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2.
Proc Natl Acad Sci U S A ; 117(29): 17195-17203, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32606248

RESUMO

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.


Assuntos
Actinobacteria/genética , Antivirais/farmacologia , Genoma Bacteriano , Macrolídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antivirais/química , Antivirais/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Sirolimo/química , Sirolimo/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
3.
Cell ; 168(4): 571-574, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28187279

RESUMO

Curative therapies are most successful when cancer is diagnosed and treated at an early stage. We advocate that technological advances in next-generation sequencing of circulating, tumor-derived nucleic acids hold promise for addressing the challenge of developing safe and effective cancer screening tests.


Assuntos
DNA/sangue , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Análise de Sequência de DNA/métodos , Detecção Precoce de Câncer , Humanos
4.
Cancer Res ; 66(14): 7216-24, 2006 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16849569

RESUMO

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Rim/fisiologia , Regeneração/fisiologia , Animais , Carcinoma de Células Renais/genética , Feminino , Expressão Gênica , Neoplasias Renais/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Regeneração/genética
6.
Hum Mutat ; 23(1): 40-6, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14695531

RESUMO

von Hippel Lindau disease (VHL) is an autosomal dominant familial cancer syndrome linked to alteration of the VHL tumor suppressor gene. Affected patients are predisposed to develop pheochromocytomas and cystic and solid tumors of the kidney, CNS, pancreas, retina, and epididymis. However, organ involvement varies considerably among families and has been shown to correlate with the underlying germline alteration. Clinically, we observed a paradoxically lower prevalence of renal cell carcinoma (RCC) in patients with complete germline deletion of VHL. To determine if a relationship existed between the type of VHL deletion and disease, we retrospectively evaluated 123 patients from 55 families with large germline VHL deletions, including 42 intragenic partial deletions and 13 complete VHL deletions, by history and radiographic imaging. Each individual and family was scored for cystic or solid involvement of CNS, pancreas, and kidney, and for pheochromocytoma. Germline deletions were mapped using a combination of fluorescent in situ hybridization (FISH) and quantitative Southern and Southern blot analysis. An age-adjusted comparison demonstrated a higher prevalence of RCC in patients with partial germline VHL deletions relative to complete deletions (48.9 vs. 22.6%, p=0.007). This striking phenotypic dichotomy was not seen for cystic renal lesions or for CNS (p=0.22), pancreas (p=0.72), or pheochromocytoma (p=0.34). Deletion mapping revealed that development of RCC had an even greater correlation with retention of HSPC300 (C3orf10), located within the 30-kb region of chromosome 3p, immediately telomeric to VHL (52.3 vs. 18.9%, p <0.001), suggesting the presence of a neighboring gene or genes critical to the development and maintenance of RCC. Careful correlation of genotypic data with objective phenotypic measures will provide further insight into the mechanisms of tumor formation.


Assuntos
Carcinoma de Células Renais/genética , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Deleção de Sequência , Doença de von Hippel-Lindau/complicações , Adulto , Carcinoma de Células Renais/diagnóstico , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , Humanos , Neoplasias Renais/diagnóstico , Fenótipo
7.
J Exp Med ; 198(6): 851-62, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12975453

RESUMO

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.


Assuntos
Perfilação da Expressão Gênica , Doença de Hodgkin/genética , Linfoma de Células B/diagnóstico , Linfoma de Células B/genética , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/diagnóstico , Neoplasias do Mediastino/genética , Adulto , Cromossomos Humanos Par 19 , Diagnóstico Diferencial , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/patologia , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/diagnóstico , Neoplasias do Mediastino/tratamento farmacológico , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Taxa de Sobrevida , Resultado do Tratamento , Células Tumorais Cultivadas
8.
Proc Natl Acad Sci U S A ; 100(12): 6958-63, 2003 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-12777628

RESUMO

To identify potential molecular determinants of tumor biology and possible clinical outcomes, global gene-expression patterns were analyzed in the primary tumors of patients with metastatic renal cell cancer by using cDNA microarrays. We used grossly dissected tumor masses that included tumor, blood vessels, connective tissue, and infiltrating immune cells to obtain a gene-expression "profile" from each primary tumor. Two patterns of gene expression were found within this uniformly staged patient population, which correlated with a significant difference in overall survival between the two patient groups. Subsets of genes most significantly associated with survival were defined, and vascular cell adhesion molecule-1 (VCAM-1) was the gene most predictive for survival. Therefore, despite the complex biological nature of metastatic cancer, basic clinical behavior as defined by survival may be determined by the gene-expression patterns expressed within the compilation of primary gross tumor cells. We conclude that survival in patients with metastatic renal cell cancer can be correlated with the expression of various genes based solely on the expression profile in the primary kidney tumor.


Assuntos
Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Adulto , Idoso , Carcinoma de Células Renais/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Taxa de Sobrevida , Estados Unidos/epidemiologia , Molécula 1 de Adesão de Célula Vascular/genética
9.
Science ; 300(5628): 2036-9, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12829768

RESUMO

A new collaborative model of research is needed to increase resources, to prioritize the R (ii) to increase the pace, reduce the overlap, and more systematically explore the elements of and delivery systems for vaccines; (iii) to use common standards for the prompt comparative testing of vaccine candidates; (iv) to expand resources for manufacturing vaccine candidates to speed their use in human trials; and (v) to increase the capacity for international clinical trials and to focus this effort toward quickly measuring the effectiveness of vaccine protection as prototype vaccine candidates are identified.


Assuntos
Vacinas contra a AIDS , Saúde Global , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Política Pública , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/economia , Vacinas contra a AIDS/imunologia , Academias e Institutos/economia , Academias e Institutos/organização & administração , Biotecnologia/economia , Ensaios Clínicos como Assunto/normas , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/normas , Indústria Farmacêutica/economia , Apoio Financeiro , Humanos , Propriedade Intelectual , Cooperação Internacional , Estudos Multicêntricos como Assunto , Setor Privado , Setor Público , Apoio à Pesquisa como Assunto , Vacinação
10.
Cancer Cell ; 3(2): 185-97, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12620412

RESUMO

We used gene expression profiling to establish a molecular diagnosis of mantle cell lymphoma (MCL), to elucidate its pathogenesis, and to predict the length of survival of these patients. An MCL gene expression signature defined a large subset of MCLs that expressed cyclin D1 and a novel subset that lacked cyclin D1 expression. A precise measurement of tumor cell proliferation, provided by the expression of proliferation signature genes, identified patient subsets that differed by more than 5 years in median survival. Differences in cyclin D1 mRNA abundance synergized with INK4a/ARF locus deletions to dictate tumor proliferation rate and survival. We propose a quantitative model of the aberrant cell cycle regulation in MCL that provides a rationale for the design of cell cycle inhibitor therapy in this malignancy.


Assuntos
Ciclina D1/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Genes Neoplásicos/genética , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/mortalidade , Proteínas de Neoplasias/genética , Fatores de Ribosilação do ADP/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Regiões não Traduzidas/genética
11.
Proc Natl Acad Sci U S A ; 99(26): 16899-903, 2002 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-12477932

RESUMO

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).


Assuntos
DNA Complementar/química , Análise de Sequência de DNA , Algoritmos , Animais , DNA Complementar/análise , Biblioteca Gênica , Humanos , Camundongos , Fases de Leitura Aberta
12.
Proc Natl Acad Sci U S A ; 99(16): 10353-8, 2002 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-12149480

RESUMO

The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Linhagem Celular Transformada , DNA Complementar , Dimerização , Elonguina , Proteínas Fúngicas/metabolismo , Humanos , Ligases/genética , Fígado/metabolismo , Mamíferos , Complexo Mediador , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Ratos , Spodoptera , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau
13.
N Engl J Med ; 346(25): 1937-47, 2002 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-12075054

RESUMO

BACKGROUND: The survival of patients with diffuse large-B-cell lymphoma after chemotherapy is influenced by molecular features of the tumors. We used the gene-expression profiles of these lymphomas to develop a molecular predictor of survival. METHODS: Biopsy samples of diffuse large-B-cell lymphoma from 240 patients were examined for gene expression with the use of DNA microarrays and analyzed for genomic abnormalities. Subgroups with distinctive gene-expression profiles were defined on the basis of hierarchical clustering. A molecular predictor of risk was constructed with the use of genes with expression patterns that were associated with survival in a preliminary group of 160 patients and was then tested in a validation group of 80 patients. The accuracy of this predictor was compared with that of the international prognostic index. RESULTS: Three gene-expression subgroups--germinal-center B-cell-like, activated B-cell-like, and type 3 diffuse large-B-cell lymphoma--were identified. Two common oncogenic events in diffuse large-B-cell lymphoma, bcl-2 translocation and c-rel amplification, were detected only in the germinal-center B-cell-like subgroup. Patients in this subgroup had the highest five-year survival rate. To identify other molecular determinants of outcome, we searched for individual genes with expression patterns that correlated with survival in the preliminary group of patients. Most of these genes fell within four gene-expression signatures characteristic of germinal-center B cells, proliferating cells, reactive stromal and immune cells in the lymph node, or major-histocompatibility-complex class II complex. We used 17 genes to construct a predictor of overall survival after chemotherapy. This gene-based predictor and the international prognostic index were independent prognostic indicators. CONCLUSIONS: DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.


Assuntos
Perfilação da Expressão Gênica , Linfoma de Células B/genética , Linfoma de Células B/mortalidade , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/mortalidade , Antibióticos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Feminino , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Análise de Sobrevida
14.
Cancer Cell ; 1(4): 315-8, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12086845

RESUMO

The Cancer Molecular Analysis Project (CMAP) of the NCI is integrating diverse cancer research data to elucidate fundamental etiologic processes, enable development of novel therapeutic approaches, and facilitate the bridging of basic and clinical science.


Assuntos
Neoplasias/etiologia , Pesquisa , Ciclo Celular/fisiologia , Ciclinas/metabolismo , Humanos , National Institutes of Health (U.S.) , Neoplasias/metabolismo , Transdução de Sinais , Estados Unidos
15.
Cancer Cell ; 1(3): 247-55, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12086861

RESUMO

Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.


Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Ligases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor , Animais , Sítios de Ligação , Western Blotting , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Feminino , Genes Supressores de Tumor , Transportador de Glucose Tipo 1 , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Técnicas Imunoenzimáticas , Neoplasias Renais/genética , Neoplasias Renais/patologia , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos SCID , Proteínas de Transporte de Monossacarídeos/metabolismo , Fenótipo , Plasmídeos , Proteínas Recombinantes de Fusão , Transcrição Gênica , Transfecção , Ubiquitina-Proteína Ligases , Proteína Supressora de Tumor Von Hippel-Lindau
17.
Cancer Res ; 62(11): 3014-9, 2002 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12036906

RESUMO

Renal cell carcinoma is associated with mutation of the von Hippel-Lindau (VHL) tumor suppressor gene. Cell lines derived from these tumors cannot exit the cell cycle when deprived of growth factors, and the ability to exit the cell cycle can be restored by the reintroduction of wild-type protein VHL (pVHL). Here, we report that cyclin D1 is overexpressed and remains inappropriately high in during contact inhibition in pVHL-deficient cell lines. In addition, hypoxia increased the expression of cyclin D1 specifically in pVHL-negative cell lines into which pVHL expression was restored. Hypoxic-induction of cyclin D1 was not observed in other pVHL-positive cell lines. This suggests a model whereby in some kidney cell types, pVHL may regulate a proliferative response to hypoxia, whereas the loss of pVHL leads to constitutively elevated cyclin D1 and abnormal proliferation under normal growth conditions.


Assuntos
Carcinoma de Células Renais/metabolismo , Ciclina D1/biossíntese , Neoplasias Renais/metabolismo , Ligases/fisiologia , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Hipóxia Celular/fisiologia , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Ligases/genética , Transfecção , Proteína Supressora de Tumor Von Hippel-Lindau
18.
Microbiology (Reading) ; 148(Pt 1): 29-40, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11782496

RESUMO

High-affinity iron uptake by a ferrous permease in the opportunistic pathogen Candida albicans is required for virulence. Here this iron uptake system has been characterized by investigating three distinct activities: an externally directed surface ferric reductase, a membrane-associated PPD (p-phenylenediamine) oxidase and a cellular ferrous iron transport activity. Copper was required for the PPD oxidase and ferrous transport activities. In contrast, copper was not required for iron uptake from siderophores. Addition of iron to the growth medium repressed ferric reductase and ferrous transport, indicating homeostatic regulation. To identify the genes involved, orthologous mutants of Saccharomyces cerevisiae were transformed with a genomic library of C. albicans. CFL95, a gene with sequence similarity to ferric reductases, restored reductase activity to the orthologous S. cerevisiae mutant. CaFTR2 and CaFTR1, genes with homology to ferrous permeases, conferred ferrous transport activity to the orthologous S. cerevisiae mutant. However, neither a genomic library nor CaFET99, a multicopper oxidase homologue and candidate gene for the PPD oxidase, complemented the S. cerevisiae mutant, possibly because of problems with targeting or assembly. Transcripts for CFL95, CaFTR1 and CaFET99 were strongly repressed by iron, whereas the CaFTR2 transcript was induced by iron. Deletion of the TUP1 regulator perturbed the homeostatic control of reductive iron uptake. Incidentally, iron starvation was noted to induce flavin production and this was misregulated in the absence of TUP1 control. The opposite regulation of two iron permease genes and the role of TUP1 indicate that the process of iron acquisition by C. albicans may be more complex and potentially more adaptable than by S. cerevisiae.


Assuntos
Candida albicans/metabolismo , Cobre/metabolismo , FMN Redutase , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Ferro/metabolismo , NADH NADPH Oxirredutases/metabolismo , Proteínas Nucleares , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Meios de Cultura , Compostos Ferrosos/metabolismo , Flavinas/metabolismo , Proteínas Fúngicas/genética , Teste de Complementação Genética , NADH NADPH Oxirredutases/genética , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Sideróforos/metabolismo
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