Biomedical subjects

Donghang Cheng

Publications and source records attributed to Donghang Cheng.

6 recordsLinked to original sources

PubMed2007

The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.

The coactivator-associated arginine methyltransferase CARM1 is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening approach for this purpose and identified CA150, SAP49, SmB, and U1C as splicing factors that are specifically methylated by CARM1. We further showed that CA150, a molecule that links transcription to splicing, interacts with the Tudor domain of the spinal muscular atrophy protein SMN in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicates this enzyme in the regulation of alternative splicing and points toward its involvement in spinal muscular atrophy pathogenesis.

Alternative Splicing↗

PubMed2006

The activity and stability of the transcriptional coactivator p/CIP/SRC-3 are regulated by CARM1-dependent methylation.

The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-(3)H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1(-/-) mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxy-terminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.

Amino Acid Sequence↗

PubMed2006

Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene.

The Cyclin E1 gene (CCNE1) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1, along with their transcriptional target CCNE1. All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

Activin Receptors, Type I↗

PubMed2004

Small molecule regulators of protein arginine methyltransferases.

Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.

Arginine↗

PubMed2004

Techniques in protein methylation.

Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening.

Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.

Amino Acid Sequence↗