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Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme.

Weirich S, Kudithipudi S, Kycia I, Jeltsch A - Clin Epigenetics (2015)

Bottom Line: Somatic mutations in epigenetic enzymes are frequently found in cancer tissues.Expression of Y4884C leads to aberrant H3K4me3 formation in cells.Our data show that different somatic cancer mutations of MLL3 affect the enzyme activity in distinct and opposing manner highlighting the importance of experimentally studying the effects of somatic cancer mutations in key regulatory enzymes in order to develop and apply targeted tumor therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Faculty of Chemistry, Stuttgart University, Pfaffenwaldring 55, Stuttgart, 70569 Germany.

ABSTRACT

Background: Somatic mutations in epigenetic enzymes are frequently found in cancer tissues. The MLL3 H3K4-specific protein lysine monomethyltransferase is an important epigenetic enzyme, and it is among the most recurrently mutated enzymes in cancers. MLL3 mainly introduces H3K4me1 at enhancers.

Results: We investigated the enzymatic properties of MLL3 variants that carry somatic cancer mutations. Asn4848 is located at the cofactor binding sites, and the N4848S exchange renders the enzyme inactive. Tyr4884 is part of an aromatic pocket at the active center of the enzyme, and Y4884C converts MLL3 from a monomethyltransferase with substrate preference for H3K4me0 to a trimethyltransferase with H3K4me1 as preferred substrate. Expression of Y4884C leads to aberrant H3K4me3 formation in cells.

Conclusions: Our data show that different somatic cancer mutations of MLL3 affect the enzyme activity in distinct and opposing manner highlighting the importance of experimentally studying the effects of somatic cancer mutations in key regulatory enzymes in order to develop and apply targeted tumor therapy.

No MeSH data available.


Related in: MedlinePlus

Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).
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Fig4: Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).

Mentions: To further confirm the change in substrate methylation state preference and product pattern of the Y4884C mutant, unmethylated and monomethylated H3K4 peptides were incubated in solution with MLL3 wild-type and the variants in the presence of unlabeled AdoMet. The samples were collected at the defined time intervals, and the methylation was analyzed by mass spectrometry. Wild-type MLL3 showed fast monomethylation of the H3K4me0 peptide, followed by weak dimethylation after long incubation times (Figure 4A, upper panel). No methylation was detectable with the H3K4me1 peptide even after the long incubation times (Figure 4B, upper panel) which suggests that the MLL3 wild-type protein is inactive on the H3K4me1 peptide and it is majorly a H3K4 monomethyltransferase. The weak dimethylation of the H3K4me0 peptide likely is due to a processive methylation of bound peptide after the first methylation cycle. In contrast, the Y4884C variant exhibited only weak activity on the H3K4me0 peptide (Figure 4A, lower panel). However, this did not result in an accumulation of monomethylated products indicating that they were rapidly converted into the di- and trimethylated state. Strikingly, Y4884C showed strong methylation activity on the H3K4me1 peptide resulting in the generation of dimethylated and later trimethylated products (Figure 4B, lower panel).Figure 4


Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme.

Weirich S, Kudithipudi S, Kycia I, Jeltsch A - Clin Epigenetics (2015)

Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).
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Fig4: Substrate specificity and product pattern of MLL3 protein variants. H3K4 unmethylated (H3; 1 to 19) and monomethylated H3 (1 to 17) peptides were methylated by MLL3-SET wild-type and MLL3-SET Y4884C mutant protein in the presence of complex members using unlabeled AdoMet. The samples were collected at different time points, and methylation was analyzed by mass spectrometry using the relative areas of the corresponding unmethylated and methylated peaks. (A) Matrix-assisted laser desorption/ionization (MALDI) spectra of the methylation of the H3K4me0 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,423.4 Da (H3K4me0), 2,437.4 Da (H3K4me1), 2,451.4 Da (H3K4me2), and 2,465.4 Da (H3K4me3). (B) MALDI spectra of the methylation of the H3K4me1 peptide with MLL3 wild-type (upper panel) and Y4884C (lower panel). The masses of the corresponding peptides are 2,181.2 Da (H3K4me1), 2,195.2 Da (H3K4me2), and 2,209.2 Da (H3K4me3).
Mentions: To further confirm the change in substrate methylation state preference and product pattern of the Y4884C mutant, unmethylated and monomethylated H3K4 peptides were incubated in solution with MLL3 wild-type and the variants in the presence of unlabeled AdoMet. The samples were collected at the defined time intervals, and the methylation was analyzed by mass spectrometry. Wild-type MLL3 showed fast monomethylation of the H3K4me0 peptide, followed by weak dimethylation after long incubation times (Figure 4A, upper panel). No methylation was detectable with the H3K4me1 peptide even after the long incubation times (Figure 4B, upper panel) which suggests that the MLL3 wild-type protein is inactive on the H3K4me1 peptide and it is majorly a H3K4 monomethyltransferase. The weak dimethylation of the H3K4me0 peptide likely is due to a processive methylation of bound peptide after the first methylation cycle. In contrast, the Y4884C variant exhibited only weak activity on the H3K4me0 peptide (Figure 4A, lower panel). However, this did not result in an accumulation of monomethylated products indicating that they were rapidly converted into the di- and trimethylated state. Strikingly, Y4884C showed strong methylation activity on the H3K4me1 peptide resulting in the generation of dimethylated and later trimethylated products (Figure 4B, lower panel).Figure 4

Bottom Line: Somatic mutations in epigenetic enzymes are frequently found in cancer tissues.Expression of Y4884C leads to aberrant H3K4me3 formation in cells.Our data show that different somatic cancer mutations of MLL3 affect the enzyme activity in distinct and opposing manner highlighting the importance of experimentally studying the effects of somatic cancer mutations in key regulatory enzymes in order to develop and apply targeted tumor therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Faculty of Chemistry, Stuttgart University, Pfaffenwaldring 55, Stuttgart, 70569 Germany.

ABSTRACT

Background: Somatic mutations in epigenetic enzymes are frequently found in cancer tissues. The MLL3 H3K4-specific protein lysine monomethyltransferase is an important epigenetic enzyme, and it is among the most recurrently mutated enzymes in cancers. MLL3 mainly introduces H3K4me1 at enhancers.

Results: We investigated the enzymatic properties of MLL3 variants that carry somatic cancer mutations. Asn4848 is located at the cofactor binding sites, and the N4848S exchange renders the enzyme inactive. Tyr4884 is part of an aromatic pocket at the active center of the enzyme, and Y4884C converts MLL3 from a monomethyltransferase with substrate preference for H3K4me0 to a trimethyltransferase with H3K4me1 as preferred substrate. Expression of Y4884C leads to aberrant H3K4me3 formation in cells.

Conclusions: Our data show that different somatic cancer mutations of MLL3 affect the enzyme activity in distinct and opposing manner highlighting the importance of experimentally studying the effects of somatic cancer mutations in key regulatory enzymes in order to develop and apply targeted tumor therapy.

No MeSH data available.


Related in: MedlinePlus