Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design

Lanouette, Sylvain; Davey, James A.; Elisma, Fred; Ning, Zhibin; Figeys, Daniel; Chica, Roberto A.; Couture, Jean-François

doi:10.1016/j.str.2014.11.004

Cited by 35 publications

(49 citation statements)

References 68 publications

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“…[LYK] ϩ2 substrate specificity derived from SMYD2 biochemical activity (30). However, we also observed a general lack of enrichment of specific amino acids at the ϩ2 positions, suggesting that SMYD2 may recognize a more diverse collection of substrates in cells than originally predicted by biochemical and structural analyses.…”

Section: Quantitative Profiling Of Smyd2 Activity In Cellsmentioning

confidence: 62%

“…These findings suggest that these motifs reflect the substrate specificity of SMYD2 in cells. consensus specificity for SMYD2 (30), although only seven Kme1 sites that we identified in cells fit within the confines of this specificity signature. Moreover, this consensus specificity signature does not account for the novel Kme1 sites in BTF3, PDAP1, AHNAK, or AHNAK2 that we characterized in our study, suggesting that characterizing the activity of SMYD2 toward a single substrate (i.e.…”

Section: Discussionmentioning

confidence: 80%

“…n.s., not significant; *p value Յ 0.0005; **p value Յ 0.00005, ***p value Յ 0.000005. 1, Lanouette et al, 2015;2, Rathert et al, 2008;3, Dhayalan et al, 2011. NAK2 relative to the rest of the proteome. These findings show that AHNAK and AHNAK2 are unique among SMYD2 methylation substrates-and mono-methylated proteins in general-in that they are extensively mono-methylated at multiple sites and primarily at sites containing the LKGP motif.…”

Section: Quantitative Profiling Of Smyd2 Activity In Cellsmentioning

confidence: 99%

“…[LYK] ϩ2 as a consensus sequence for SMYD2 substrate specificity (30); however, whether the substrate specificity derived from such biochemical analyses encompasses all SMYD2 substrates in cells is unknown. In addition to substrates identified using biochemical approaches, an investigation into proteins that stably physically associate with SMYD2 led to the discovery of HSP90AA1-K615me1 as a Kme1 site in cells directly regulated by SMYD2 activity (31,32).…”

mentioning

confidence: 99%

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Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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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. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

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Section: Quantitative Profiling Of Smyd2 Activity In Cellsmentioning

confidence: 62%

Section: Discussionmentioning

confidence: 80%

Section: Quantitative Profiling Of Smyd2 Activity In Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

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“…This is often called multi-state protein design [32,33 ,34]. Lanouette et al have successfully applied multi-state protein design to predict the preferred substrates for a lysine methytransferase [35]. They used a computationally generated ensemble of peptide structures docked into the enzyme active site as target structures for design.…”

Section: Structural Flexibilitymentioning

confidence: 99%

Computational protein design for given backbone: recent progresses in general method-related aspects

Chen

2016

Current Opinion in Structural Biology

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Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non‐Histone Substrates

et al. 2019

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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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Discovery of Substrates for a SET Domain Lysine Methyltransferase Predicted by Multistate Computational Protein Design

Cited by 35 publications

References 68 publications

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Computational protein design for given backbone: recent progresses in general method-related aspects

Analysis of the Substrate Specificity of the SMYD2 Protein Lysine Methyltransferase and Discovery of Novel Non‐Histone Substrates

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