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1.
Arthritis Care Res (Hoboken) ; 74(10): 1585-1592, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-33938161

RESUMO

OBJECTIVE: The aim of this study was to quantify costs of hospital-associated care for juvenile idiopathic arthritis (JIA), provide insights in patient-level variation in costs, and investigate costs over time from the moment of JIA diagnosis. Results were reported for all JIA patients in general and by subtype. METHODS: This study was a single-center, retrospective analysis of prospective data from electronic medical records of children with JIA, ages 0-18 years, between April 1, 2011 and March 31, 2019. Patient characteristics (age, sex, JIA subtype) and hospital-based resource use (consultations, medication, radiology procedures, laboratory testing, surgeries, emergency department [ED] visits, hospital stays) were extracted and analyzed. Unit prices were obtained from Dutch reimbursement lists and pharmaceutical and hospital list prices. RESULTS: The analysis included 691 patients. The mean total cost of hospital care was €3,784/patient/year, of which €2,103 (55.6%) was attributable to medication. Other costs involved pediatric rheumatologist visits (€633/patient/year [16.7%]), hospital stays (€439/patient/year [11.6%]), other within-hospital specialist visits (€324/patient/year [8.6%]), radiology procedures (€119/patient/year [3.1%]), laboratory tests (€114/patient/year [3.0%]), surgeries (€46/patient/year [1.2%]), and ED visits (€6/patient/year [0.2%]). Mean annual total costs varied between JIA subtypes and between individuals and were the highest for systemic JIA (€7,772/patient/year). Over the treatment course, costs were the highest in the first month after JIA diagnosis. CONCLUSION: Hospital care costs of JIA vary substantially between individuals, between subtypes, and over the treatment course. The highest annual costs were for systemic JIA, primarily attributable to medication (i.e., biologics). Costs of other hospital-associated care were comparable regardless of subtype.


Assuntos
Artrite Juvenil , Produtos Biológicos , Adolescente , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/terapia , Produtos Biológicos/uso terapêutico , Criança , Pré-Escolar , Custos de Cuidados de Saúde , Hospitais , Humanos , Lactente , Recém-Nascido , Preparações Farmacêuticas , Estudos Prospectivos , Estudos Retrospectivos
2.
Mol Oncol ; 15(12): 3348-3362, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34510716

RESUMO

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh-frozen tumor tissue of 239 patients with stage I-III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group-regularized logistic ridge regression with post hoc group-weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.


Assuntos
Neoplasias Colorretais , Metilação de DNA , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilação de DNA/genética , Humanos
4.
World J Surg Oncol ; 19(1): 96, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33820567

RESUMO

BACKGROUND: Colorectal cancer is the third most common type of cancer in the world. We characterize a cohort of patients who survived up to 5 years without recurrence and identify factors predicting the probability of cure. METHODS: We analyzed data of patients who underwent curative intent surgery for stage I-III CRC between 2007 and 2012 and who had had been included in a large multicenter study in the Netherlands. Cure was defined as 5-year survival without recurrence. Survival data were retrieved from a national registry. RESULTS: Analysis of data of 754 patients revealed a cure rate of 65% (n = 490). Patients with stage I disease and T1- and N0-tumor had the highest probability of cure (94%, 95% and 90%, respectively). Those with a T4-tumor or N2-tumor had the lowest probability of cure (62% and 50%, respectively). A peak in the mortality rate for older patients early in follow-up suggests early excess mortality as an explanation. A similar trend was observed for stage III disease, poor tumor grade, postoperative complications, sarcopenia, and R1 resections. Patients with stage III disease, poor tumor grade, postoperative complications, sarcopenia, and R1 resections show a similar trend for decrease in CSS deaths over time. CONCLUSION: In the studied cohort, the probability of cure for patients with stage I-III CRC ranged from 50 to 95%. Even though most patients will be cured from CRC with standard therapy, standard therapy is insufficient for those with poor prognostic factors, such as high T- and N-stage and poor differentiation grade.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Humanos , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Estadiamento de Neoplasias , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos
5.
Cancers (Basel) ; 13(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920880

RESUMO

Circular RNAs (circRNAs) appear important in tumor progression of colon cancer (CC). We identified an extensive catalog of circRNAs in 181 chemonaive stage I/II colon tumors, who underwent curative surgery between 2007 and 2014. We identified circRNAs from RNAseq data, investigated common biology related to circRNA expression, and studied the association between circRNAs and relapse status, tumor stage, consensus molecular subtypes (CMS), tumor localization and microsatellite instability (MSI). We identified 2606 unique circRNAs. 277 circRNAs (derived from 260 genes) were repeatedly occurring in at least 20 patients of which 153 showed a poor or even negative (R < 0.3) correlation with the expression level of their linear gene. The circular junctions for circSATB2, circFGD6, circKMT2C and circPLEKHM3 were validated by Sanger sequencing. Multiple correspondence analysis showed that circRNAs were often co-expressed and that high diversity in circRNAs was associated with favorable disease-free survival (DFS), which was confirmed by Cox regression analysis (Hazard Ratio (HR) 0.60, 95% CI 0.38-0.97, p = 0.036). Considering individual circRNAs, absence of circMGA was significantly associated with relapse, whereas circSATB2, circNAB1, and circCEP192 were associated with both MSI and CMS. This study represents a showcase of the potential clinical utility of circRNAs for prognostic stratification in patients with stage I-II colon cancer and demonstrated that high diversity in circRNAs is associated with favorable DFS.

6.
J Surg Oncol ; 123(4): 1005-1014, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33368279

RESUMO

BACKGROUND: Although the prognostic relevance of KRAS status in metastatic colorectal cancer (CRC) depends on tumor laterality, this relationship is largely unknown in non-metastatic CRC. METHODS: Patients who underwent resection for non-metastatic CRC between 2000 and 2018 were identified from institutional databases at six academic tertiary centers in Europe and Japan. The prognostic relevance of KRAS status in patients with right-sided (RS), left-sided (LS), and rectal cancers was assessed. RESULTS: Of the 1093 eligible patients, 378 had right-sided tumors and 715 had left-sided tumors. Among patients with RS tumors, the 5-year overall (OS) and recurrence-free survival (RFS) for patients with KRASmut versus wild-type tumors was not shown to differ significantly (82.2% vs. 83.2% and 72.1% vs. 76.7%, respectively, all p > .05). Among those with LS tumors, KRAS mutation was associated with shorter 5-year OS and RFS on both the univariable (OS: 79.4% vs. 86.1%, p = .004; RFS: 68.8% vs. 77.3%, p = .005) and multivariable analysis (OS: HR: 1.52, p = .019; RFS: HR: 1.32, p = .05). CONCLUSIONS: KRAS mutation status was independently prognostic among patients with LS tumors, but this association failed to reach statistical significance in RS and rectal tumors. These findings confirm reports in metastatic CRC and underline the possible biologic importance of tumor location.


Assuntos
Neoplasias Colorretais/patologia , Cirurgia Colorretal/mortalidade , Repetições de Microssatélites , Mutação , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Idoso , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/cirurgia , Feminino , Seguimentos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
7.
Pediatr Res ; 59(3): 365-70, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16492973

RESUMO

Children with hereditary tyrosinemia type 1 (HT1) suffer from liver failure, renal tubular dysfunction, and rickets. The disease is caused by deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of tyrosine catabolism, and leads to accumulation of the toxic substrate fumarylacetoacetate (FAA) in hepatocytes and renal proximal tubular cells. Patients are treated with 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione (NTBC), which prevents accumulation of FAA by blocking an enzyme upstream of FAH. Liver transplantation is performed when patients do not respond to NTBC or develop hepatocellular carcinoma. This reduces the tyrosine load for the kidney but does not abolish renal exposure to locally produced FAA. To investigate the pathogenesis of liver and kidney damage induced by tyrosine metabolites, we challenged FAH-deficient mice with various doses of homogentisic acid (HGA), a precursor of FAA. Injecting NTBC-treated Fah-/- mice with low doses of HGA caused renal damage and death of renal tubular cells, as was shown by histologic analyses and deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) assay but did not lead to liver damage. In addition, kidney function, but not liver function, was affected after exposure to low doses of HGA. Administration of high doses of HGA led to massive cell death in both the liver and kidneys. Resistance to HGA-induced cell death was seen after withdrawing NTBC from Fah-/- mice. The finding that the kidneys of Fah-/- mice are especially sensitive to damage induced by low doses of HGA underscores the need to perform careful monitoring of the kidney function of tyrosinemia patients undergoing any form of treatment.


Assuntos
Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , Ácido Homogentísico/farmacologia , Rim/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Tirosinemias/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , Animais , Cicloexanonas/metabolismo , Cicloexanonas/uso terapêutico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Ácido Homogentísico/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Marcação In Situ das Extremidades Cortadas , Rim/citologia , Rim/metabolismo , Rim/patologia , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrobenzoatos/metabolismo , Nitrobenzoatos/uso terapêutico , Tirosinemias/tratamento farmacológico
8.
J Pediatr ; 146(5): 706-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15870679

RESUMO

Fatty acids play an important role in regulating insulin secretion, but the mechanisms are unclear. We report a case of a novel splice site mutation in the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) gene associated with hyperinsulinism. This mutation resulted in a nearly complete absence of immunoreactive protein and a decrease in fibroblast SCHAD activity.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Hiperinsulinismo/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Clorotiazida/uso terapêutico , Diazóxido/uso terapêutico , Diuréticos , Humanos , Hiperinsulinismo/tratamento farmacológico , Lactente , Masculino , Mutação Puntual , Inibidores de Simportadores de Cloreto de Sódio/uso terapêutico
9.
Pediatr Res ; 56(6): 981-7, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15496606

RESUMO

Mutations in ATP8B1 are associated with FIC1 disease, an autosomal recessive disorder in which intrahepatic cholestasis is the predominant manifestation. ATP8B1 encodes FIC1, which is expressed in several tissues, most prominently in the intestine, pancreas, and stomach and, to a much lesser extent, in the liver. In this study, Fic1 localization and expression during postnatal development was examined in healthy mice. Immunoblot and RT-PCR analysis indicated Fic1 is expressed abundantly in regions of the adult gastrointestinal tract of humans and mice. Immunohistochemistry revealed that Fic1 was localized to the apical membranes of enterocytes, pancreatic acinar cells, gastric pit epithelial cells, and hepatocytes and cholangiocytes. Subsequent analysis of early postnatal expression revealed that Fic1 expression in the small intestine was limited or absent at the age of 7 and 14 d and increased significantly with maturation. In contrast, pancreatic, hepatic, and gastric Fic1 expression was not diminished during the first 3 wk of postnatal development. In conclusion, these data show that Fic1 is expressed in a tissue-specific and developmentally regulated fashion at the apical membranes of epithelial cells. We speculate that the developing bile salt pool in the maturing intestine accounts for the increase in Fic1 protein expression in this tissue.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Células Epiteliais/fisiologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/fisiologia , Animais , Ductos Biliares/citologia , Ductos Biliares/crescimento & desenvolvimento , Ductos Biliares/fisiologia , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mucosa Intestinal/citologia , Intestinos/crescimento & desenvolvimento , Intestinos/fisiologia , Fígado/crescimento & desenvolvimento , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/crescimento & desenvolvimento , Pâncreas/fisiologia , Proteínas de Transferência de Fosfolipídeos , Estômago/crescimento & desenvolvimento , Estômago/fisiologia
10.
Kidney Int ; 66(3): 990-1000, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15327392

RESUMO

BACKGROUND: Hereditary tyrosinemia type 1 (HT1), which is associated with severe liver and kidney damage, is caused by deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine breakdown cascade. HT1-associated liver and kidney failure can be prevented by blocking an enzyme upstream of FAH in the tyrosine breakdown pathway with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). FAH knockout mice develop the HT1 phenotype when NTBC treatment is discontinued. METHODS: The occurrence of cell death was investigated in kidneys of Fah(-/-) mice on and off NTBC either unchallenged or injected with 800 mg/kg of homogentisic acid (HGA), an intermediate of tyrosine breakdown. RESULTS: No cell death could be detected in kidneys of Fah(-/-) mice on NTBC. A slight increase of cleaved caspase-3 was the only apoptosis-related feature that could be detected in kidneys of Fah(-/-) mice off NTBC. Challenge of Fah(-/-) mice on NTBC with HGA led to massive death of renal proximal tubular cells, with positive terminal deoxynucleotidyl transferase-mediated deoxyuridine diphosphate (dUDP) nick-end labeling (TUNEL) and DNA fragmentation assays, but hardly any cleavage of caspase-9 and caspase-3. Fah(-/-) mice off NTBC acquired resistance to HGA-induced renal cell death and the kidneys exhibited relatively few features of apoptosis upon challenge with HGA, with a small increase in expression of cleaved caspase-9 and caspase-3. CONCLUSION: Kidneys of adult Fah(-/-) mice, withdrawn from NTBC for 15 days, reveal limited characteristics of apoptosis, and have acquired resistance to a caspase-9- and caspase-3-independent form of cell death provoked by HGA.


Assuntos
Fragmentação do DNA/fisiologia , Hidrolases/genética , Túbulos Renais Proximais/patologia , Tirosinemias/patologia , Animais , Caspase 3 , Caspase 9 , Caspases/metabolismo , Cicloexanonas/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Hepatócitos/transplante , Ácido Homogentísico/farmacologia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Nitrobenzoatos/farmacologia , Taxa de Sobrevida , Tirosina/metabolismo , Tirosinemias/genética , Tirosinemias/mortalidade
11.
Gastroenterology ; 127(2): 379-84, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15300568

RESUMO

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are hereditary liver disorders; PFIC is characterized by severe progressive liver disease whereas BRIC patients have intermittent attacks of cholestasis without permanent liver damage. Mutations in ATP8B1 are present in PFIC type 1 and in a subset of BRIC patients. We hypothesized that a genetically distinct form of BRIC is associated with mutations in ABCB11. This gene encodes the bile salt export pump (BSEP) and is mutated in PFIC type 2. METHODS: Patients from 20 families were included; all had a normal ATP8B1 sequence. Sequencing of all 27 coding exons including the splice junctions of ABCB11 revealed 8 distinct mutations in 11 patients from 8 different families: one homozygous missense mutation (E297G) previously described in PFIC2 patients, 6 novel missense mutations, and one putative splice site mutation. RESULTS: In 12 families, no mutations in ATB8B1 or ABCB11 were detected. Pancreatitis is a known extrahepatic symptom in BRIC caused by ATP8B1 mutations, but was not present in BRIC patients with mutations in ABCB11. In contrast, cholelithiasis was observed in 7 of 11 BRIC patients with mutations in ABCB11, but has not been described in ATP8B1-affected BRIC patients. CONCLUSIONS: Mutations in ABCB11 are associated with BRIC, and consistent with the genetic classification of PFIC into 2 subtypes, we propose that this disorder be named BRIC type 2.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Colestase Intra-Hepática/genética , Mutação de Sentido Incorreto , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Adolescente , Adulto , Processamento Alternativo , Colestase Intra-Hepática/classificação , Colestase Intra-Hepática/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Recidiva
12.
Hepatology ; 39(2): 433-43, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14767996

RESUMO

The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo. In healthy fumarylacetoacetate hydrolase deficient mice (Fah(-/-)), protected from liver injury by the drug 2-(2- nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah(-/-) animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo-2 and to necrosis-like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up-regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c-Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N-terminal kinase (JNK) stress-activated kinase pathways were markedly impaired in the cell-death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo. Stress-induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases.


Assuntos
Hidrolases/genética , Falência Hepática Aguda/metabolismo , Tirosinemias/metabolismo , Tirosinemias/patologia , Acetaminofen , Analgésicos não Narcóticos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Depressores do Sistema Nervoso Central/farmacologia , Doença Crônica , Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , Etanol/farmacologia , Proteína Ligante Fas , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Ácido Homogentísico/metabolismo , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , Necrose , Nitrobenzoatos/farmacologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regulação para Cima , Receptor fas/metabolismo
13.
J Hepatol ; 39(6): 901-9, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14642604

RESUMO

BACKGROUND: Hereditary Tyrosinemia type I, caused by deficiency of fumarylacetoacetate hydrolase (FAH), is characterized by liver and kidney damage. Administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) corrects the tyrosinemia phenotype, but does not prevent development of hepatocellular carcinoma. AIM: To gain insight into the pathophysiological changes associated with liver damage induced by tyrosinemia and the preventive action of NTBC on these changes. METHODS: Differential gene expression patterns in livers of tyrosinemia-affected and healthy mice, and of tyrosinemia-affected and NTBC-treated Fah-/- mice were investigated by suppression subtractive hybridization. RESULTS: Transcripts encoding proteins playing a role in protein turnover, growth and proliferation, RNA processing, and signal transduction were primarily induced in tyrosinemia-affected livers. Transcripts mainly contributing to the profile of suppressed genes encode proteins that are secreted by the liver, or are necessary for intermediate metabolism. NTBC treatment fails to normalize the tyrosinemia-induced alterations in expression of transcripts encoding proteins involved in protein turnover, signal transduction, and cell growth and proliferation. CONCLUSIONS: The failure of NTBC to normalize liver gene expression of Fah-/- mice may play a role in rendering the tyrosinemia-affected liver susceptible to development of hepatocellular carcinoma under NTBC treatment.


Assuntos
Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fígado/fisiologia , Nitrobenzoatos/farmacologia , Tirosinemias/tratamento farmacológico , Tirosinemias/genética , Animais , Northern Blotting , DNA Complementar , Feminino , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Reprodutibilidade dos Testes , Tirosinemias/fisiopatologia
14.
Biochem J ; 370(Pt 3): 881-9, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12466020

RESUMO

We have used indirect immunofluorescense studies and glycosylation-site insertion and deletion mapping to characterize the topology of human copper transporter 1 (hCTR1), the putative human high-affinity copper-import protein. Both approaches indicated that hCTR1 contains three transmembrane domains and that the N-terminus of hCTR1, which contains several putative copper-binding sites, is localized extracellularly, whereas the C-terminus is exposed to the cytosol. Based on previous observations that CTR1 proteins form high-molecular-mass complexes, we investigated directly whether CTR1 proteins interact with themselves. Yeast two-hybrid studies showed that interaction of yeast, mouse, rat and human CTR1 occurs at the sites of their N-terminal domains, and is not dependent on the copper concentration in the growth media. Analysis of deletion constructs indicated that multiple regions in the N-terminus are essential for this self-interaction. In contrast, the N-terminal tail of the presumed low-affinity copper transporter, hCTR2, does not interact with itself. Taken together, these results suggest that CTR1 spans the membrane at least six times, permitting formation of a channel, which is consistent with its proposed role as a copper transporter.


Assuntos
Proteínas de Transporte de Cátions , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Animais , Linhagem Celular , Cobre/metabolismo , Transportador de Cobre 1 , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Humanos , Proteínas de Membrana/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
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