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Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

ABSTRACT: A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out. The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix. The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted. The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8. Two proteinase-resistant mutants of PDGF [( Ser28]- and [Pro32]-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB. These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

SUBMITTER: Craig S

PROVIDER: S-EPMC1130641 | biostudies-other | 1992 Jan

REPOSITORIES: biostudies-other

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Project description:Drug resistance is a consequence of how most modern medicines work. Drugs exert pressure on cells that causes death or the evolution of resistance. Indeed, highly specific drugs are rendered ineffective by a single DNA mutation. In this study, we apply the drug methotrexate, which is widely used in cancer and rheumatoid arthritis, and perform evolution experiments on Baker's yeast to ask the different ways in which cells become drug resistant. Because of the conserved nature of biological pathways between yeast and man, our results can inform how the same mechanism may operate to render human cells resistant to treatment. Exposure of cells to small molecules and drug therapies imposes a strong selective pressure. As a result, cells rapidly acquire mutations in order to survive. These include resistant variants of the drug target as well as those that modulate drug transport and detoxification. To systematically explore how cells acquire drug resistance in an unbiased manner, rapid cost-effective approaches are required. Methotrexate, as one of the first rationally designed anticancer drugs, has served as a prototypic example of such acquired resistance. Known methotrexate resistance mechanisms include mutations that increase expression of the dihydrofolate reductase (DHFR) target as well as those that maintain function yet reduce the drug's binding affinity. Recent evidence suggests that target-independent, epistatic mutations can also result in resistance to methotrexate. Currently, however, the relative contribution of such unlinked resistance mutations is not well understood. To address this issue, we took advantage of Saccharomyces cerevisiae as a model eukaryotic system that combined with whole-genome sequencing and a rapid screening methodology, allowed the identification of causative mutations that modulate resistance to methotrexate. We found a recurrent missense mutation in SEC21 (orthologous to human COPG1), which we confirmed in 10 de novo methotrexate-resistant strains. This sec21 allele (S96L) behaves as a recessive, gain-of-function allele, conferring methotrexate resistance that is abrogated by the presence of a wild-type copy of SEC21 These observations indicate that the Sec21p/COPI transport complex has previously uncharacterized roles in modulating methotrexate stress.

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Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae.

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