The overall experimental questions to be addressed during the period of this grant are: What are the structural determinants for the correct folding of the subunits of the glycoprotein hormone hCG and is there any clinical relevance to the production of misfolded or unfolded hCG subunits? The Specific Aims of this proposal, designed to provide some answers to the questions posed, are: (1.) To determine the effect of mutations in key folding domains of the hCG-beta subunit on the folding of the subunit and its ability to assembly with the alpha subunit to form a biologically active heterodimer, (2.) to determine if there is a disulfide bond rearrangement during the folding of the hCG-beta subunit, (3.) to determine the folding pathway of the common glycoprotein hormone alpha subunit, employing techniques similar to those developed in our laboratory to study the folding of the hCG-beta subunit, (4.) to determine whether there are genetic polymorphisms of the hCG-beta gene in the human population and whether misfolded or unfolded subunits are detectable in the serum of cancer patients, and (5.) to determine the structure by NMR and/or crystallography of hCG-beta folding intermediates identified in the in vitro folding pathway. Methods to carry out the Specific Aims include (1.) The use of site-directed mutagenesis to create alterations in key motifs involved in the native structure of the hCG-beta subunit, (2.) use of specific proteinases and introduction of cyanogen bromide cleavage sites to determine the rearrangements of disulfide bond patterns during folding of the beta subunit, (3.) pulse-chase, immunopurification, HPLC separation, and tryptic peptide mapping of radiolabeled beta-mutant forms and alpha subunit forms to follow folding and subunit assembly, (4.) analysis of DNA from human lymphocytes by single-stranded conformational polymorphism (SSCP) to look for genetic polymorphisms, and (5.) use of NMR and X-ray crystallography techniques to carry out structural analysis of hCG-beta subunit folding intermediates. The health related significance relates to the fact that misfolded or unfolded forms of the hCG-beta subunit appear to be present in serum samples of women with ovarian, cervical, and endometrial cancer. Thus, characterization of these forms may provide a more sensitive diagnostic marker for these cancers. Moreover, understanding the fundamental mechanisms of protein folding has broad implications in biology and medicine. Defects in protein folding and processing occur in a number of human diseases, including alpha/1- antitrypsin deficiency, Alzheimer's disease, cystic fibrosis, and cancer. Thus, what we learn about protein folding in our system may be applicable to our understanding of a number of disease processes.
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