the first n - terminal acetylation is catalyzed a variety of n - terminal acetyltransferases ( nats ) , which co - translationally transfer acetyl moieties from acetyl - coenzyme a ( acetyl - coa ) to the -amino
although n - terminal acetylation is rare in prokaryotes , it was estimated that 85% of eukaryotic proteins are n - terminally modified ( 1,2 ) .
the second type is n - lysine acetylation , which specifically modifies -amino group of protein lysine residues ( 38 ) .
although n - lysine acetylation is less common , it s one of the most important and ubiquitous post - translational modifications ( ptms ) conserved in prokaryotes and eukaryotes ( 1,2 )
. moreover , the acetylation and deacetylation are dynamically and temporally regulated by histone acetyltransferases ( hats ) and histone deacetylases ( hdacs ) , respectively ( 48 ) . in 1964 , allfrey et al .
( 9 ) first observed that lysine acetylation of histones plays an essential role in regulation of gene expression .
later and recent studies in epigenetics solidified this seminal discovery , and proposed acetylation as a key component of the  histone code ( 6 ) . beyond histones , a wide - range of non - histone proteins
can also be lysine acetylated , and involved in a variety of biological processes , such as transcription regulation ( 10 ) , dna replication ( 11 ) , cellular signaling ( 12 ) , stress response ( 13 ) and so on .
aberrance of lysine acetylation and deacetylation is associated with various diseases and cancers ( 5,7,14 ) . in particular ,
acetylation was demonstrated to be implicated in cellular metabolism and aging ( 1517 ) , while one class of nad dependent hdacs of sirtuins might be potent drug target for promoting longevity ( 13,17 ) .
although a great number of efforts have been carried out during the past four decades , the functional contents of lysine acetylation are still far from fully understood . in this
regard , identification of acetylated substrates with their sites is fundamental for understanding the molecular mechanisms and regulatory roles of acetylation .
in contrast with labor - intensive and time - consuming conventional experimental approaches , recent progresses in acetylome with high - throughput mass spectrometry ( ms ) have detected thousands of acetylation sites . in 2006 , kim et al .
( 14 ) performed a large - scale identification of acetylome with an anti - acetyllysine antibody .
there were 195 acetylated proteins with 388 sites detected in hela cells and mouse liver mitochondria ( 14 ) . with a similar strategy , choudhary et al .
( 11 ) experimentally identified 3600 acetylation sites in human . in 2010 , zhao et al .
( 16 ) discovered 1047 acetylated substrates in human liver , and demonstrated acetylation playing a major role in metabolic regulation .
furthermore , two acetylomic studies revealed that the functions of lysine acetylation are conserved in escherichia coli ( 18 ) and salmonella enterica ( 15 ) .
since the number of known acetylation sites has rapidly increased , it is an urgent topic to collect the experimental data and provide an integrated resource for the community .
recently , several public databases , such as phosphositeplus ( 19 ) , hprd ( 20 ) , sysptm ( 21 ) and dbptm ( 22 ) , have already contained protein acetylation information . in these databases ,
both of n - terminal and n - lysine acetylation data were curated , while lysine acetylation sites are usually only a limited part of total sites .
for example , sysptm 1.1 contains 3001 acetylation sites in 2000 proteins , with only 345 lysine sites ( 11.5% ) in 397 substrates ( 21 ) . in dbptm 2.0 ,
2071 experimentally verified acetylation sites were collected in 1525 proteins , with only 792 lysine sites ( 38.2% ) in 299 targets ( 22 ) .
interestingly , hprd release 9 contains 4691 total sites in 1987 proteins , with 4420 lysine sites ( 94.2% ) in 1821 substrates ( 20 ) .
however , hprd database only focuses on human protein information ( 20 ) , while thousands of lysine acetylation sites in other species still remain to be collected . with the motivation to meet the desire for complete acetylomes , here we developed a novel database of compendium of protein lysine acetylation ( cpla ) . from the scientific literature in pubmed , we manually curated 3311 acetylated proteins with 7151 lysine sites ( table 1 ) .
in cpla database , the primary references and other annotations of these substrates were provided , while the protein  protein interaction ( ppi ) information was also integrated .
based on the gene ontology ( go ) and interpro annotations , we carried out an analysis of functional diversities and regulatory roles of lysine acetylation .
as 75.64% of total lysine acetylation sites are taken from homo sapiens , a potential human lysine acetylation network ( hlan ) among hats , substrates and hdacs was constructed , with 1019 ppis among 199 proteins .
interestingly , we revealed 1862 potential triplet relationships of hat - substrate - hdac , while at lease 13 were previously experimentally verified .
taken together , the cpla database might be an integrated resource for protein lysine acetylation and provide useful information for further experimental or computational considerations . 

( % ) homo sapiens2585540975.64salmonella typhimurium1902603.64mus musculus1875527.72escherichia coli1492593.62saccharomyces cerevisiae44851.19others1565868.20total33117151100sub . , number of substrates ; site , number of acetylation sites ; per . , percentiles of acetylation sites .
to ensure the quality of cpla database , we searched the pubmed with a major keyword  acetylation and collected experimentally identified lysine acetylated proteins with their sites from more than 18  500 published articles ( before 1 march 2010 ) . to avoid missing data
after all substrates with unambiguous acetylation lysines were collected , we searched the uniprot knowledgebase ( 23 ) to obtain the corresponding protein sequences and associated annotation information .
the theoretical pi ( isoelectric point ) and mw ( molecular weight ) were calculated for each protein ( http://www.expasy.org/tools/pi_tool.html ) ( 24,25 ) . in cpla database ,
we took experimental ppis from several major public databases ( on 10 april 2010 ) , such as hprd ( 20 ) , biogrid ( 26 ) , dip ( 27 ) , mint ( 28 ) and intact ( 29 ) .
in addition , a well - known pre - predicted database of string ( 30 ) was also used .
all proteins were mapped to the uniprot sequences by blast . for human , we collected a total of 59  481 experimental ppis in 12  221 proteins and 1  212  607 predicted ppis in 16  523 proteins , respectively .
the cpla 1.0 database contains 7151 lysine acetylation sites in 3311 substrates ( table 1 ) .
particularly , 1742 ( 24.4% ) acetylation sites in 726 proteins are collected from non - human species ( table 1 ) .
the online service and local packages were implemented in php  +  mysql  +  javascript and java 1.5 ( j2se 5.0 ) , separately . moreover ,
the search option ( http://cpla.biocuckoo.org/search.php ) provides an interface for querying the cpla 1.0 database with one or several keywords such as gene / protein names , uniprot i d or cpla i d , etc .
for example , if the keyword  stat3 is inputted and submitted ( figure 1a ) , the result will be shown in a tabular format , with the features of cpla i d , uniprot accession and protein / gene names / aliases ( figure 1b ) . by clicking on the cpla
i d ( cpla-000136 ) , the detailed information for human stat3 will be shown ( figure 1c ) .
the acetylation information , including acetylated positions , flanking peptides , experimental reagents or upstream hats , and primary references are provided . the protein sequence , go annotation , domain organization , molecular weight , computed/ theoretical ip and ppi information are also presented . 

users could click on the cpla i d ( cpla-000025 ) to visualize the detailed information .
users could click on the cpla i d ( cpla-000025 ) to visualize the detailed information .
furthermore , we provided three additional advance options , including ( i ) advance search , ( ii ) browse and ( iii ) blast search ( supplementary figure s1 ) .
( i ) advance search : in this option , users could use relatively complex and combined keywords to locate the precise information , with up to two search terms . the interface of search - engine permits the querying by different database fields and the linking of queries through three operators of  and , 
( ii ) browse : instead of searching for a specific protein , all entries of cpla database could be listed by species name ( supplementary figure s1b ) .
( iii ) blast search : this option was designed for finding related information in cpla database quickly . the blastall program of ncbi blast packages ( 31 ) was included in cpla 1.0 database ( supplementary figure s1c ) .
users can input a protein sequence in fasta format for searching identical or homologous proteins .
recent progresses toward understanding the full functional content of acetylome have experimentally revealed several thousands of lysine acetylated substrates with their sites . besides experimental efforts ,
the current available computational resources were summarized and listed in supplementary table s2 . among these researches ,
database development is particularly important for integrating experimental data from heterogeneous sources , and providing a high quality benchmark for further experimental or computational designs .
although several public databases ( 1922 ) have already maintained the acetylation information , the lysine acetylation is usually collected together with another less controlled n - terminal acetylation . in this work
, we only focused on protein lysine acetylation and manually curated 7151 lysine acetylation sites in 3311 proteins . since a large proportion of acetylation sites were taken from homo sapiens , we had the opportunity to analyze abundance and functional diversity of lysine acetylation in an acetylomic level .
we surveyed the go terms of 2585 acetylated proteins from uniprot annotations . using the human proteome as the background , we statistically calculated over - represented biological processes , molecular functions and cellular components in acetylome with the hypergeometric distribution ( p  <  0.01 ) .
the top five most enriched go entries in each category were shown in table 2 .
for example , the three most abundant biological processes such as translational elongation , rna splicing and mrna processing suggest that acetylation predominantly regulates gene expression in a post - transcriptional manner ( table 2 ) .
also , four most over - represented molecular functions such as atp binding , protein binding , rna binding and nucleotide binding suggest that acetylation modulates enzyme activity and protein interaction ability ( table 2 ) . in addition
, the statistical analysis of cellular components revealed acetylated proteins to be highly enriched in distinct cellular compartments .
for instance , 30 and 62% of cytosol and mitochondrial matrix proteins are acetylated , respectively ( table 2 ) . for more detailed information ,
the top 15 most over - represented go terms and interpro domains were shown in supplementary tables s3 and s4 . 

table 2.the top five most enriched go terms of biological processes , molecular functions and cellular components in human acetylomedescription of go termacetylomeproteomee - ratiop - valuen ( % ) n ( % ) the top five most enriched biological processes  translational elongation ( go:0006414)69 ( 2.80)93 ( 0.52)5.443.35e-40  nucleosome assembly ( go:0006334)43 ( 1.75)79 ( 0.44)3.991.17e-17  rna splicing ( go:0008380)70 ( 2.84)188 ( 1.04)2.733.50e-16  interspecies interaction between organisms ( go:0044419)100 ( 4.06)333 ( 1.85)2.203.97e-15  mrna processing ( go:0006397)65 ( 2.64)193 ( 1.07)2.479.06e-13the top five most enriched molecular functions  atp binding ( go:0005524)398 ( 16.17)1473 ( 8.17)1.984.67e-46  protein binding ( go:0005515)798 ( 32.06)3996 ( 22.16)1.459.00e-35  rna binding ( go:0003723)186 ( 7.56)557 ( 3.09)2.459.49e-34  structural constituent of ribosome ( go:0003735)71 ( 2.89)155 ( 0.86)3.361.99e-22  nucleotide binding ( go:0000166)85 ( 3.45)255 ( 1.41)2.445.75e-16the top five most enriched cellular components  cytosol ( go:0005829)298 ( 12.11)1009 ( 5.60)2.164.71e-42  mitochondrial matrix ( go:0005759)83 ( 3.37)139 ( 0.77)4.386.33e-37  nucleoplasm ( go:0005654)124 ( 5.04)333 ( 1.85)2.731.12e-27  nucleolus ( go:0005730)133 ( 5.40)425 ( 2.36)2.291.84e-21  cytosolic small ribosomal subunit ( go:0022627)29 ( 1.18)36 ( 0.20)5.902.24e-19num . , number of proteins annotated ; per .
the top five most enriched go terms of biological processes , molecular functions and cellular components in human acetylome num .
the acetylation and deacetylation of proteins are carried out by hats and hdacs , which antagonistically and dynamically control protein function .
combined with experimental and predicted ppis , we constructed a potential hlan among hats , substrates and hdacs , with 1019 ppis of 199 proteins ( supplementary table s5 ) .
if only experimental ppis are considered , the core hlan contained 369 ppis among 77 proteins , including 12 hats and 12 hdacs ( figure 2 ) . from the whole hlan
if a substrate is a hat or hdac , it should be acetylated or deacetylated by a different hat or hdac .
we carefully surveyed scientific literature and found that at least 13 triplet interactions were experimentally identified ( supplementary table s6 ) .
for example , gaughan et al . ( 32 ) observed that tip60 ( kat5 ) and histone deacetylase 1 ( hdac1 ) regulate the transcriptional activity of androgen receptor ( ar ) through changing its acetylation status , and form a kat5-ar - hdac1 relation ( supplementary table s6 ) .
for instance , ep300 acetylates bcl6 at k379 and inhibits its function , while deacetylases were not clearly identified ( 33 ) . in our results , the ep300-bcl6-hdac5 , ep300-bcl6-sirt2 , ep300-bcl6-hdac11 , ep300-bcl6-hdac3 , ep300-bcl6-hdac2 and ep300-bcl6-hdac8 suggested that bcl6 might be deacetylated by multiple hdacs ( supplementary table s6 ) .
moreover , human gcma / gcm1 was reported to be acetylated by cbp / crebbp at k367 , k406 and k409 ( 34 ) . in our results , the relations of crebbp - gcm1-hdac3 , crebbp - gcm1-hdac3 ,
crebbp - gcm1-hdac1 and crebbp - gcm1-hdac4 proposed that at least four hdacs might deacetylate gcm1 ( supplementary table s6 ) . 

green node : hat ; blue node : hdac ; yellow node : hat that is acetylated ; purple node : hdac that is acetylated ; pink node : substrate . a core hlan identified from experimentally identified ppi data .
green node : hat ; blue node : hdac ; yellow node : hat that is acetylated ; purple node : hdac that is acetylated ; pink node : substrate . taken together
the statistical analyses revealed functional diversity and enrichment of acetylation , while network studies generated a large number of potentially useful results for further experimental or computational researches . the cpla database will be routinely updated if new acetylated substrates are reported .
funding for open access charge : national basic research program ( 973 project ) ( 2010cb945400 , 2007cb947401 ) ; national natural science foundation of china ( 90919001 , 30700138 , 30900835 , 30830036 , 31071154 ) ; chinese academy of sciences ( info-115-c01-sdb4 - 36 ) .
 as a reversible post - translational modification ( ptm ) discovered decades ago , protein lysine acetylation was known for its regulation of transcription through the modification of histones . 
 recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation . 
 although acetylation is comparable with other major ptms such as phosphorylation , an integrated resource still remains to be developed . in this work 
 , we presented the compendium of protein lysine acetylation ( cpla ) database for lysine acetylated substrates with their sites . from the scientific literature , we manually collected 7151 experimentally identified acetylation sites in 3311 targets . 
 we statistically studied the regulatory roles of lysine acetylation by analyzing the gene ontology ( go ) and interpro annotations . 
 combined with protein  
 protein interaction information , we systematically discovered a potential human lysine acetylation network ( hlan ) among histone acetyltransferases ( hats ) , substrates and histone deacetylases ( hdacs ) . 
 in particular , there are 1862 triplet relationships of hat - substrate - hdac retrieved from the hlan , at least 13 of which were previously experimentally verified . 
 the online services of cpla database was implemented in php  +  mysql  +  javascript , while the local packages were developed in java 1.5 ( j2se 5.0 ) . 
 the cpla database is freely available for all users at : http://cpla.biocuckoo.org . 