Investigation of the specificity of protein lysine methyltransferases

  • We followed the specificity profile based method to identify the best substrate and/or to find novel non-histone targets of the MLL1, NSD2 and SET2 protein lysine methyltransferases. Specificity profile is the specific amino acid sequence motif recognized as substrate by the respective methyltransferase's active site. The MLL1 methyltransferase plays its role in development and leukemia. It exists in vivo, in a multisubunit complex. We showed that MLL1 recognizes a five amino acid residue sequence in which the target lysine is in the center. We investigated the specificity profile to find possible non-histone targets for MLL1, first at the peptide level and then at the protein level in vitro. We found one novel non-histone target at the protein level, the MLL1 protein itself. This auto-methylation of MLL1 could have functional consequences. Of the two MLL1 constructs used in this study, the shorter MLL13812 was significantly more catalytically active than the longer MLL13745. The NSD2 methyltransferase plays a role in some types of cancer. We showed that NSD2 methylates the peptides H3 (1-20), H3 (15-35), H3 (28-48), H4 (38-52) and H1.5 (161-175) which harbour the potential methylation sites H3K4, H3K9, H3K18, H3K27, H3K36, H4K44 and H1.5K168, but it did not methylate the peptide H4 (10-30) which harbours the H4K20 site. Since, the NSD2 methyltransferase was not specific for any of the histone tail peptides, we devised an unbiased random peptide array approach to investigate its substrate specificity and/or non-histone targets. We found that NSD2 methylated those substrates in which a hydrophobic residue preceded the target lysine. This led us to find a novel and stronger histone site, K168 of histone H1.5 than the original K36 of histone H3 in vitro at the peptide level. The methylation of H1.5K168 could have roles in the regulation of gene expression. It may have connections with nucleosome compaction. The SET2 methyltransferase has tumor suppressive functions. We derived the substrate specificity profile for SET2 using peptide arrays. We showed that SET2 recognizes a six amino acid residue sequence in which the target lysine is at position 4. We report that SET2 is a robust H3K36 mono- and di-methyltransferase at the peptide level in vitro. We found three non-histone targets for SET2 at the peptide level in vitro. We report that lysine 119 of Chloride intracellular channel 1 (CLIC1, NCC27), lysine 2245 of A kinase anchoring protein 13 and lysine 683 of ASHIL (another H3K36 methyltransferase) are methylated by SET2 at the peptide level in vitro.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Qazi Muhammad Raafiq
Referee:Albert Jeltsch, Sebastian Springer, Muhammed Naseem
Advisor:Albert Jeltsch
Persistent Identifier (URN):urn:nbn:de:101:1-201307119383
Document Type:PhD Thesis
Language:English
Date of Successful Oral Defense:2013/02/22
Date of First Publication:2013/04/12
PhD Degree:Biochemistry
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QP Physiology / QP501-801 Animal biochemistry / QP525-801 Special substances / QP550-801 Organic substances / QP551-619 Proteins, amino acids, etc. / QP561-563 Amino acids / QP562.A-Z Constituents of proteins, A-Z / QP562.L8 Lysine
Call No:Thesis 2013/3

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