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Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.aging ͉ label-free ͉ proteomics ͉ small molecules

The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione Stransferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form N G -monomethyl and N G ,N Gdimethylarginine (asymmetric). We term the proteinarginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1," for protein-arginine methyltransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.The protein products of the immediate-early/primary response genes are thought to act as "third messengers," mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation . Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitricoxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 1995).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 1991) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 1991). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 1994). Moreover, the half-life of both mitogen-and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 1994). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and ...

No abstract

Methylation is one of the many post-translational modifications that modulate protein function. Although asymmetric N G ,N G -dimethylation of arginine residues in glycine-arginine-rich domains of eucaryotic proteins, catalyzed by type I protein arginine N-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 S-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 rmt1 mutant that lacks type I arginine methyltransferase activity; PRMT3 is thus a functional type I protein arginine N-methyltransferase. However, rat PRMT1 and PRMT3 glutathione S-transferase fusion proteins have distinct enzyme specificities for substrates present in both hypomethylated rmt1 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.Protein function is often modulated by post-translational covalent modifications. One such modification is the N-methylation of the side chain of guanidino arginine residues (1-3). Type I protein arginine N-methyltransferase (PRMT) 1 enzymes catalyze the formation of asymmetric N G ,N G -dimethylarginine residues in proteins by transferring methyl groups from Sadenosylmethionine (AdoMet) to the guanidino nitrogen atoms of arginine residues. These enzymes generally methylate arginines found in RGG consensus sequences in the context of GAR (glycine and arginine-rich) domains (1, 4 -6). The first cDNA for a mammalian type I PRMT enzyme to be cloned, PRMT1, was identified by a yeast two-hybrid screen as a rat cDNA encoding a protein that interacts with the product of the mammalian TIS21 immediate-early gene (7). TIS21 (also known as PC3 and BTG2) is a member of a family proteins (TIS21, BTG1, BTG3, and TOB) thought to be involved in negative control of the cell cycle (8, 9). Association of recombinant TIS21 protein with a fusion protein between glutathione S-transferase and rat PRMT1, GST-PRMT1, modulates PRMT1 activity in vitro (7), suggesting that interaction between transiently induced TIS21 (10 -14) and constitutively expressed PRMT1 (7) may modulate PRMT1 enzyme activity in vivo. ...