Associate Professor, Department of Chemistry
Kansas State University
Engineering Human NRMT1 for its Substrate Profiling (2015-16)
NRMT1 is a newly discovered protein methyltransferase that has been suggested to play important roles in oncogenesis and progression of several major cancers. Additionally, it is overexpressed in patients with colon cancers and the level increases as the cancer advances. This proposal aims to engineer NRMT1 to profile its substrates so that the roles of NRMT1 in tumorigenesis can be revealed.
Methylation is a commonly observed protein posttranslational modification that plays important roles in a variety of signaling pathways and epigenetic regulation. Defects in the methylation have been demonstrated to cause various major cancers. Protein methylation is catalyzed by protein methyltransferases (PMTs) using S-adenosyl-L-methionine (SAM) as the methyl donor. So far, three classes of PMTs have been discovered, which include protein lysine methyltransferases (PKMTs), protein arginine methyltransferases (PRMTs), and protein N-terminus methyltransferases. While mechanisms and functions of PKMT and PRMT have been studied in detail, knowledge of protein N-terminus methyltransferases is largely unexplored. The α-N-terminal RCC1 methyltransferase (NRMT, now renamed as NRMT1) discovered in 2010 is the first eukaryotic protein N-terminus methyltransferase, which methylates proteins containing a N-terminal sequence consisting of MXPK. Upon the cleavage of end methionine, the N-terminal α-NH2 is methylated to form mono-, di-, and trimethylated products. Based on the conserved sequence, more than 300 human proteins have been predicted to be NRMT1 substrates, in which ~40 proteins are related to cancers. Four of them have been validated in vitro, which includes SET, retinoblastoma protein (Rb), damaged DNA-binding protein 2 (DDB2), and RCC1. These validated NRMT1 targets are known to be involved in the oncogenesis and progression of various cancers. Additional NRMT1 substrates such as centromere protein A (CENP-A) that do not contain the conserved sequence have also been discovered. Dysfunction of the CENP-A are linked to colon cancers.
In addition, NRMT1 is found to overexpress in patients with colon cancers and the level of expression increases as the cancer progresses. Thus, NRMT1 may serve as a new therapeutic target or biomarker of cancers. The ultimate goal of this research is to characterize the roles of NRMTs (including isoforms of NRMT1 and NRMT2) and α-N-methylation in maintaining the normal cellular states so that their links with cancers can be revealed. The current one-year pilot proposal will focus on engineering NRMT1 to identify its substrates using modified SAM analogs and click-chemistry.
Expression and purification of an obesity-important enzyme hGOAT in E.coli (2012-13)
Ghrelin O-acyltransferase (GOAT) is the only enzyme responsible for the formation of biologically active acylated ghrelin that has been linked to obesity. Sufficient amount of human GOAT (hGOAT) is a prerequisite to study its molecular mechanisms, which will lead to new treatment for obesity. Development of expressing and purifying hGOAT heterologously in E. coli will ensure that enough enzymes can be obtained.
Ghrelin is a peptide hormone that consists of 28 amino acids and regulates our appetite and adiposity in humans. The active acylated ghrelin that has an octanoyl group attached to Ser-3 will increase the appetite and slow down metabolism, causing the body to feel hungrier and to burn fat slowly, thus predisposing the person to obesity. The formation of acylated ghrelin is catalyzed by ghrelin O-acyltransferase (GOAT), which was discovered recently in 2008. The US is currently facing obesity pandemic, which costs more than 150 billion dollars per year.
Obesity has become a major contributing factor in human deaths from diabetes, cancers, and cardiovascular diseases. Therefore, it is more urgent than ever for us to develop effective ways to treat obesity. Mouse model has suggested that inhibition of GOAT could provide promising therapeutics for treating obesity. However, studies of human GOAT (hGOAT) are hampered due to the insufficient amount of pure enzymes that can be obtained.
The goal of this pilot project is to express and purify membrane protein hGOAT heterologously from Escherichia coli. The specific aims are: (1) To develop protocols for the overexpression of hGOAT in E. coli with high yields. Strategies will be developed on how to design constructs and to choose the appropriate expression promoters and host strains. In order to increase the expression levels of soluble hGOAT, focus will be placed on the design of fusion proteins and the reasons why they are selected are described; and (2) To develop protocols for the renaturation of hGOAT from inclusion bodies. Two methods will be employed to obtain the soluble active hGOAT from its inclusion bodies. Different detergents will also be tested for extracting hGOAT and maintaining its activity. Although this project will involve lots of trials and errors, we believe, by working closely with the experts from COBRE facilities (Protein Production Group and Protein Structure Laboratory at KU), it will lead to obtain sufficient amount of active hGOAT in soluble form from the E. coli recombinant expression system.