Date of Award:

5-2009

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Joan M. Hevel

Committee

Joan M. Hevel

Committee

Scott A. Ensign

Committee

Sean J. Johnson

Committee

Alvan C. Hengge

Committee

Brett A. Adams

Abstract

Protein arginine methyltransferases (PRMTs) posttranslationally modify protein arginine residues. Type I PRMTs catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) via methyl group transfer from S-adenosyl methionine onto protein arginine residues. Type II PRMTs generate MMA and symmetric dimethylarginine. PRMT-methylation affects many biological processes. Although PRMTs are vital to normal development and function, PRMT-methylation is also linked to cardiovascular disease, stroke, multiple sclerosis, and cancer.

Thus far, nine human PRMT isoforms have been identified with orthologues present in yeast, plants, and fish. PRMT1 predominates, performing an estimated 85% of all protein arginine methylation in vivo. Yet, the substrate specificity and catalytic mechanism of PRMT1 remain poorly understood.

Most PRMT1 substrates are methylated within repeating `RGG' and glycine-arginine rich motifs. However, PRMT1 also methylates a single arginine on histone-H4 that is not embedded in a glycine-arginine motif, indicating that PRMT1 protein substrates are not limited to proteins with `RGG' sequences. In order to determine if PRMT1 displays broader substrate selectivity, I first developed a continuous spectrophotometric assay to measure AdoMet-dependent methyltransferase activity. Using this assay and a focused peptide library based on a sequence derived from the in vivo PRMT1 substrate fibrillarin, we observed that PRMT1 demonstrates amino acid sequence selectivity in peptide and protein substrates. PRMT1 methylated eleven substrate motifs that went beyond the `RGG' and glycine-arginine rich paradigm, suggesting that the methyl arginine proteome may be larger and more diverse than previously thought.

PRMT1 methylates multiple arginine residues within the same protein to form protein-associated MMA and ADMA. Interestingly, ADMA is the dominant biological product formed and is a predictor of mortality and cardiovascular disease. To understand why PRMT1 preferentially forms ADMA in vivo, we began to 1) probe the mechanism of ADMA formation and 2) examine the catalytic role of certain active site residues and their involvement in ADMA formation. We found that PRMT1 dissociatively methylated several peptide substrates and preferred to methylate mono-methylated substrates over their non-methylated counterparts. Methylation of a multiple arginine-containing substrate was systematic (not random), a phenomenon that may be important biologically. All in all, our data help explain how PRMT1 generates ADMA in vivo.

Checksum

88ca8e9971bd21af00244d888ba3ecf3

Download

Share

COinS

Copyright for this work is retained by the student. If you have any questions regarding the inclusion of this work in the Digital Commons, please email us at .