Jie Tang

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.

Antioxidants were administered to diabetic rats and experimentally galactosemic rats to evaluate the ability of these agents to inhibit the development of diabetic retinopathy. Alloxan diabetic rats and nondiabetic rats that were fed 30% galactose randomly received standard diets or the diets supplemented with ascorbic acid and alpha-tocopherol (vitamins C+E diet) or a more comprehensive mixture of antioxidants (multi-antioxidant diet), including Trolox, alpha-tocopherol, N-acetyl cysteine, ascorbic acid, beta-carotene, and selenium. Diabetes or galactose feeding of at least 12 months resulted in pericyte loss, acellular capillaries, and basement membrane thickening. Compared with diabetic controls, the development of acellular capillaries was inhibited by 50% (P < 0.05) in diabetic rats that received supplemental vitamins C+E, and the number of pericyte ghosts tended to be reduced. The vitamins C+E supplement had no beneficial effect in galactosemic rats, but these rats consumed only approximately half as much of the antioxidants as the diabetic rats. The multi-antioxidant diet significantly inhibited ( approximately 55-65%) formation of both pericyte ghosts and acellular capillaries in diabetic rats and galactosemic rats (P < 0.05 vs. controls), without affecting the severity of hyperglycemia. Parameters of retinal oxidative stress, protein kinase C activity, and nitric oxides remained elevated for at least 1 year of hyperglycemia, and these abnormalities were normalized by multi-antioxidant therapy. Thus, long-term administration of antioxidants can inhibit the development of the early stages of diabetic retinopathy, and the mechanism by which this action occurs warrants further investigation. Supplementation with antioxidants can offer an achievable and inexpensive adjunct therapy to help inhibit the development of retinopathy in diabetes.

Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric<i>N</i>^G,<i>N</i>^G-dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine<i>N</i>-methyltransferases (PRMT), has been known for some time, members of this enzyme class have only recently been cloned. The first example of this type of enzyme, designated PRMT1, cloned because of its ability to interact with the mammalian TIS21 immediate-early protein, was then shown to have protein arginine methyltransferase activity. We have now isolated rat and human cDNA orthologues that encode proteins with substantial sequence similarity to PRMT1. A recombinant glutathione <i>S</i>-transferase (GST) fusion product of this new rat protein, named PRMT3, asymmetrically dimethylates arginine residues present both in the designed substrate GST-GAR and in substrate proteins present in hypomethylated extracts of a yeast<i>rmt1</i> mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine<i>N</i>-methyltransferase. However, rat PRMT1 and PRMT3 glutathione <i>S</i>-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated<i>rmt1</i> yeast extract and hypomethylated RAT1 embryo cell extract. TIS21 protein modulates the enzymatic activity of recombinant GST-PRMT1 fusion protein but not the activity of GST-PRMT3. Western blot analysis of gel filtration fractions suggests that PRMT3 is present as a monomer in RAT1 cell extracts. In contrast, PRMT1 is present in an oligomeric complex. Immunofluorescence analysis localized PRMT1 predominantly to the nucleus of RAT1 cells. In contrast, PRMT3 is predominantly cytoplasmic.