A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity

Cornett, Evan M.; Dickson, Bradley M.; Krajewski, Krzysztof; Spellmon, Nicholas; Umstead, Andrew; Vaughan, Robert M.; Shaw, Kevin M.; Versluis, Philip; Cowles, Martis W.; Brunzelle, J.S.; Yang, Zhe; Vega, Irving E.; Sun, Zu Wen; Rothbart, Scott B.

doi:10.1126/sciadv.aav2623

Cited by 25 publications

(28 citation statements)

References 38 publications

(51 reference statements)

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“…Our studies also included an advance of the previously performed OPAL (oriented peptide array library) technology. In this work, we used more quantitative flashplate scintillation counting of PRMT activity towards immobilized degenerate peptides (Cornett et al 2018). As these peptides contained a fixed central arginine substrate along with a single other fixed amino acid position (excluding Cysteine), we were able to derive in vitro substrate preferences for the three most abundant enzymes: PRMT1, PRMT4/CARM1, and PRMT5.…”

Section: Discussionmentioning

confidence: 99%

“…We then, in the presence of 3 H-SAM, incubated recombinant human PRMT1, human PRMT4 (CARM1), C. elegans PRMT5, and Xenopus laevis PRMT5-MEP50 complex on the OPAL arrays. Using scintillation counting on the OPAL peptides, we determined relative methyltransferase activity and for each enzyme plotted the results as a heatmap (Figure 2b-e) (Cornett et al 2018). To determine consensus motifs, we generated Weblogo probability plots (Figure 2f-i, bottom panels).…”

Section: Substrate Specificity Of Prmtsmentioning

confidence: 99%

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Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Lehman

Chen

Burgos

et al. 2020

Preprint

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Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (~0.1% of total arginine), Rme2a was highly abundant (~1.1%), and Rme2s was intermediate (~0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

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Section: Discussionmentioning

confidence: 99%

Section: Substrate Specificity Of Prmtsmentioning

confidence: 99%

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Lehman

Chen

Burgos

et al. 2020

Preprint

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“…Associated histone mark MPP8 chromo H3K9me2/3 (Kokura et al, 2010) L3MBTL1 3xMBT H1bK26me1/2 (Trojer et al, 2007) H4K20me1/2 (Min et al, 2007) DIDO1 PHD H3K4me3 (Gatchalian et al, 2016) PHF20 Tudor H3K4me2 (Klein et al, 2016) H4K20me2 (Klein et al, 2016) L3MBTL3 3xMBT Many Kme2 (Nady et al, 2012) 53BP1 TTD H4K20me1/2 (Botuyan et al, 2006;Hartlerode et al, 2012) H3K18me2 (Shanle et al, 2017) H3K36me2 (Tong et al, 2015) PCL1 Tudor H3K36me3 (Cai et al, 2013) CDYL1b chromo H3K9me2/3 (Franz et al, 2009;Escamilla-Del-Arenal et al, 2013) H3K27me3 (Vermeulen et al, 2010) CDYL2 chromo H3K9me3 (Fischle et al, 2008) H3K27me3 (Fischle et al, 2008) (Cornett et al, 2018) to predict where these readers may bind in the proteome ( Figure 1B). For these studies, MPP8 and CDYL2 chromodomains were chosen because aspects of their amino acid binding preferences are reported elsewhere (Li et al, 2011;Barnash et al, 2016), and these data were consistent with Kme-OPL profiles generated with these readers.…”

Section: Protein Domainmentioning

confidence: 99%

“…In order to relate Kme-OPL profiles to the human proteome, we used our previously developed LoB scoring function (Cornett et al, 2018). LoB scoring ranks all lysine centered seven-mers in the proteome from most to least likely to bind to a given reader.…”

Section: Protein Domainmentioning

confidence: 99%

A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions

Kupai

Vaughan

Dickson

et al. 2020

Front. Cell Dev. Biol.

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Lysine methylation facilitates protein-protein interactions through the activity of methyllysine (Kme) "reader" proteins. Functions of Kme readers have historically been studied in the context of histone interactions, where readers aid in chromatin-templated processes such as transcription, DNA replication and repair. However, there is growing evidence that Kme readers also function through interactions with non-histone proteins. To facilitate expanded study of Kme reader activities, we developed a high-throughput binding assay to reveal the sequence determinants of Kme-driven protein interactions. The assay queries a degenerate methylated lysine-oriented peptide library (Kme-OPL) to identify the key residues that modulate reader binding. The assay recapitulated methyl order and amino acid sequence preferences associated with histone Kme readers. The assay also revealed methylated sequences that bound Kme readers with higher affinity than histones. Proteome-wide scoring was applied to assay results to help prioritize future study of Kme reader interactions. The platform was also used to design sequences that directed specificity among closely related reader domains, an application which may have utility in the development of peptidomimetic inhibitors. Furthermore, we used the platform to identify binding determinants of site-specific histone Kme antibodies and surprisingly revealed that only a few amino acids drove epitope recognition. Collectively, these studies introduce and validate a rapid, unbiased, and high-throughput binding assay for Kme readers, and we envision its use as a resource for expanding the study of Kme-driven protein interactions.

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“…[19][20][21][22] However, antibody based methyl-enrichments have a number of challenges and limitations: (1) they require a large amount of sample and multiple antibodies to cover all methyl forms; (2) they often contain a sequence bias for antigen recognition, thus selectively assaying a portion of the methylome while masking an unknown but likely significant portion of methyl-sites; and (3) by nature these approaches do not enrich the unmodified form of the peptide, hindering quantification of all peptidoforms together and thus impairing the ability to study the relative abundance of PTMs. 23,24 To overcome these limitations and facilitate a comprehensive and technically straightforward quantitative survey of the methylome in biological samples, we leveraged small precursor acquisition window DIA-MS, coupled with careful curation of a combined low pH SCX fractionated and methyl peptide immunoprecipitation enriched peptide library consisting of both unmodified and methylated peptides against which each experimental sample is extracted and compared. In addition to methylation, lysine residues can be modified by various other PTM's, including acetylation and succinylation.…”

Section: Graphical Abstract Introductionmentioning

confidence: 99%

Quantitative Method for Assessing the Role of Lysine & Arginine Post-Translational Modifications in Nonalcoholic Steatohepatitis

Robinson

Binek

Venkatraman

et al. 2020

Preprint

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Arg and Lys undergo multiple enzymatically driven post-translational modifications (PTMs), including methylation, which may be linked to key cellular processes. Today there is not a method that simultaneously quantifies proteins and the methylome at the site level. We have developed a method that can differentiate an unmodified peptide from its mono-, di-or tri-methylated Arg or Lys counterpart through a data independent acquisition approach that is suitable for both triple TOF and Orbitrap mass spectrometers. This method was further expanded to include Lys acetylation and succinylation, which in conjunction with methylation allow for simultaneous quantification of Lys PTMs in a single measurement. Our approach leverages small precursor mass windows to physically separate the various methylated species from each other during MS2 acquisition and shows linearity in the quantitation of low abundant peptides. We did this by first creating a biologically hyper-methylated peptide assay library, for mono-, di-and tri-methylated Lys and mono-and di-methylated Arg to which each experimental sample is compared. We further expanded the peptide assay library to include Lys acetylation and succinylation, providing the opportunity to multiplex five Lys and two Arg modification without the need for sample enrichment. To assess the validity of PTMs quantified with this method, we added an algorithm to determine the false localization rate for each potential modified amino acid residue. To evaluate its biological relevance, we applied this method to complex liver lysate from differentially methylated invivo non-alcoholic steatohepatitis mouse models and show that differential methylation potential altered not only protein quantity but also acetylation and to some degree succinylation. Of interest, acetylation data together with total protein changes drive strong novel hypothesis of a regulatory function of posttranslational modifications in protein synthesis and mRNA stability. In conclusion: our method, which is suitable for different mass spectrometers along with the corresponding publicly available mouse liver PTM enriched peptide assay library, provides an easily adoptable framework needed for studying global protein methylation, acetylation, and succinylation in any proteomic experiment when total protein quantification is being carried out. Wide adoption of this easy approach will allow more rapid integration of PTM studies and knowledge.

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A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity

Abstract: Mapping lysine methyltransferase substrate selectivity reveals gaps in the proteome-wide curation of lysine methylomes.

Cited by 25 publications

References 38 publications

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions

Quantitative Method for Assessing the Role of Lysine & Arginine Post-Translational Modifications in Nonalcoholic Steatohepatitis

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