| Autor: |
Mendoza M; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Mendoza M; Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States., Lubrino T; Schmid College of Science and Technology, Keck Center for Science and Engineering, Chapman University, 450 N. Center Street, Orange, California 92866, United States., Briski S; Schmid College of Science and Technology, Keck Center for Science and Engineering, Chapman University, 450 N. Center Street, Orange, California 92866, United States., Osuji I; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Cuala J; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Ly B; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Ocegueda I; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Peralta H; Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, California 90033, United States., Garcia BA; Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, United States., Zurita-Lopez CI; Schmid College of Science and Technology, Keck Center for Science and Engineering, Chapman University, 450 N. Center Street, Orange, California 92866, United States. |
| Abstrakt: |
We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α. |
