03 Fakultät Chemie

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Author: search.filters.author.Jeltsch, AlbertSubject: search.filters.subject.570

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    Analysis of the substrate specificity of the SMYD2 protein lysine methyltransferase and discovery of novel non-histone substrates
    (2019) Weirich, Sara; Schuhmacher, Maren Kirstin; Kudithipudi, Srikanth; Lungu, Cristiana; Ferguson, Andrew D.; Jeltsch, Albert
    The SMYD2 protein lysine methyltransferase methylates various histone and non-histone proteins and is overexpressed in several cancers. Using peptide arrays, we investigated the substrate specificity of the enzyme, revealing a recognition of leucine (or weaker phenylalanine) at the -1 peptide site and disfavor of acidic residues at the +1 to +3 sites. Using this motif, novel SMYD2 peptide substrates were identified, leading to the discovery of 32 novel peptide substrates with a validated target site. Among them, 19 were previously reported to be methylated at the target lysine in human cells, strongly suggesting that SMYD2 is the protein lysine methyltransferase responsible for this activity. Methylation of some of the novel peptide substrates was tested at the protein level, leading to the identification of 14 novel protein substrates of SMYD2, six of which were more strongly methylated than p53, the best SMYD2 substrate described so far. The novel SMYD2 substrate proteins are involved in diverse biological processes such as chromatin regulation, transcription, and intracellular signaling. The results of our study provide a fundament for future investigations into the role of this important enzyme in normal development and cancer.
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    Mechanistic insights into the allosteric regulation of the Clr4 protein lysine methyltransferase by autoinhibition and automethylation
    (2020) Khella, Mina S.; Bröhm, Alexander; Weirich, Sara; Jeltsch, Albert
    Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.
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    Globally altered epigenetic landscape and delayed osteogenic differentiation in H3.3-G34W-mutant giant cell tumor of bone
    (2020) Lutsik, Pavlo; Baude, Annika; Mancarella, Daniela; Öz, Simin; Kühn, Alexander; Toth, Reka; Hey, Joschka; Toprak, Umut H.; Lim, Jinyeong; Nguyen, Viet Ha; Jiang, Chao; Mayakonda, Anand; Hartmann, Mark; Rosemann, Felix; Breuer, Kersten; Vonficht, Dominik; Grünschläger, Florian; Lee, Suman; Schuhmacher, Maren Kirstin; Kusevic, Denis; Jauch, Anna; Weichenhan, Dieter; Zustin, Jozef; Schlesner, Matthias; Haas, Simon; Park, Joo Hyun; Park, Yoon Jung; Oppermann, Udo; Jeltsch, Albert; Haller, Florian; Fellenberg, Jörg; Lindroth, Anders M.; Plass, Christoph
    The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.
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    Somatic cancer mutations in the MLL1 histone methyltransferase modulate its enzymatic activity and dependence on the WDR5/RBBP5/ASH2L complex
    (2017) Weirich, Sara; Kudithipudi, Srikanth; Jeltsch, Albert
    Somatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1–WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1–WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1–WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered.
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    Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme
    (2015) Weirich, Sara; Kudithipudi, Srikanth; Kycia, Ina; Jeltsch, Albert
    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.
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    Structure, activity and function of the Suv39h1 and Suv39h2 protein lysine methyltransferases
    (2021) Weirich, Sara; Khella, Mina S.; Jeltsch, Albert
    SUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment.
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    Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate
    (2020) Schuhmacher, Maren Kirstin; Beldar, Serap; Khella, Mina S.; Bröhm, Alexander; Ludwig, Jan; Tempel, Wolfram; Weirich, Sara; Min, Jinrong; Jeltsch, Albert
    SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.
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    Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites
    (2017) Lungu, Cristiana; Pinter, Sabine; Broche, Julian; Rathert, Philipp; Jeltsch, Albert
    Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing target epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.
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    Investigation of the methylation of Numb by the SET8 protein lysine methyltransferase
    (2015) Weirich, Sara; Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert
    It has been reported that the Numb protein is methylated at lysine 158 and 163 and that this methylation is introduced by the SET8 protein lysine methyltransferase [Dhami et al., (2013) Molecular Cell 50, 565-576]. We studied this methylation in vitro using peptide arrays and recombinant Numb protein as substrates. Numb peptides and protein were incubated with recombinant SET8 purified after expression in E. coli or human HEK293 cells. However, no methylation of Numb by SET8 was detectable. SET8 methylation of Histone H4 and p53 peptides and proteins, which were used as positive controls, was readily observed. While SET8 methylation of Numb in cells cannot be ruled out, based on our findings, more evidence is needed to Support this claim. It appears likely that another not yet identified PKMT is responsible for the reported methylation of Numb in cells.