RESUMO
Gingival fibroblast cell lines were derived from Sorsby's fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.
Assuntos
Gengiva/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mutação Puntual , Inibidor Tecidual de Metaloproteinase-3/genética , Animais , Sequência de Bases , Células Cultivadas , DNA Complementar/genética , Gelatinases/metabolismo , Expressão Gênica , Genes Dominantes , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.
Assuntos
Metaloendopeptidases/genética , Epitélio Pigmentado Ocular/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas ADAM , Proteína ADAMTS7 , Proteína ADAMTS9 , Northern Blotting , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Metaloendopeptidases/biossíntese , Epitélio Pigmentado Ocular/efeitos dos fármacos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
The ADAMTS (A Disintegrin and metalloproteinase with thrombospondin-1 type repeats) family of enzymes have been implicated in turnover of extracellular matrix. We previously showed that levels of ADAMTS6 mRNA in ARPE-19 cells were markedly increased following treatment with tumour necrosis factor alpha (TNFalpha). This study shows that the ADAMTS6 transcript contains unusually large untranslated regions (UTRs) at both the 5' and 3'end. The 5'UTR contains 11 AUG codons upstream of the predicted ADAMTS6 start codon and potently inhibits translation of a downstream reporter gene. However some translation can be restored by truncating the 5'UTR from the 5'end. The 5'UTR was tested for internal ribosome entry site activity using a bicistronic luciferase reporter plasmid, but none was detected. Using the 5' and 3'UTR sequences to screen the GenBank database we identified a full length ADAMTS6 cDNA of 7262 bp. This transcript is alternatively spliced at the 3'end of the open reading frame (ORF), resulting in an extended ORF containing 3 additional tsp-1 type repeats. Quantitative RT-PCR showed that the long and short form of the ADAMTS6 ORF are co-expressed in ARPE-19 cells, but the relative levels of the two forms is modulated by TNFalpha. The region of the transcript encoding the catalytic domain also contains several notable differences compared to the previously published ADAMTS6 cDNA sequence, including a redefinition of the predicted active site motif.
Assuntos
Regiões 5' não Traduzidas/genética , Proteínas ADAM/genética , Processamento Alternativo , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Proteínas ADAMTS , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Domínio Catalítico/genética , Linhagem Celular , Códon de Iniciação/genética , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/genética , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Mutação Puntual , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5 x 10(9) International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1.04 g x ml(-1) with 73 % of virus below 1.08 g x ml(-1). Full-length HCV RNA was detected by Northern blotting and the ratio between positive- and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn(2+) and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7.