Developing Novel Arginine Biased Arrays for Epigenetic Research

DESCRIPTION provided by applicant Histone modifications play causal roles in regulating chromatin structure and nuclear processes in general There is clear interplay between different epigenetic histone modifying enzymes affecting the activity of nuclear and chromatin associated proteins which leads to specific cellular effects such as gene transcription or DNA repair Although histone lysine methylation has garnered much attention in the research community it is clear that arginine methylation is an essential biological process and perturbation can result i disease e g cancer and neurodegenerative diseases However there is a lack of cutting edge tools to investigate the role of arginine methylation in biology and human disease For example the absence of array based proteomic platforms focused on arginine methylation presents a roadblock in identifying and understanding the function of arginine methylases arginine demethylases and methylarginine binding proteins This study will focus on the development of a novel screening platform that will enable the identification of the substrates of arginine methylation characterization of effector proteins that bind specific methylated arginine motifs and characterization of molecular reagents to study epigenetic pathways involving arginine methylation Each of these research applications will provide academic and pharmaceutical investigators for the first time a set of tools to facilitate studies defining therole of arginine methylation in the context of signal transduction and chromatin in normal and diseased cells Specifically we will focus our efforts on developing a random fixed arginine and methylarginine Oriented Peptide Array Library OPAL which will allow the rapid screening of methylarginine binding proteins effectors and protein arginine methyltransferases enzymes for their substrate or binding specificities In order to interrogate all effectors and enzymes we will synthesize four oriented KGXXXRXXXGK biotin peptide libraries X represents random amino acids any amino acid except cysteine in which the central R is unmethylated central R is mono methylated central R is asymmetric dimethylarginine and central R is symmetric dimethylarginine The OPAL platform will be validated by determining the binding specificities of known methylarginine effector proteins SMN TDRD SND and TDRKH The OPAL platform will also be used to map the epitope recognition motifs of a methylarginine specific antibody F In addition we will use this platform to confirm the established consensus substrates recognition sequences for the methyltransferases PRMT and CARM Success will be achieved and progression to Phase justified if we observe that the consensus targets for the effectors Aim and enzymes Aim are consistent with published results Given that these proteins will be screened against high complexity arrays the results of the OPAL screen will very likely reveal novel targets for these effectors and enzymes which will provide new insights into the function of these factors In the long term EpiCypher will create a suite of OPAL platforms that include PTMs other than arginine methylation such as lysine acetylation which will enable the company to serve as a valuable resource to the large portion research community that has an interest in signal transduction and epigenetics More generally it is becoming clear that arginine methylation is not only found on histones but on a large number of non histone proteins as well Thus the proposed OPAL platforms would for the first time provide the resources necessary to define the function of andquot orphanandquot effectors and enzymes and help the research community predict which non histone proteins are possible substrates and interacting partners for enzymes and effectors that have been traditionally associated with the histone code The OPAL platforms will serve as an important tool to help us understanding the biological roles of human arginine methylome PUBLIC HEALTH RELEVANCE For cells to respond to environmental cues they make use a process called signal transduction This process is mediated by a vast array of post translational modifications PTMs which initiate at the cell membrane travels through the cytoplasm into the nucleus and culminates in the deposition of chromatin modifications that epigenetically regulate cellular function The characterization of the enzymes that catalyze these modifications and the proteins that recognize and transduce specific PTMs to biological outcomes remains largely underdeveloped particularly in the field of protein methylation Given the association of aberrant PTM dynamics with disease there is a compelling need to develop innovative tools to characterize proteins involved in signaling and epigenetic regulation We propose to develop a new technology utilizing protein arrays for high throughput coverage of the human methylome so that the link between protein methylation and disease can be more effectively investigated These arrays will also be useful for identifying new therapeutic targets by revealing the binding and substrate specificities of `orphanandapos effectors and methyltransferases whose dysregulation is linked to human disease