Emerging Roles of Non-Histone Protein Crotonylation in Biomedicine

Hou et al. Cell Biosci (2021) 11:101 https://doi.org/10.1186/s13578-021-00616-2 Cell & Bioscience

REVIEW Open Access Emerging roles of non‑histone protein crotonylation in biomedicine Jia‑Yi Hou1,2, Lan Zhou1, Jia‑Lei Li1, De‑Ping Wang1 and Ji‑Min Cao1*

Abstract Crotonylation of proteins is a newly found type of post-translational modifcations (PTMs) which occurs leadingly on the lysine residue, namely, lysine crotonylation (Kcr). Kcr is conserved and is regulated by a series of enzymes and co-enzymes including lysine crotonyltransferase (writer), lysine decrotonylase (eraser), certain YEATS proteins (reader), and crotonyl-coenzyme A (donor). Histone Kcr has been substantially studied since 2011, but the Kcr of non-histone proteins is just an emerging feld since its fnding in 2017. Recent advances in the identifcation and quantifcation of non-histone protein Kcr by mass spectrometry have increased our understanding of Kcr. In this review, we summa‑ rized the main proteomic characteristics of non-histone protein Kcr and discussed its biological functions, including gene transcription, DNA damage response, enzymes regulation, metabolic pathways, cell cycle, and localization of heterochromatin in cells. We further proposed the performance of non-histone protein Kcr in diseases and the pros‑ pect of Kcr manipulators as potential therapeutic candidates in the diseases. Keywords: Lysine crotonylation, Post-translational modifcation, Protein, Cell biology, Disease

Introduction and ubiquitination, have been extensively studied and Post-translational modifcations (PTMs) of proteins are considered to be related to chromatin remodeling, make up most of the proteome and to a large extent, and gene transcriptional regulation, and other key biological establish the impressive level of functional diversity in processes. higher multi-cellular organisms. Mounting evidence sug- Among the above PTMs, lysine crotonylation (Kcr) gests that PTMs provide an elegant mechanism to govern represents a newly discovered and often elusive PTM protein function in diverse biological processes includ- that has nonetheless the potential to alter the function ing cell diferentiation and organismal development, and of the modifed protein. Histone crotonylation was frst aberrant protein modifcation may contribute to diseases reported as a special marker of sex-related genes and was such as cancer. As the only amino acid with a side chain directly related to gene transcription [7]. Subsequently, amine [1], lysine can be covalently modifed by glycosyl some studies found that lysine crotonylation can play [2], propionyl [3], butyryl [4], acetyl [5], hydroxyl [6], an important role in many diseases such as acute renal crotonyl [7], ubiquitinyl and ubiquitinyl-like [8], formyl injury, depression, HIV latency, and cancer process by [9], malonyl [10], succinyl [11], and methyl [12] groups. regulating the structure and function of histone [13–16]. Some modifcations, such as acetylation, methylation, In the past decade, mass spectrometry (MS)-based proteomics has greatly accelerated the discovery and identifcation of endogenous crotonylated proteins, and *Correspondence: [email protected] revealed the regulatory process of non-histone croto- 1 Key Laboratory of Cellular Physiology At Shanxi Medical University, Ministry of Education, Key Laboratory of Cellular Physiology of Shanxi nylation. Te discovery and identifcation of mammalian Province, and the Department of Physiology, Shanxi Medical University, lysine crotonyltransferases (KCTs), lysine decrotonylases Taiyuan, China (KDCRs), and the crotonylation reader domain pave the Full list of author information is available at the end of the article way for the study of non-histone protein crotonylation.

© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons.org/ licen ses/ by/4. 0/ . The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons. org/ publi cdoma in/ zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hou et al. Cell Biosci (2021) 11:101 Page 2 of 16

Te identifcation of thousands of crotonylation sites have quantifed the relative efects of tens of thousands has arisen great interest of biomedical researchers. More of crotonylation sites on genetic and metabolic activities, importantly, a growing number of studies have shown and provided insight into the dynamic regulation of Kcr. that non-histone protein crotonylation is involved in almost all major biological processes [17–21]. Regulation of crotonylation Here, we provide an overview of the expanding land- Tere are structural similarities between lysine croto- scape of non-histone protein crotonylation, including the nylation and lysine acetylation, and in fact, many of the regulation, large-scale identifcation, biological function “modulators” of crotonylation are the same as those of of protein crotonylation, and the crosstalk of Kcr with acetylation [27, 28]. However, crotonylation is unique other PTMs. We further discuss the connection between in its π-electron conjugation, resulting in being rigid protein crotonylation and diseases in which Kcr may be and planar in shape. Crotonyllysine but not acetyllysine potential therapeutic targets of the diseases. Although is found to more robustly neutralize the positive charge there are not many reported in-depth investigations on because of a longer carbon chain [29]. Histone crotonyla- the crotonylation of non-histone proteins, a comprehen- tion have been found sharing the similar set of regulatory sive review on this rapidly developing feld may help to machineries as histone acetylation [19, 30]. In the process uncover its biomedical signifcance and prospects. of crotonylation, crotonyl is added to the substrate proteins by lysine crotonyltransferase (KCT) (“writer”) [17, Uncovering lysine crotonylation 31–33] and then histone crotonylation can be recognized It is increasingly appreciated that combinations of PTMs by another family of proteins such as YEATS2, AF9, and can generate distinct protein isoforms with varying func- Taf14 (“reader”) [29, 34, 35], which usually initiates a tions, which vastly expand the functional diversity of series of downstream signals [29]. At the end of PTM- mammalian proteomes. Te widespread occurrence of induced signal transduction, most crotonylation can also PTMs only started to become clear in the frst years of be removed by lysine decrotonylase (KDCR) (“eraser”) the twenty-frst century, when advances in high-resolu- [17, 36–38]. Part of the efect of either KCT or KDCR can tion mass spectrometry enabled detection of thousands be selectively counteracted by drug inhibitors (Fig. 1). of low-abundance PTM sites. In this context, protein Kcr Te crotonyltranferase, decrotonylase, reader, and donor on histones was frst described in 2011 by Zhao and col- of non-histone and histone crotonylation do not com- leagues [7], who designed a comprehensive method to pletely overlap, which are respectively described below. systematically analyze histone PTM, using PTMap, an algorithm that can recognize all possible PTMs of pro- Lysine crotonyltranferase (writer), decrotonylase (eraser), teins [22]. In this method, mass spectrometry (MS) anal- and reader ysis of histone hydrolyzed peptides maximizes sequence Te modulators of histone crotonylation have been coverage and sensitivity, resulting in recognition of many extensively studied and summarized earlier [19, 39], and new PTM sites, including Kcr identifed as a new type of some of them can also regulate non-histone crotonyla- histone modifcation. tion. Six KCTs have been identifed in metazoans and Histone crotonylation is an evolutionarily conserved are classifed into three major families: P300/CBP (p300/ histone post-translational modifcation appearing in CREB-binding protein) [40], MYST (Moz, Ybf2, Sas2, eukaryotic cells from a wide range of species. Using a pan and Tip60) [32], and GNAT (GCN5-related N-acetyl- antibody against Kcr, Kcr signals in the core histones of transferase) [33]. So far, p300 and hMOF in MYST sapiens (HeLa) cells, mouse, cerevisiae, elegans, mela- family and PCAF in GNAT family were revealed to be nogaster, as well as plant, have been detected [7, 23–26]. non-histone crotonyltransferase (Table 1). P300 and its Te modifed proteins and sites vary and overlap among homologue CBP are two highly related KCTs that func- diferent species, such as H2AK125cr in humans and tion as general transcriptional coactivators. Te human mice [26]. males absent on the frst (hMOF) protein which belongs Tese groundbreaking discoveries set the stage for the to the MYST family, and the P300/CBP-associated fac- feld of non-histone protein crotonylation. In 2017, Xu tor (PCAF) which is a member of the GNAT family, et al. [17] frst reported that in addition to histones, Kcr both possess evolutionarily conserved KCT activity [17, also occurs on a variety of non-histone proteins. Increas- 32]. CBP, p300, and hMOF have the same enzyme reac- ing proteomic analyses have demonstrated that the fre- tion centers which catalyze acetylation and crotonyla- quency of non-histone protein crotonylation is very high, tion at multiple sites. It is found that the activity of each and these proteins are involved in many key cellular KCT varies from substrate to substrate (Table 1). Te processes which are related to physiology and disease in non-histone NPM1 can be strongly crotonylated by CBP eukaryotic cells. Tese mass spectrometry-based studies and hMOF, but only moderately crotonylated by PCAF. Hou et al. Cell Biosci (2021) 11:101 Page 3 of 16

Fig. 1 The metabolic pathways and enzymes and modulators for lysine crotonylation (Kcr). Colon microbiota is a source of short chain fatty acids (SCFAs) which may be metabolized to acetyl/crotonyl/propionyl/butyryl-CoA in host cells. These CoAs are precursors used by enzymes to promote lysine acetylation (Kac), crotonylation (Kcr), propionylation, and butyrylation of histone and non-histone proteins. Currently characterized crotonyltransferases (KCTs) (writer) include CBP/P300, MYST, and GNAT family, and histone decrotonylases (KDCRs) (eraser) include HDAC I and HDAC III. When substrates are histone proteins, Kcr readers, including bromodomains, YEATS, and DPF domain, recognize the Kcr. Chromodomain Y-like (CDYL), acting as a crotonyl-CoA hydratase which converts crotonyl-CoA to β-hydroxybutyryl-CoA, negatively regulates crotonylation. ACCS2, acyl-CoA synthetase short chain family member 2. Inhibitors of P300/CBP include C646 and SGC-CBP30. Inhibitors of HDACs include trichostatin A (TSA), nicotinamide (NAM), suberoylanilide hydroxamic acid (SAHA), and LBH589

Table 1 Lysine crotonyltranferases and their histone and non-histone substrates Family Enzyme name Substrates and modifed lysine residues Histone References Non-histone References p300/CBP family p300, CBP H3K18 [31] NPM1, DDX5 [17] MYST family hMOF H3K4, H3K9, H3K18, H3K23, H4K8, and [32] NPM1 [17] H4K12 Esa1 H4K5, H4K8, H4K12, and H4K16 [33] N/A N/A GNAT family GCN5 H3K9, K3K14, and H3K18A [33] N/A N/A PCAF N/A N/A NPM1 [17]

DDX5 DEAD-box helicase 5, Esa1 essential Sas2-related acetyltransferase 1, GCN5 general control nonrepressed-protein 5, GNAT GCN5-related N-acetyltransferase, hMOF human males absent on the frst, MYST Moz, Ybf2, Sas2, and Tip60, NPM1 nucleophosmin-1, N/A not available, PCAF P300/CBP-associated factor, P300/CBP P300/ CREB-binding protein

However, crotonylation of DDX5 can be catalyzed by Zn2+-dependent KDCRs and NAD+-dependent sirtuin CBP, but not by other KCTs [17]. decrotonylases (Table 2). Te Zn 2+-dependent KDCRs Most lysine decrotonylases have a wide range of sub- are often referred to as classical HDACs and belong to strates, including histone and non-histone proteins. class I [41]. In class I HDACs, HDAC1 and HDAC3, but KDCRs can be grouped into two major categories: not HDAC2, can decrotonylase non-histone NPM1, and Hou et al. Cell Biosci (2021) 11:101 Page 4 of 16

Table 2 Diferent classes of lysine decrotonylases, their co-factors, cellular locations, and substrates Co-factor Class Members Location Histone Substrate References Non-histone References

2 Zn +-dependent HDAC I HDAC1 Nucleus H3K4, H3K9, H3K18, H3K23, H4K8, and [36] NPM1 [17] H4K12 HDAC2 Nucleus H3K9, H3K18, and H4K8 [36] N/A N/A HDAC3 Nucleus H3K9, H3K18, and H4K8 [36] NPM1 [17] HDAC8 Nucleus H3K4, H3K9, H3K18, H4K8, and H4K12 [36] N/A N/A

NAD+-dependent HDAC III SIRT1 Nucleus/ cytoplasm H3K4, H3K9, and H4K8 [36–38] N/A N/A SIRT2 cytoplasm H3K4 and H3K9 [37, 38] N/A N/A SIRT3 Mitochondria H3K4 [38] N/A N/A

N/A not available this efect can be reversed by treatment with HDACs lead to a signifcant increase in the crotonylation of many inhibitors such as trichostatin A (TSA). Similarly, two non-histone proteins, including ubiquitin ligase RNF2 other HDAC inhibitors, suberoylanilide hydroxamic acid and the binding enzymes UBE2E1, NCOR1, and RBBP4, (SAHA) and LBH589, can promote the crotonylation of which are components of histone deacetylase complex 12 NPM1 [17] (Fig. 1). Sirtuin family deacetylases (SIRTs), [49]. Crotonylation of proteins induced by NaCr may also which are also referred to as class III HDACs, localize to be catalyzed by enzymes. NaCr can specifcally enhance diferent cellular compartments, including the nucleus protein crotonylation but not acetylation in wild HeLa (SIRT1), cytoplasm (SIRT2), and mitochondria (SIRT3) cells, and this induction can be nearly completely elimi- [42, 43]. So far, it has not been reported that Sirtuin fam- nated by C646 and SGC-CBP30, two selective P300/CBP ily can decrotonylase non-histone protein. In the func- inhibitors [49]. tion and regulation of protein crotonylation, Kcr must Second, enzymatic regulation of crotonyl-CoA level. be recognized by certain proteins possessing special Te enzymes involved in the conversion of crotonate to structures, these proteins are called reader, including crotonyl-CoA can afect the crotonylation. Chromodo- bromodomain, double plant homeodomain (PHD) fnger main Y-like (CDYL) protein, which contains an N-ter- (DPF), and YEATS domains [29, 34, 35, 44–46]. Reader minal chromodomain and a C-terminal enoyl-coenzyme originally refers to a chromatin binding protein module A hydratase/isomerase catalytic domain (also known that recognizes the covalent modifcation of histone. At as CoA pocket or CoAP), has been implicated in epige- present, there is no evidence that non-histone KCR also netic regulation and transcription repression by targeting has a reader. chromatin through its N-terminal chromodomain [50]. Liu et al. [51] reported that CDYL, acting as a crotonyl- Crotonyl‑CoA (donor) CoA hydratase, can add a water molecule to the double Crotonyl-CoA is presumed a crotonyl donor in the pro- bond at the position between the second and third car- cess of Kcr. Crotonate, mainly produced by the colon bon, and negatively regulate histone Kcr (Fig. 1). Yu et al. microbiota, is the short-chain fatty acid (SCFA) precur- [52] reported that CDYL can negatively regulate the Kcr sor of crotonyl-CoA [47]. Circulating short-chain fatty of RPA1 on multiple lysine sites, which is a reaction to acids (SCFA), such as acetate, crotonate, butyrate, and DNA-damaging insults. propionate, are taken up by tissues and converted to acyl-CoA by the acyl-CoA synthetase short chain fam- Large‑scale identifcation of non‑histone protein ily member 2 (ACSS2) or eventually yield crotonyl-CoA crotonylation through diferent metabolic pathways such as fatty acid Te breakthroughs in high-resolution mass spectrometry β-oxidation pathway and lysine degradation [48] (Fig. 1). (MS)-based proteomics have enabled both the identifca- Intracellular and intercellular metabolism can afect Kcr tion of thousands of crotonylation sites and the relative by changing the concentration of crotonyl-CoA in the quantifcation of these sites in a single experiment, ush- following two ways: ering in the age of crotonylomics (proteome-wide char- First, direct elevation of crotonyl-CoA by sodium cro- acteristics of crotonylation). Crotonylomics mapping is tonate. Sodium crotonate (NaCr) enhances non-histone a prerequisite for systems-level analyses and provides an crotonylation most likely through conversion of NaCr to important resource for discovering novel properties and crotonyl-CoA. Adding NaCr to the cell culture media can regulatory functions of crotonylation. Hou et al. Cell Biosci (2021) 11:101 Page 5 of 16

Quantitative mass spectrometry for crotonylomics analysis In the next workfow, proteins are extracted from bio- Various MS instruments and methodological approaches logical samples and digested into large numbers of pep- can be used to perform crotonylomics analysis for pro- tides by enzymatic hydrolysis (usually trypsin), only teins and peptides. Nearly all large-scale crotonyla- a small part of the peptides is crotonylated. In order to tion studies use the “shotgun” or bottom‑up proteomics identify the modifed sites under specifc conditions, approach, which involves enzymatic digestion of all pro- quantitative analysis should be used. Te most com- teins (the proteome) followed by liquid chromatography monly used methods are to identify modifed sites by coupled to tandem MS (LC–MS/MS) (Fig. 2). comparing the intensity of crotonylated peptides among Crotonylation of some protein sites cannot be detected diferent samples, including metabolic labeling, chemical in wild types cells. In order to fnd out which lysine sites labeling, and label-free quantifcation. Each method has may be crotonylated, some studies use drugs that pro- its advantages and disadvantages. SILAC (stable isotope mote crotonylation or knock down the expression of labelling by amino acids in cell culture), which belongs “modulators” of crotonylation during cell culture, such as to metabolic labeling, is one of the most commonly used sodium crotonate (NACR) which promotes non-histone methods in quantitative proteomics [53]. Natural iso- crotonylation by conversion to crotonyl-coenzyme A topes (light) or stable isotopes (medium/heavy) are used [49], and HDAC inhibitor SAHA [21] or crotonyl-CoA to replace the corresponding amino acids during cell cul- hydrolase CDYL [52] which negatively regulate Kcr. But ture. Its advantage is the high efciency for protein labe- more researchers collect biological samples under inter- ling, while its disadvantage is time consuming. Metabolic ested conditions for modifcation (Table 3). labeling has been successfully used to reveal the relative

Fig. 2 Schematic diagram of the experimental procedures for mass spectrometry-based global analysis. Proteins are extracted from cells or tissues and digested into peptides with a protease such as trypsin. The tryptic peptides are then separated and fractionated by high pH reverse-phase high performance liquid chromatography (HPLC). Proteolysis of whole-cell protein extracts generates numerous peptides, but only a small fraction is crotonylated. To enrich lysine crotonylated peptides, pan-Kcr antibodies are applied to identify the crotonylated peptides in complex peptide mixtures using immunoafnity purifcation. The resulting peptides are ionized in the electrospray source before entering the mass spectrometer. MS and MS/MS spectra are then computationally processed to deduce peptide sequences, including the presence and location of crotonylation, and to quantify the abundance of crotonylated peptides and proteins Hou et al. Cell Biosci (2021) 11:101 Page 6 of 16 References [ 68 ] [ 52 ] [ 61 ] [ 56 ] Year 2020 2020 2018 2018 Validated proteins proteins Validated WB by N/A RPA1, APEX1, and HP1 RPA1, LMNB2 N/A ‑ ‑ - ‑ - translational substrate sion, cell - substrate junction, cell - adherens substrate junction, biological of regulation process of body fuid levels, and calcium ion bind ‑ ing.Downregulated involved: proteins cellular component of blood microparticle, process biological of humoral immune and response, ubiquitin - like protein ligase binding involved: focal adhe focal involved: lism RNA splicing, and RNA splicing, amino acid metabo transcribed mRNA and catabolic process, translational initiation protein targeting to to targeting protein nuclear membrane, teins enrichedteins in cellular metabolic and cellular process outside to response and downregulated localized proteins in cellular functions and such as migration development Biological functionBiological Upregulated proteins proteins Upregulated Translation initiation, Translation Co Upregulated pro Upregulated ‑ ‑ ‑ regions and mem ‑ regions branes (69%) cytoplasm and nucleus (roughly equal) ‑ (89%), and mitochon dria (19%) nylated proteins: cyto proteins: nylated plasm (46%), nucleus (14%), mitochondria (12%), and extracel ‑ (11%). lular region Downregulated cytoplasm proteins: (57%), extracellular (11%), mito region chondria (9%), and nucleus (8%) Subcellular Subcellular localization Organelles, extracellular Organelles, Mitochondria (18%), Nucleus (69%), cytosol Upregulated croto Upregulated − 1 + 2 + 10 + 5 to + 4 position; + 1, and + 4 posi ‑ + 1 positions + 1 positions

acid (D), and lysine (K) at the residues to tamic acid (E) at − 1 and − 1, valine(V)tions, at position glutamine (E) at − 1 to lysine (K) at − 10 to − 5 and positions Amino acids fankingAmino sites Kcr the identifed Alanine (A), asparticAlanine (A), Negatively charged glu ‑ charged Negatively Glutamic acid (E) at − 4, Aspartic acid (D) and Kcr sites Kcr 770 14,311 816 1109 Identifed Identifed proteins 353 3734 392 347 and compared with and compared normal controls and compared with and compared normal controls Experiment Experiment condition PBMCs were obtained PBMCs were CDYL KO CDYL P300 KO PBMCs were obtained PBMCs were histone crotonylation based on LC–MS/MS analysis based on LC–MS/MS crotonylation of non - histone Global profling with hemodialysis maintenance IgAN patients HeLa cells HCT116 cells PBMCs of patients 3 Table samples Biological analyzed H. sapiens Hou et al. Cell Biosci (2021) 11:101 Page 7 of 16 References [ 17 ] [ 49 ] [ 49 ] [ 21 ] [ 71 ] [ 60 ] Year 2017 2017 2017 2017 2019 2019 Integrin β1, Vinculin, ERK2, CDK1, GAPDH, and OTUB1 Cul4B, and HDAC1 Cul4B, Validated proteins proteins Validated WB by NPM1, FHL1, ACTN1,NPM1, MTA2, CBX3, CBX5 、 MTA2, HDAC1 N/A N/A N/A proteins are involved involved are proteins in the cell process, in involved 14% are metabolism, 13% single biological are and 11% proteins, biomodulatoryare proteins acid metabolism, ‑ organi chromosome ‑ zation, gene expres ‑ sion, DNA conforma tional change and packaging, chromatin and cell organization cycle tions and biological tions and biological closely processes DNA and to related RNA metabolism and cell cycle ‑ fold scription, protein and a varietying, of metabolic processes lation, compound metabolism, and biosynthesis fxation, and amino acid metabolism Biological functionBiological 18% of crotonylated 18% of crotonylated RNA processing, nucleic RNA processing, Various molecular func ‑ Various Gene expression, tran ‑ Gene expression, Related to protein trans ‑ protein to Related Photosynthesis, carbon Photosynthesis, ‑ ‑ nucleus (27%), and mitochondria (13%) (62.3%), nucleo plasmic localization (9.4%), other proteins cellular components including plasma cytomembrane, appara ‑ plasm, Golgi and unclassifed tus, (28.3%) proteins nucleus (27.4%) and mitochondria (13.5%) around the nucleus around cytoplasm (33.0%), and nucleus (13.7%) Subcellular Subcellular localization Cytoplasm (40%), Cytoplasm Nuclear proteins Nuclear proteins N/A Cytoplasm (35.7%), Cytoplasm Concentrated in and Concentrated Chloroplast (36.8%), Chloroplast + 1 − 6 positions + 3 or at the − 1 and positions at lysine (K) occurred leucine (L), upstream; lysine (K)‑ and pheny lalanine (F) occurred downstream (E), and lysine (K) ‑ at surround residues ing positions Amino acids fankingAmino sites Kcr the identifed Glutamate (E) residues (E) residues Glutamate Alanine (A) residues residues Alanine (A) N/A N/A Isoleucine (I) and Alanine (A), glutamate glutamate Alanine (A), Kcr sites Kcr 2696 558 N/A 8475 1,061 984 2288 Identifed Identifed proteins 1024 453 70 2021 3735 3396 971 ‑ ‑ + defciency/ ment ment resupply Experiment Experiment condition Without any treat any Without Treated with NaCr Treated Without any treat any Without Treated with SAHA Treated RH strain ME49 strain NH4 (continued) The tea plants tea The H1299 cells HeLa cells A549 cells T. gondii T.

3 Table samples Biological analyzed Others Hou et al. Cell Biosci (2021) 11:101 Page 8 of 16 References [ 70 ] [ 69 ] [ 18 ] [ 54 ] [ 25 ] Year 2018 2018 2018 2018 2017 Validated proteins proteins Validated WB by N/A N/A N/A N/A N/A ‑ ‑ ‑ ‑ organism pro organism - ics, carbon metabo ics, lism, biosynthesis and of amino acids, glycolysis process, and diverse and diverse process, of skeletal regulation muscle contraction cellular process, and cellular process, single cess with upregulated cess with upregulated or downregulated proteins sis the biosynthesis of amino acids ‑ cycle, glycoly citrate and photosynthe sis, Biological functionBiological Photosynthesis Biosynthesis of antibiot Translation, metabolic Translation, Metabolic process, Metabolic process, Carbon metabolism, the ‑ ‑ cytoskeleton (1%), endoplasmic (1%), and reticulum extracellularly located (2%) tides), cytosol (691 peptides), nucleus (290 peptides), and mitochondria (138 peptides) chondria (11%), extracellular matrix (8%), and plasma membrane (6%) cytosol (32%), and nucleus (14%) cytosol (30%), nucleus ‑ (12%), and mitochon dria (5%) Subcellular Subcellular localization Chloroplast (51%), Chloroplast Chloroplast (713 pep Chloroplast Cytosol (58%), mito Cytosol Mitochondria (39%), Chloroplast (37%), Chloroplast ‑ + 1 + 5 + 10 to toxoplasma gondii toxoplasma gondii T. acid, hydroxamic SAHA suberoylanilide blood mononuclear cells, peripheral PBMCs patulin, + 5 positions, + 10

tamine (E), isoleucine (I), lysine (K), leucine (R), and (L), arginine valine (V)‑ at surround ing positions (D), and glutamine (E) − 1 and at − 5 to to while lysine(K) and (R) at − 10 to arginine − 5 and positions acidic (D and E) amino acids at surrounding positions residues from − 10 from residues to glutamine (E), at sur positions rounding Amino acids fankingAmino sites Kcr the identifed Aspartic acid (D), glu ‑ Alanine (A), asparticAlanine (A), acid Hydrophobic (L, I, V) (L, I, Hydrophobic and Negatively charged charged Negatively Aspartic acid (D) and Kcr sites Kcr 1265 5995 557 1691 2044 Identifed Identifed proteins 690 2120 218 629 637 ‑ ‑ ‑ ‑ ‑ human lung adenocarcinoma cells, IgAN cells, H1299 human lung adenocarcinoma human cervical HeLa cells, adenocarcinoma cells, cancer HCT116 human colon protein, Y-like chromodomain not available, PAT N/A not available, knockout, NaCr sodium crotonate ment ment ment ‑ mucilagi dotorula nosa ment Experiment Experiment condition Without any treat any Without Without any treat any Without Without any treat any Without Degradation in RhoDegradation Without any treat any Without (continued) Oryza L. japonica sativa Carica papaya L. papaya Carica Zebrafsh embryos Zebrafsh PAT Nicotiana tabacum Nicotiana parasites 3 Table samples Biological analyzed CDYL cells, A549 human lung adenocarcinoma KO immunoglobulin A nephropathy, Hou et al. Cell Biosci (2021) 11:101 Page 9 of 16

changes of crotonylation of non-histone proteins in that crotonylated proteins are widely located in the cyto- homologous recombination-mediated DNA repair [52]. plasm and nuclei of H1299 and HeLa cells [17]. In addi- Chemical labeling can be used in samples that can- tion, crotonylated proteins are widely found in a variety not be metabolically labeled, and several samples can be of tissues of mice, including lung, kidney, liver, colon, quantifed in parallel, for example, by tandem mass tags uterus, and ovary. Although these methods are not as (TMTs) [54] or by isobaric tags for relative and absolute efcient as MS, they can be used to verify the conclu- quantifcation (iTRAQ) [55, 56]. However, due to the sions of large-scale experiments, such as NPM1, FHL1, infuence of labeled groups, the identifcation fux was ACTN1, integrin β1, ERK2, and CDK1, which are consid- lower than the label-free approach. ered to be crotonylated as assayed by MS and can also be Label-free quantifcation allows direct identifca- detected by western blotting [17]. tion and quantifcation of proteins in a large scale, usually requiring analysis of a sample in triplicate to ensure Proteomic characteristics of crotonylation that the measured diferences are statistically signifcant Due to the progress of technology, the feld of lysine cro- [17, 20]. However, the accuracy of label-free quantifca- tonylation has developed rapidly in the past few years. tion is slightly worse than that of the labeled quantifca- Crotonylation was initially found to occur mainly in his- tion, because the former may be afected by the stability tones. However, a signifcant conclusion from recent pro- of mass spectrometry and other factors. In recent years, teomic studies is that most crotonylation events occur mass spectrometry techniques with increased stabil- on non-histone proteins [17, 52, 56, 61, 68]. MS-based ity and repeatability, as well as greatly improved com- proteomics methods are now used in a variety of organ- putational algorithms for quantifcation of MS data, isms not limited to humans, resulting in the identifcation have made label-free quantifcation an attractive option of thousands of new crotonylation sites (Table 3). Tese [57–59]. studies show that crotonylation sites are often conserved To reduce the complexity of samples, after digestion, in diferent organisms [18, 60, 69–71], thus it is obvious the trypsin polypeptides can be divided into several com- that crotonylation can be regarded as a protein modi- ponents by high-pH reversed-phase fractionation (RPF), fcation that exists prevalently in all felds of life. Cro- known as sample fractionation. To increase the depth of tonylated non-histone proteins are widely distributed in the crotonylation analysis, immunoafnity purifcation subcellular compartments and participate in a variety of is usually used, in which the pan-Kcr antibody is immo- important cellular functions, signal pathways, and variant bilized to a resin bead and selectively bound to the cro- biological activities (Table 3). tonylated tryptic peptides and is then eluted [17, 52, 60, Motifs refer to some specifc amino acids sequences 61]. Enrichment of crotonylated peptides is usually com- which localize near the lysine acylation site and are bined with sample fractionation to improve the efciency generally highly conserved. Te identifcation of the of the next mass spectrometry (MS). Finally, the enriched sequence of modifcation sites and the study of the cor- peptides are usually separated by liquid chromatography responding model peptides provides clues for predict- and ionized in the electrospray source, and entered into ing the potential modifcation sites of new proteins. For the mass spectrometer for analysis. High-resolution and instance, motif analysis is often used to predict potential high-quality precision