Author

Mahta Nili

Date

September 2011

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

Iron homeostasis is a tightly regulated process that is maintained by several key factors that ensure proper iron distribution and storage throughout the body. Disruption in homeostatic processes can lead to inadequate iron levels, which impairs hemoglobin synthesis and red cell production, while chronic excess of iron causes tissue damage. Excessive iron accumulation in organs such as the liver, heart and pancreas is a hallmark characteristic of hereditary hemochromatosis. Mutations in RGMc/HJV cause juvenile hemochromatosis (JH), an early onset aggressive form of this disorder. Patients with juvenile hemochromatosis, and RGMc knockout mice, have reduced expression of the critical iron-regulatory peptide, hepcidin. Although RGMc plays a key role in the regulation of iron homeostasis, many of the mechanisms of RGMc actions are unknown. This dissertation explores RGMc actions through a structure-function approach. More specifically, functional interactions with members of the bone morphogenetic family (BMP) are explored, and disulfide mapping studies provide insight into the three-dimensional structure of RGMc isoforms. Several significant findings are described. First, RGMc is demonstrated to be a potent antagonist of BMP-mediated signaling and gene expression with a specificity that encompasses two distinct BMP sub-families. Full-length and PC-cleaved soluble RGMc isoforms, and selected mutants that retain the ability to bind BMPs, are shown to antagonize the actions of both BMP2 and BMP6. Secondly, by using a newly-described MS3 disulfide bond mapping method combining ETD and CID, a definitive map has been established of several disulfide bonds of the growth factor binding protein, insulin-like growth factor binding protein-5 (IGFBP-5), representing the first use of this method to define disulfide bonds of a protein with unknown structure. Finally, through the same MS3-based investigative approach coupled with ab initio molecular modeling, two disulfide bonding patterns representing two distinct oxidation states of RGMc are uncovered. In summary, the structure-function studies reported in this dissertation provide major insight into the iron-regulatory mechanisms of action of RGMc.

Identifier

doi:10.6083/M4668B6M

School

School of Medicine

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