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Example of a peptide array stained with Bromophenol blue to confirm the synthesis of peptides. Please click here to view a larger version of this figure.
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Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferas...
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... Preparation of Membrane, Amino Acids and Reagents 1. Pre-swell the SPOT membrane in DMF ( N,N -Dimethylformamide) for 10 min and then place the wet membrane on the SPOT synthesizer frame without air bubbles or wrinkles. 2. Wash the membrane three times with 100% ethanol. Dry the membranes completely for at least 10 min before starting the synthesis program. 3. In parallel, freshly prepare 0.5 M working concentrations of commercially available Fmoc-protected amino acids by dissolving them in NMP and also prepare the activator and base as described in Table 1 . 4. Make sure that all the waste solution containers of the SPOT synthesizer were emptied and refill the solvent reservoirs with the DMF, ethanol and piperidine. NOTE: Now the machine is ready for the synthesis. 3. Spotting of the First Amino Acid 1. To generate the amide bond with the free amino group on the membrane, activate the carboxy group of the incoming amino acid by adding the activator ( N,N’ -Diisopropylcarbodiimide) and base solution (Ethyl hydroxyimino cyanoacetate) to the amino acid derivative. 2. Spot the desired volumes of activated amino acids on the Amino-PEG functionalized membrane using the programmable robot. NOTE: Thereby, the C-terminus of the amino acid is coupled to the amino group of the membrane. 3. Repeat this step three times to ensure better coupling of the first activated amino acid. Incubate the membrane for 20 min and then wash with DMF to remove uncoupled amino acids. 4. Blocking of Free Spaces on Non-spot Areas NOTE: After the coupling of the first C-terminal amino acid of all peptides to the amino group of the membrane, the amino groups between the spots and also some of the amino groups within the spot areas do not form bonds with the amino acids. 1. Block these free amino groups by incubating with the capping solution (20% acetic anhydride in DMF) for 5 min. NOTE: This avoids the coupling of amino acids in the further cycles with the membrane instead of the growing peptide chain. 5. Removal of Fmoc Group 1. After the blocking, wash the membrane with DMF 4 times and then incubate with 20% piperidine in DMF for 20 min to remove the Fmoc protection group. NOTE: This step leads to removal of the amino-protecting Fmoc groups and it is called deprotection. 2. Afterwards, wash the membrane twice with DMF and twice with ethanol and allow it to dry. NOTE: Now the membrane is ready for the coupling of the next amino acids. 6. Chain Elongation NOTE: Addition of each amino acid is called a cycle. Apart from the coupling of first amino acid, the cycle begins with the deprotection of Fmoc group of the coupled amino acid and it is followed by the coupling of the activated carboxyl group of an incoming amino acid to the amino group of the growing peptide chain. 1. Repeat steps 1.3 and 1.5 until the desired peptide length is achieved. 7. Staining NOTE: After the final cycle, the peptide arrays are stained with bromophenol blue to confirm the successful synthesis of peptides. Bromophenol blue binds with the free amino groups of the peptides. Peptide spots show light blue color after the staining but the intensity of the spots varies depending on the peptide sequence. This staining allows confirmation of the complete removal of piperidine, because in presence of piperidine bromophenol blue does not stain the peptides. In addition, this also helps to mark the peptide arrays for further usage. 1. After the Fmoc group deprotection in the last synthesis cycle, wash the membrane with DMF and 100% ethanol. Then, treat the membrane with bromophenol blue (0.02% bromophenol blue in DMF) for a minimum of 5 min until it completely turns blue. 2. Afterwards, wash the membrane with DMF and ethanol followed by drying for 10 min. Now take out the membrane from the synthesizer and perform side chain deprotection (section 1.8) manually. If needed, photograph the membrane to document the results of the peptide synthesis ( Fig. 2 ). 8. Side Chain Deprotection 1. Treat the membranes with 20 to 25 ml of side chain deprotection mixture consisting of 95% Trifluoroacetic acid (TFA) to cleave the side chain protection groups and scavenger reagents (2.5% water and 2.5% triisopropylsilane) to protect the side chains of amino acids from modification during this step. Take a sufficient volume of deprotection mixture to ensure that it covers the membrane completely in a tightly closed chemical resistant box and incubate it for 1 to 2 hr while shaking gently. 2. Wash the membrane six times with 20-25 ml of DCM (Dichloromethane) for 2 min each and finally twice with 100% ethanol and dry it in a desiccator overnight. NOTE: Now the membrane can be used for experiments. A scheme of the peptide array synthesis is provided in Figure 1 . 1. Protein Expression of PKMTs as GST Fusions 1. Using standard techniques, clone the gene encoding the full length PKMT or its catalytic domain into a bacterial expression vector (like pGEX-6p2) to express it as GST-fusion protein. 2. Transfer the vector with the desired PKMT insert into BL21 or BL21 codon plus E. coli cells by the heat shock method or any other method. 3. Prepare a pre-culture with 30 ml of Luria-Bertani (LB) media on the day of expression and incubate at 37 °C for 7 to 8 hr with continuous shaking. 4. Next, transfer 10 ml of the pre-culture into a 2 L big baffled flasks containing 1 L of LB media and incubate at 37 °C in the incubator with continuous shaking until the culture reaches a defined optical density at 600 ...
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The aim of our research was to design an efficient method for the solid phase synthesis of carbonylated peptides. For this purpose, we designed and synthesized a fully protected derivative Fmoc-amino(2,5,5-trimetyhyl-1,3-dioxolan-2-yl)acetic acid (Fmoc-Atda-OH) of a novel unnatural amino acid (Thr(O)-2-amino-3-oxo-butanoic acid). To obtain the ment...
Ubiquitination is a versatile posttranslational modification that elicits signaling roles to impact on various cellular processes and disease states. The versatility is a result of the complexity of ubiquitin conjugates, ranging from a single ubiquitin monomer to polymers with different length and linkage types. Recent studies have revealed the abu...
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... One approach to determine the full substrate spectrum of PKMTs and identify novel substrates is to characterize the sequence specificity of the enzyme and use this information to identify novel potential substrate proteins 11 . For this task, peptide SPOT arrays can be employed, as they allow to study the methylation of several hundred peptides in one experiment at moderate costs 33,34 . NSD2 is an essential PKMT which generates H3K36 mono-and dimethylation and has essential roles in chromatin regulation, cell physiology and cancer biology 12 . ...
... Based on the successful design of an NSD2-specific ssK36 at peptide level, we next intended to investigate its methylation using soluble H3K36 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) and ssK36 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) peptides. Therefore, H3K36 and different dilutions of the ssK36 peptide were incubated with NSD2 in the presence of radioactively labeled AdoMet, separated by a tricine gel and the methylation was analyzed by autoradiography. ...
... Based on the successful design of an NSD2-specific ssK36 at peptide level, we next intended to investigate its methylation using soluble H3K36 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) and ssK36 (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) peptides. Therefore, H3K36 and different dilutions of the ssK36 peptide were incubated with NSD2 in the presence of radioactively labeled AdoMet, separated by a tricine gel and the methylation was analyzed by autoradiography. ...
The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.
... Synthesis of the peptide arrays was performed with the SPOT synthesis method (Frank, 2002) using an Autospot Multipep synthesizer (Intavis AG). Each spot contained approximately 9 nmol peptide (Autospot Reference Handbook, Intavis AG) and the successful synthesis of the peptides on the cellulose membrane was qualitatively confirmed by bromophenol blue staining (Kudithipudi et al., 2014b;Weirich & Jeltsch, 2022). Peptide arrays were prepared using the sequences of H4K12 (amino acid 6-19, with an additional A at the N-terminus to place K12 in the center and avoid starting the peptide with a K) and ERF1 (amino acid [179][180][181][182][183][184][185][186][187][188][189][190][191][192][193] as template sequence with the respective Lys (Yang et al., 2004) or Gln 185 in the center. ...
The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1‐Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q‐ and K‐methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q‐methylation preferentially occurs in a G‐Q‐X3‐R context, while K‐methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K‐methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000‐times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.
... While the mechanisms underpinning substrate selectively of SET domain enzymes have been widely studied, substrate selectivity for 7βS enzymes is less well understood. SET domain enzymes tend to recognise their substrates as linear amino acid sequences, and thus studies of their substrate selectivity are amenable to peptide-based approaches (12,13). In contrast, 7βS enzymes tend to recognise the three-dimensional structural features of their substrates, making investigations into the mechanisms unpinning their substrate selection more difficult. ...
Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferases methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases after histone H3. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. To reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316, we have used AlphaFold-Multimer modeling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for the methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. We further show that the eEF1A guanine nucleotide exchange factor, eEF1Bα, inhibits Efm4 methylation of eEF1A in vitro, likely due to competitive binding. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be highly selective towards a single residue on a single protein in the cell.
... SETD6 potential enzymatic activity on E2F1 was first tested on peptide substrates. To this end, 15 amino acids long peptides were synthesized on a cellulose membrane using the SPOT technology (33,34) to contain the 14 lysines within the E2F1 sequence and the corresponding K to A mutants. The RelA peptide (7), served as positive control. ...
The protein lysine methyltransferase SETD6 has been shown to influence different cellular activities and to be critically involved in the regulation of diverse developmental and pathological processes. However, the upstream signals which regulate the mRNA expression of SETD6 are not known. Bioinformatic analysis revealed that the SETD6 promoter has a binding site for the transcription factor E2F1. Using various experimental approaches, we show that E2F1 binds to the SETD6 promoter and regulates SETD6 mRNA expression. Our further observation that this phenomenon is SETD6 dependent, suggested that SETD6 and E2F1 are linked. We next demonstrate that SETD6 mono-methylates E2F1 specifically at K117 in-vitro and in cells. Finally, we show that E2F1 methylation at K117 positively regulates the expression level of SETD6 mRNA. Depletion of SETD6 or overexpression of E2F1 K117R mutant, which cannot be methylated by SETD6, reverses the effect. Taken together, our data provide evidence for a positive feedback mechanism which regulates the expression of SETD6 by E2F1 in a SETD6 methylation dependent manner and highlight the importance of protein lysine methyltransferases and lysine methylation signaling in the regulation of gene transcription.
... This part is an independent folding unit as illustrated by structural studies [pdb 7E8D (Sato et al., 2021)]. A corresponding GST-tagged mouse NSD1 catalytic domain (amino acids 1701-1987 containing an additional C1920S mutation was taken from (Kudithipudi et al., 2014a). The SET domains of human and mouse NSD1 share >95% identity and none of the residues that are affected in this study or known to be catalytically relevant resides is different. ...
... The antibodies used in the Western-Blot analysis were validated by peptide array binding. Peptide arrays were synthesized with an Autospot peptide array synthesizer (Intavis AG, Köln, Germany) using the SPOT method (Kudithipudi et al., 2014a;Weirich and Jeltsch, 2022). Four spots corresponding to 4 different K36 methylation states of the H3K36 peptide (29-43) (unmodified-H3K36me1-H3K36me2-(which was not certified by peer review) is the author/funder. ...
Somatic mutations in protein lysine methyltransferases are frequently observed in cancer cells. We show here that the NSD1 mutations Y1971C, R2017Q and R2017L observed mostly in solid cancers are catalytically inactive suggesting that NSD1 acts as tumor suppressor gene in these tumors. In contrast, the frequent T1150A in NSD2 and its T2029A counterpart in NSD1, both observed in leukemia, are hyperactive and introduce up to H3K36me3 in biochemical and cellular assays, while wildtype NSD2 and NSD1 only generate up to H3K36me2. MD simulations with NSD2 revealed that H3K36me3 formation is possible due to an enlarged active site pocket of T1150A and loss of direct contacts of T1150 to critical residues which regulate the product specificity of NSD2. Bioinformatic analyses of published data suggest that the NSD2 T1150A mutation in lymphocytic leukemia could alter gene regulation by antagonizing H3K27me3 finally leading to the upregulation of oncogenes.
... Lysis Buffer: 30 mL is stable for 24 months when stored at À20 C. Can be stored at 4 C for 1-2 weeks. Note: Multimedia presentation for procedure of peptide synthesis can be obtained from (Kudithipudi et al., 2014) and freely accessible at PubMed Central portal. ...
Identification of peptides mediating protein-protein interaction (PPI) is crucial for understanding the function of interlinked proteins in cellular processes and amino acid-associated diseases. Traditional PPI assays are laborious, involving the generation of many truncated proteins. SPOT peptide assay allows high-throughput detection of domains essential for PPI by synthesizing several hundred peptides on a cellulose membrane. Here, we present a rapid SPOT peptide protocol for identifying the binding motifs, which mediate interaction between the chromatin remodeling factors BAF155/BAF170 and the epigenetic factor Kdm6b.
For complete details on the use and execution of this protocol, please refer to Narayanan et al. (2015).
... Selected examples include the substrate exploration of methyl-regulatory (i.e., methyltransferase or demethylase) or phosphor-regulatory (i.e., kinase or phosphatase) PTM-modifying enzymes (Fig. 2a). These approaches are often complemented with radioactive labelling of the donor group or methyl-/phospho-specific antibodies (Kudithipudi et al., 2014;Rowe and Biggar, 2018;Thiele et al., 2011). Such PTM-modifying enzymes specifically recognise their substrates based on a sequence of conserved recognizable amino acids (i.e., motif), making them a subject of study for both computational and wet-lab scientists (Hamey et al., 2018). ...
... These novel methods involve arrays of proteins immobilized on a surface (Fig. 2b), or by using arrays of peptides containing lysine predicted to be modified (Rowe and Biggar, 2018;Szymczak et al., 2018). Lysine-oriented peptide libraries (K-OPL) are a novel modification of peptide arrays, used to identify a sequence motif for substrate selectivity from libraries of systematically degenerate amino acid sequences surrounding a methylatable lysine (Fig. 2c) (Cornett et al., 2018;Kudithipudi et al., 2014). Cornett and colleagues used this systematic approach to determine the substrate selectivity of lysine methyltransferases (KMTs) G9a, SET7 and SMYD2 (Cornett et al., 2018). ...
Following the decoding of the first human genome, researchers have vastly improved their understanding of cell biology and its regulation. As a result, it has become clear that it is not merely genetic information, but the aberrant changes in the functionality and connectivity of its encoded proteins that drive cell response to periods of stress and external cues. Therefore, proper utilization of refined methods that help to describe protein signalling or regulatory networks (i.e., functional connectivity), can help us understand how change in the signalling landscape effects the cell. However, given the vast complexity in ‘how and when’ proteins communicate or interact with each other, it is extremely difficult to define, characterize, and understand these interaction networks in a tangible manner. Herein lies the challenge of tackling the functional proteome; its regulation is encoded in multiple layers of interaction, chemical modification and cell compartmentalization. To address and refine simple research questions, modern reductionist strategies in protein biochemistry have successfully used peptide-based experiments; their summation helping to simplify the overall complexity of these protein interaction networks. In this way, peptides are powerful tools used in fundamental research that can be readily applied to comparative biochemical research. Understanding and defining how proteins interact is one of the key aspects towards understanding how the proteome functions. To date, reductionist peptide-based research has helped to address a wide range of proteome-related research questions, including the prediction of enzymes substrates, identification of posttranslational modifications, and the annotation of protein interaction partners. Peptide arrays have been used to identify the binding specificity of reader domains, which are able to recognise the posttranslational modifications; forming dynamic protein interactions that are dependent on modification state. Finally, representing one of the fastest growing classes of inhibitor molecules, peptides are now begin explored as “disruptors” of protein-protein interactions or enzyme activity. Collectively, this review will discuss the use of peptides, peptide arrays, peptide-oriented computational biochemistry as modern reductionist strategies in deconvoluting the functional proteome.
... Generation and methylation of peptide arrays. Peptide arrays of 15-mer peptides were generated using the SPOT method 44,45 . Methylation reactions were conducted by incubating the arrays in Reaction Buffer (50 mM Tris-HCl pH 7.8, 50 mM NaCl, 5 mM EDTA) with 0.76 μM [ 3 H]-AdoMet (PerkinElmer) and 0.5-1 µM of His 6 -hMETTL9. ...
Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where “x” is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
... PKMT target residues are usually surrounded by a defined short sequence motif of amino acids, which are specifically recognized in a binding cleft located in the SET domain of the enzymes. The resulting substrate sequence specificities have been analyzed for many PKMTs using peptide and protein libraries as methylation substrates [28][29][30][31][32][33][34][35][36][37][38]. In a study investigating the Clr4 specificity sequence motif using peptide array methylation experiments, it was observed that Clr4 prefers R at the −1 position of its target site (related to the target lysine), and it shows some additional more relaxed preferences at the +1 (S > K, R >T) and +2 (T >> C > S) positions [39]. ...
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.
... Substrate specificity analysis of SETD2 in an H3K36 context. After confirmation of the SETD2 activity, its substrate specificity was determined by using SPOT peptide arrays 27 . The H3K36 (aa 29-43) fragment was used as a template sequence in this analysis, and each single position of the template sequence was exchanged against 18 other natural amino acids creating single amino acid mutants for each position. ...
... The standard deviations (SD) of the methylation activity of each single spot were calculated, which showed that most spots had an SD smaller than ±10% ( Supplementary Fig. 2a) indicating a high reproducibility of the results. For visualization of the substrate specificity motif of SETD2, discrimination factors were calculated as previously described 27 . The discrimination factors show the preference of SETD2 for one specific amino acid over all other amino acids in each single position of the peptide sequence (Fig. 1c). ...
... The signals of both independent replicates were quantified, normalized and averaged. c Discrimination factors showing the preference of SETD2 for specific amino acids over all others at each position 27 and visualization of the substrate specificity motif of SETD2 as Weblogo (https://weblogo.berkeley.edu/logo.cgi) 29 . ...
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.
