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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA032949-16
Application #
2683422
Study Section
Biochemical Endocrinology Study Section (BCE)
Program Officer
Mohla, Suresh
Project Start
1982-08-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
16
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Nebraska Medical Center
Department
Type
Schools of Medicine
DUNS #
City
Omaha
State
NE
Country
United States
Zip Code
68198
Wilken, Jason A; Matsumoto, Kikuko; Laughlin, Lisa S et al. (2002) Comparison of chorionic gonadotropin expression in human and macaque (Macaca fascicularis) trophoblasts. Am J Primatol 56:89-97
Darling, R J; Wilken, J A; Miller-Lindholm, A K et al. (2001) Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits. J Biol Chem 276:10692-9
Darling, R J; Wilken, J A; Ruddon, R W et al. (2001) Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit. Biochemistry 40:577-85
Darling, R J; Ruddon, R W; Perini, F et al. (2000) Cystine knot mutations affect the folding of the glycoprotein hormone alpha-subunit. Differential secretion and assembly of partially folded intermediates. J Biol Chem 275:15413-21
Miller-Lindholm, A K; Bedows, E; Bartels, C F et al. (1999) A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient. Endocrinology 140:3496-506
Silva, R A; Sherman, S A; Keiderling, T A (1999) Beta-hairpin stabilization in a 28-residue peptide derived from the beta-subunit sequence of human chorionic gonadotropin hormone. Biopolymers 50:413-23
Muyan, M; Ruddon, R W; Norton, S E et al. (1998) Dissociation of early folding events from assembly of the human lutropin beta-subunit. Mol Endocrinol 12:1640-9
Ruddon, R W; Bedows, E (1997) Assisted protein folding. J Biol Chem 272:3125-8
Miller-Lindholm, A K; LaBenz, C J; Ramey, J et al. (1997) Human chorionic gonadotropin-beta gene expression in first trimester placenta. Endocrinology 138:5459-65
Feng, W; Bedows, E; Norton, S E et al. (1996) Novel covalent chaperone complexes associated with human chorionic gonadotropin beta subunit folding intermediates. J Biol Chem 271:18543-8

Showing the most recent 10 out of 36 publications