletrofisiológicos , Feminino , Genes Fúngicos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Vacúolos/metabolismo , Xenopus laevis
6.
J Biol Chem ; 287(14): 11489-97, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22334707

RESUMO

Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.


Assuntos
Biologia Computacional , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/química , Simportadores/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Indóis/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Projetos Piloto , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Relação Estrutura-Atividade , Simportadores/genética
7.
Proc Natl Acad Sci U S A ; 109(5): E210-7, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22232659

RESUMO

Secondary active transporters use electrochemical gradients provided by primary ion pumps to translocate metabolites or drugs "uphill" across membranes. Here we report the ion-coupling mechanism of cystinosin, an unusual eukaryotic, proton-driven transporter distantly related to the proton pump bacteriorhodopsin. In humans, cystinosin exports the proteolysis-derived dimeric amino acid cystine from lysosomes and is impaired in cystinosis. Using voltage-dependence analysis of steady-state and transient currents elicited by cystine and neutralization-scanning mutagenesis of conserved protonatable residues, we show that cystine binding is coupled to protonation of a clinically relevant aspartate buried in the membrane. Deuterium isotope substitution experiments are consistent with an access of this aspartate from the lysosomal lumen through a deep proton channel. This aspartate lies in one of the two PQ-loop motifs shared by cystinosin with a set of eukaryotic membrane proteins of unknown function and is conserved in about half of them, thus suggesting that other PQ-loop proteins may translocate protons.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Lisossomos/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Mutagênese , Prótons , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
8.
Biochem J ; 439(1): 113-28, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21692750

RESUMO

DIRC2 (Disrupted in renal carcinoma 2) has been initially identified as a breakpoint-spanning gene in a chromosomal translocation putatively associated with the development of renal cancer. The DIRC2 protein belongs to the MFS (major facilitator superfamily) and has been previously detected by organellar proteomics as a tentative constituent of lysosomal membranes. In the present study, lysosomal residence of overexpressed as well as endogenous DIRC2 was shown by several approaches. DIRC2 is proteolytically processed into a N-glycosylated N-terminal and a non-glycosylated C-terminal fragment respectively. Proteolytic cleavage occurs in lysosomal compartments and critically depends on the activity of cathepsin L which was found to be indispensable for this process in murine embryonic fibroblasts. The cleavage site within DIRC2 was mapped between amino acid residues 214 and 261 using internal epitope tags, and is presumably located within the tentative fifth intralysosomal loop, assuming the typical MFS topology. Lysosomal targeting of DIRC2 was demonstrated to be mediated by a N-terminal dileucine motif. By disrupting this motif, DIRC2 can be redirected to the plasma membrane. Finally, in a whole-cell electrophysiological assay based on heterologous expression of the targeting mutant at the plasma membrane of Xenopus oocytes, the application of a complex metabolic mixture evokes an outward current associated with the surface expression of full-length DIRC2. Taken together, these data strongly support the idea that DIRC2 is an electrogenic lysosomal metabolite transporter which is subjected to and presumably modulated by limited proteolytic processing.


Assuntos
Catepsina L/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Catepsina L/genética , Biologia Computacional , Eletrofisiologia , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Transportadoras/genética , Camundongos , Proteínas de Neoplasias/genética , Ligação Proteica , Xenopus
9.
J Mol Med (Berl) ; 88(5): 459-66, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20174775

RESUMO

Vitamin B(12) (cobalamin) is essential in animals and humans for metabolism of methylmalonic acid, for the remethylation of homocysteine to methionine and, consequently, for all S-adenosylmethionine-dependent methylation reactions, including DNA synthesis. In man, cobalamin deficiency leads to anemia and neurologic and cognitive impairment. In the cblF inborn error of vitamin B(12) metabolism, free vitamin accumulates in lysosomes and cannot be converted to cofactors for mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase. Recent work has shown that this defect is caused by mutations in the lysosomal membrane protein LMBD1, which shows significant homology to lipocalin membrane receptors, thereby indicating that LMBD1 is a lysosomal membrane exporter for cobalamin.


Assuntos
Proteínas de Membrana Lisossomal/genética , Lisossomos/metabolismo , Erros Inatos do Metabolismo/metabolismo , Vitamina B 12/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Erros Inatos do Metabolismo/genética , Dados de Sequência Molecular , Proteínas de Transporte Nucleocitoplasmático/genética , Alinhamento de Sequência
10.
Biochim Biophys Acta ; 1793(4): 636-49, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19146888

RESUMO

Lysosomal membrane proteins act at several crucial steps of the lysosome life cycle, including lumen acidification, metabolite export, molecular motor recruitment and fusion with other organelles. This review summarizes the molecular mechanisms of lysosomal storage diseases caused by defective transport of small molecules or ions across the lysosomal membrane, as well as Danon disease. In cystinosis and free sialic acid storage diseases, transporters for cystine and acidic monosaccharides, respectively, are blocked or retarded. A putative cobalamin transporter and a hybrid transporter/transferase of acetyl groups are defective in cobalamin F type disease and mucopolysaccharidosis type IIIC, respectively. In neurodegenerative forms of osteopetrosis, mutations of a proton/chloride exchanger impair the charge balance required for sustained proton pumping by the V-type ATPase, thus resulting in bone-resorption lacuna neutralization. However, the mechanism leading to lysosomal storage and neurodegeneration remains unclear. Mucolipidosis type IV is caused by mutations of a lysosomal cation channel named TRPML1; its gating properties are still poorly understood and the ion species linking this channel to lipid storage and membrane traffic defects is debated. Finally, the autophagy defect of Danon disease apparently arises from a role of LAMP2 in lysosome/autophagosome fusion, possibly secondary to a role in dynein-based centripetal motility.


Assuntos
Doenças por Armazenamento dos Lisossomos/patologia , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Animais , Autofagia , Transporte Biológico , Humanos , Doenças por Armazenamento dos Lisossomos/metabolismo
11.
Biol Cell ; 100(9): 551-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18399798

RESUMO

BACKGROUND INFORMATION: Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non-Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla-like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane. RESULTS: The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5-fold decrease of sialic acid transport. CONCLUSIONS: The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss-of-function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.


Assuntos
Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/genética , Polimorfismo Genético , Doença do Armazenamento de Ácido Siálico/genética , Simportadores/genética , Linhagem Celular , Criança , Humanos , Lisossomos/química , Lisossomos/metabolismo , Modelos Moleculares , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/metabolismo , Doença do Armazenamento de Ácido Siálico/metabolismo , Simportadores/química , Simportadores/metabolismo
12.
EMBO J ; 23(23): 4560-70, 2004 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-15510212

RESUMO

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.


Assuntos
Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , Endocitose , Humanos , Concentração de Íons de Hidrogênio , Mutação , Transportadores de Ânions Orgânicos/genética , Transporte Proteico , Doença do Armazenamento de Ácido Siálico/genética , Ácidos Siálicos/metabolismo , Simportadores/genética
13.
Hum Mol Genet ; 13(13): 1361-71, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15128704

RESUMO

Cystinosis is an inherited disorder characterized by defective lysosomal efflux of cystine. Three clinical forms (infantile, juvenile and ocular cystinosis) have been described according to the age of onset and severity of the symptoms. The causative gene, CTNS, encodes a seven transmembrane domain protein, cystinosin, which we recently identified as a H+-driven cystine transporter using an in vitro transport assay. In this study, we explored the relationship between transport activity and intracellular localization of cystinosin mutants and their associated clinical phenotype. Thirty-one pathogenic mutations (24 missense mutations, seven in-frame deletions or insertions) were analysed. Most of the mutations did not alter the lysosomal localization of cystinosin, although three partially mislocalized the protein independently of its C-terminal sorting motif, thus confirming the presence of an additional sorting mechanism. Sixteen of 19 mutations associated with infantile cystinosis abolished transport, whereas three of five mutations associated with juvenile or ocular forms strongly reduced transport, in agreement with the milder clinical phenotype. Five atypical, unclassified or misclassified mutations could be clarified using the transport data and additional genetic information. Overall, our data demonstrate that, excluding premature termination of cystinosin, impaired transport is the most frequent cause of pathogenicity, with infantile cystinosis generally resulting from a total loss of activity. Thus the transport assay could be used as a prognostic tool when novel mutations are identified.


Assuntos
Cistina/metabolismo , Cistinose , Glicoproteínas/genética , Glicoproteínas/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros , Transporte Biológico/genética , Células HeLa , Humanos , Proteínas de Membrana Transportadoras , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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