title: Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics.
abstract: The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation.
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285;
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Olsen
authors.affiliation: §Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania Philadelphia, Pennsylvania 19104;
authors.forename: Xing-Jun
authors.initials: XJ
authors.lastname: Cao
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285;
authors.forename: Bomie
authors.initials: B
authors.lastname: Han
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285;
authors.forename: Lisa Hong
authors.initials: LH
authors.lastname: Chen
authors.affiliation: ¶Advanced Testing Laboratory, Cincinnati, Ohio, 45242.
authors.forename: Alexander
authors.initials: A
authors.lastname: Horvath
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285;
authors.forename: Timothy I
authors.initials: TI
authors.lastname: Richardson
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285;
authors.forename: Robert M
authors.initials: RM
authors.lastname: Campbell
authors.affiliation: §Epigenetics Program, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania Philadelphia, Pennsylvania 19104; nguyen_anh-quan_hannah@lilly.com bgarci@mail.med.upenn.edu.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
authors.affiliation: From the ‡Lilly USA, Lilly Research Laboratories, Indianapolis, Indiana 46285; nguyen_anh-quan_hannah@lilly.com bgarci@mail.med.upenn.edu.
authors.forename: Hannah
authors.initials: H
authors.lastname: Nguyen
mesh_terms: D020816:Amino Acid Motifs; D001549:Benzamides / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004938:Esophageal Neoplasms / Q000378:metabolism*; D000077277:Esophageal Squamous Cell Carcinoma; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D011759:Pyrrolidines / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D001549:Benzamides; C000600304:LLY-507; D020543:Proteome; D011759:Pyrrolidines; D011495:Histone-Lysine N-Methyltransferase; C512569:SMYD2 protein, human; D008239:Lysine