Identifying Novel Substrates by Specificity Profile Analysis of Protein Lysine Methyltransferases

  • In the cell nucleus the DNA binds to histone proteins and forms a compact structure called chromatin. Both the components of chromatin are subjected to several post-translational modifications which regulate the gene expression. Enzymes (histone acetyltransferases and histone lysine methyltransferases) known to methylate histone protein have also been shown to act on non-histone proteins and methylation and acetylation of non-histone proteins carries many important biological signals, but not many non-histone methylation substrates of protein lysine methyltransferases are known. In this study we have characterised the substrate specificity of histone lysine methyltransferases and based on the specificity data, we identified several novel histone and non-histone substrates. The NSD1 enzyme is a histone lysine methyltransferase enzyme. Mutations of this protein cause the Sotos syndrome. We studied the substrate specificity of NSD1 using the H3 (30-50) sequence as a template. With the obtained consensus sequence motif we identified several novel histone and non-histone NSD1 substrates. We showed that NSD1 could not methylate H4K20, instead it methylates K44 in H4 protein, which is in agreement with our specificity profile. For the first time we showed NSD1 methylates H1 proteins in a variant specific manner; NSD1 methylates K168 in H1.2, H1.3 and H1.5 proteins but not in H1.4. Apart from the novel histone substrates, we also identified several non-histone proteins containing the NSD1 consensus sequence motif and confirmed methylation of 45 novel non-histone peptides and of the (ATRX and Probable U3 Small Nucleolar RNA-associated Protein) proteins. Based on the candidate screening approach, we also identified an automethylation site in NSD1 and confirmed the loss of methylation signal with the corresponding predicted lysine; NSD1-K1769R mutant protein. We also show that the NSD1 Sotos SET domain mutants impair its methyltransferase activity and thus establish a possible deregulation of signalling networks in Sotos patients. SUV39H1 is a H3K9 methyltransferase enzyme which plays a vital role in the formation of heterochromatin. We derived the specificity profile of this enzyme and showed that it mainly recognises an 'RK' motif corresponding to R8 and K9 in the H3 tail. In addition, lysine 4 of the H3 tail is very important for substrate recognition. With the derived specificity profile of SUV39H1 we identified several novel non-histone peptide substrates and confirmed methylation of RAG2, SET8, Jumonji and Sex comb on midleg protein 2 proteins at the protein level, albeit methylation on Jumonji and Sex comb on midleg protein 2 were weak. Similar to the K4 recognition on H3 tail, we have also observed lysine at -5 position with respect to the target lysine is important for SUV39H1 to methylate the newly identified targets RAG2 and SET8. We have shown that methylation of RAG2 alters its sub-nuclear localization and found that the JMJD2A tandem tudor domain interacts with the newly identified targets in a methyl specific manner. SET8 is a H4K20 specific mono-methyl transferase which acts preferentially on H4 integrated into nucleosomes. By employing peptide arrays we have shown that it has long recognition sequence motif covering 7 amino acids (R17H18R19K20V21L22R23). Based on the derived specificity profile, we identified only 4 potential non-histone substrate proteins. But after relaxing the specificity profile we identified several proteins and showed methylation of 22 non-histone peptides. However, apart from p53 and H4 proteins, none of the identified targets were methylated at the protein level. Celluspot analysis revealed that symmetric and asymmetric methylation on R17 of H4 tail further inhibits methylation on H4K20, while other modifications on K16 and R19 affected H4K20 methylation partially. In summary, our specificity analysis results and methylation assays demonstrate that SET8 as a highly specific histone H4 methyltransferase enzyme. The SMYD family of protein methyltransferases is a group of enzymes which are unique for having a characteristic MYND domain inserted into the catalytic SET domain. The SMYD proteins have roles in the regulation of the cell cycle and important development pathways such as heart and muscle differentiation. A member of this family, SMYD2, is an uncharacterised histone lysine methyltransferase enzyme, which has been shown to methylate both H3K4 and H3K36. In addition, it was also found to methylate one non-histone substrate (p53) and, thereby, repress its activity. Here we applied peptide arrays and derive a specificity profile for SMYD2 via two approaches: a "best target" approach using a p53 peptide as template and an unbiased random approach. Results revealed that SMYD2 possesses a strong preference for a 'LK' or 'FK' motif. With the derived sequence motif, we have identified 40 novel peptide substrates from human proteins and for 8 proteins we showed methylation at the protein level and confirmed the predicted target lysine by mutagenesis. Experiments to show cellular methylation and to understand the possible downstream consequences of methylation of some of the identified non-histone proteins are in progress.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Srikanth Kudithipudi
Referee:Albert Jeltsch, Mike Schutkowski, Sebastian Springer
Advisor:Albert Jeltsch
Persistent Identifier (URN):urn:nbn:de:101:1-201307119487
Document Type:PhD Thesis
Date of Successful Oral Defense:2011/08/11
Date of First Publication:2011/11/22
Full Text Embargo Until:2012/12/31
PhD Degree:Biochemistry
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QD Chemistry / QD71-142 Analytical chemistry
Call No:Thesis 2011/39

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