Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
2002-05-20
2004-06-08
Weber, Jon P. (Department: 1651)
Radiant energy
Ionic separation or analysis
Methods
C250S282000
Reexamination Certificate
active
06747273
ABSTRACT:
BACKGROUND OF THE INVENTION
Post-translational modifications can have profound effects on the activities of proteins. One type of protein modification involves the transfer of methyl groups to a specific arginine residue resulting in the formation of dimethylarginine. This reaction is catalyzed by a protein arginine methyltransferase (PRMT). PRMTs have been implicated in a variety of processes, including cell proliferation, signal transduction, and protein trafficking. Arginine dimethylation has been implicated in such cellular processes as transcription (Chen et al. 1999
Science
284:2174-7), signal transduction (Bedford et al. 2000
J Biol Chem
275:16030-6; Mowen et al. 2001
Cell
104:731-41), nuclear export (Shen et al. 1998
Genes Dev
. 12:679-91), myelin integrity (Kim et al. 1997
Int J Biochem Cell Biol
29:743-51) and possibly antigenicity (Brahms et al. 2000
J Biol Chem
275:17122-9).
Methylation of arginine residues is one of many covalent modifications of eukaryotic proteins that occur concomitant with or shortly following translation. There are two classes of PRMTs that differ in the type of dimethylarginine that they produce. Type I PRMTs produce asymmetric N
G
, N
G
dimethylarginine residues, while type II PRMTs produce symmetric N
G
, N′
G
dimethylarginines (FIG.
1
). See also Gary et al. 1998
Prog. Nucleic Acid Res
. 61:65-133).
Most substrates for type I enzymes bind nucleic acid, usually RNA. These include heterogeneous nuclear RNA binding proteins (hnRNPs), which collectively contain 65% of the nuclear asymmetric dimethylarginine, as well as fibrillarin and nucleolin (Lischwe et al. 1985
J. Biol. Chem
. 260:14304-14310; Liu et al. 1995
Mol. Cell. Biol
. 15:2800-2808; Najbauer et al. 1993
J. Biol. Chem
. 268:10501-10509). Examples of physiological substrate of symmetric (type II) arginine methyltransferase include myelin basic protein, a major protein component of the myelin sheath, as well as the Sm proteins D1 and D3, which are components of small nuclear ribonucleoproteins.
Genes encoding rat (PRMT1), human (HRMT1L2), and yeast (RMT1) type I enzymes have been characterized (Gary et al. 1996
J. Biol. Chem
. 271:12585-12594; Henry et al. 1996
Mol. Cell. Biol
. 16:3668-3678; Lin et al. 1996
J. Biol. Chem
. 271:15034-15044; Scott et al. 1998
Genomics
48:330-340). The mammalian genes appear to be ubiquitously expressed in all tissues (Lin et al. supra; Scott et al. supra; Tang et al. 1998
J. Biol. Chem
. 273:16935-16945).
Type I enzymes have been implicated in a variety of processes, including cell growth control, signal transduction, and protein trafficking. The enzymes preferentially methylate motifs rich in arginine and glycine (RGG boxes), a common feature of the RNA binding domains of hnRNPs (Liu et al. 1995
Mol. Cell. Biol
. 15:2800-2808). Arginine-methylated hnRNP A1, but not Hrp1p, has lower affinity for RNA than the native protein, suggesting a potential mechanism for modulation of protein-RNA interactions. Levels of protein methylarginine may change in response to extracellular stimuli under circumstances in which biological responses are also suppressed by methyltransferase inhibitors. These include nerve growth factor-induced neurite outgrowth in PC12 cells and mitogenic responses of lipopolysaccharide-treated B cells.
Interactions between the PRMT1 enzyme and potential signaling components have also emerged from yeast two-hybrid screens. The immediate-early gene product TIS21 (BTG2) and the leukemia-associated gene product BTG1 interact with PRMT1 and can modulate its enzymatic activity in vitro. TIS21 and BTG1 both belong to a family of mitogen-induced proteins implicated in negative regulation of the cell cycle. PRMT1 also binds to the cytoplasmic domain of the IFNAR1 chain of the alpha beta interferon receptor, while growth-inhibitory effects of interferon were suppressed by antisense oligonucleotides directed against the methyltransferase. Finally, a novel arginine methyltransferase (CARM1) associates with p160 coactivators and serves as a secondary coactivator of nuclear hormone receptors.
Other studies have identified a role for arginine methylation in protein trafficking. Shuttling of the yeast hmRNP-related proteins Np13p and Hrp1p between the nucleus and cytoplasm requires methylation by the Hmt1p methyltransferase. The human enzyme complements the shuttling defect, suggesting functional conservation between the two enzymes. Nuclear translocation of the large form of basic fibroblast growth factor may also depend on arginine methylation. In the presence of a methyltransferase inhibitor, basic fibroblast growth factor was not methylated and the protein did not localize to the nucleus.
The prevalence of N
G
,N
G
-dimethylarginine in RNA binding proteins and conservation among protein arginine N-methyltransferases underscore the potential biological importance of this posttranslational modification. However, a major issue arguing against a dynamic role for the type I enzymes in cell regulation concerns the possibility that arginine methylation is both constitutive and irreversible. While most substrates have not been characterized, some are known to exist only in a fully methylated state. Moreover, no demethylase capable of removing dimethylarginine residues has been identified and in the case of histones, turnover of dimethylarginine accompanies protein degradation.
Efforts to understand the biochemical function of mammalian arginine methyltransferases are complicated by several factors, including the existence of multiple enzymes and the fact that methyltransferase inhibitors nonspecifically target multiple processes in which S-adenosylmethionine serves as a methyl donor. In yeast, functional studies of arginine methylation have benefited greatly from genetic approaches that have led to the isolation of cells deficient in the enzyme. In principle, gene targeting strategies could be used for similar studies of the mammalian enzymes, assuming that the proteins are not required for cell viability.
It is likely that the different classes of PRMTs will regulate different cellular targets and pathways as the known substrates for class I and class II PRMTs appear to be distinct from each other. It is an object of the present invention to provide methods and systems that can detect methylarginine residues in polypeptide samples, and can differentiate between symmetric and asymmetric dimethylation.
SUMMARY OF THE INVENTION
One aspect of the present invention provides a method for identifying the structure of a dimethylarginine, e.g., to distinguish between symmetrical and asymmetrical dimethylarginine residues. In certain embodiments, the subject method includes obtaining, by mass spectroscopy, a neutral loss spectra of a peptide containing a dimethylarginine. From the neutral loss spectra, the neutral loss, if any, of monomethylamine, dimethylcarbodiimide, and/or the neutral loss of dimethylamine is determined. Neutral loss of monomethylamine and dimethylcarbodiimide indicates the presence of a symmetrically dimethylated arginine residue. On the other hand, neutral loss of dimethylamine indicates the presence of a asymmetrically dimethylated arginine residue.
Another aspect of the present invention provides a method for identifying dimethylarginine residues in a test peptide. In general, the method includes identifying the presence of dimethylarginine residues from mass spectra of a sample peptide obtained under conditions in which the spectra reveal mass modification by methylation, if any, of arginine residue in the sample peptide. For dimethylarginine residue which are identified, the method also ascertains the nature of the methylation by determining if a neutral loss spectra of the sample peptide shows one or both of neutral loss of monomethylamine, dimethylcarbodiimide, and/or neutral loss of dimethylamine. Neutral loss of monomethylamine and of dimethylcarbodiimide indicates the presence of a symmetrically dimethylated arginine residue whereas neutral loss of dimethylamine indicates the presence of a asymmetrically d
Brame Cynthia J.
McBroom Linda
MDS Proteomics Inc.
Ropes & Gray LLP
Vincent Matthew P.
Weber Jon P.
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