ORCID Profile
0000-0001-7896-4019
Current Organisation
MIR SCIENTIFIC
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Publisher: American Chemical Society (ACS)
Date: 08-12-2001
DOI: 10.1021/BI0157666
Abstract: Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins.Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites. Furthermore, the region(s) of clusterin involved in these many binding interactions remain(s) to be identified. As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris. The resultant protein had variable proteolytic truncations of the C-terminal region of the alpha-chain and the N-terminal region of the beta-chain. We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum. We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-clusterin monoclonal antibodies. Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins, and unstressed ligands, respectively, and (ii) the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules.
Publisher: American Chemical Society (ACS)
Date: 12-1997
DOI: 10.1021/BI9703507
Publisher: Wiley
Date: 02-1985
DOI: 10.1111/J.1432-1033.1985.TB08678.X
Abstract: The goal of this study is to explain the molecular basis of the marked deinduction of Xenopus albumin synthesis and secretion accompanying the activation of vitellogenin genes by estrogen. We have characterized by restriction analysis, DNA sequencing and hybrid-selected translation of mRNA, a cloned cDNA specifying the two 74-kDa albumins which constitute the predominant circulating form of albumin in Xenopus laevis. Using this recombinant DNA plasmid as a hybridization probe, we have determined the steady-state levels of albumin mRNA, the rate of transcription of the two 74-kDa albumin genes and the stability of the mRNA in male and female Xenopus hepatocytes in vivo and in primary cell cultures following estrogen treatment. In both whole liver and cultured hepatocytes estradiol caused a rapid drop in the steady-state levels of 74-kDa albumin mRNAs, which was reversed spontaneously in the continued presence of the hormone. The concentration of albumin mRNA was substantially higher in male than in female hepatocytes, the hormonal effect being more marked in male than in female hepatocytes. The decrease in steady-state levels of mRNA was anticipated in male hepatocytes by a 70% inhibition of rate of transcription of albumin genes within 2 h of exposure to estradiol, as measured by run-off transcription in liver nuclei isolated from animals treated in vivo or by determining the absolute transcription rate in cell cultures. In the latter the diminished transcription rate returned to normal within 12 h in the continued presence of the hormone. Estradiol caused a threefold destabilization of albumin mRNA in both male and female hepatocyte cultures to t 1/2 = 3 h and 2 h respectively. The combined effects on rate of or transcription and mRNA stability largely explain the changes in the steady-state levels of mRNA caused by hormone administration. Comparison of the kinetics of transcription rates of vitellogenin and albumin genes in vivo and in vitro reveals a striking reciprocity in the selective activation of the inducible genes and deinduction of the constitutively expressed genes at the early stages of response of Xenopus hepatocytes to estrogen.
Location: United States of America
No related grants have been discovered for Martin Tenniswood.