ubiquitination is an important type of post - translational modification in which an isopeptide bond is formed between the c - terminus of ubiquitin and a lysine residue from either a substrate or another ubiquitin molecule ( 1 ) . in addition to its original role in protein degradation ( 2 ) , ubiquitination regulates other cellular processes , including transcription , cell cycle , dna repair , apoptosis and receptor endocytosis ( 3 ) . thus , aberrations of ubiquitin  proteasome system function in all the above - mentioned processes have been implicated in the pathogenesis of human diseases , ranging from inflammatory , neurodegenerative muscle - wasting , to various forms of malignancies ( 4 , 5 ) . ubiprot is the first database on ubiquitination ( 6 ) , which focuses on the properties of ubiquitinated proteins per se , with data from different species combined . the data in ubiprot are mainly sourced from several high - throughput studies , and do not include information of ubiquitination cascade enzymes . the other ubiquitination database is a yeast database [ saccharomyces cerevisiae ubiquitination database ( scud ) ] focusing on ubiquitination cascades ( 7 ) . scud has collected almost all known enzymes involved in the ubiquitination process of yeast and has grouped them into reasonable classes . e3miner is a text - mining tool for literature search ( 8) , and appears to replace labor - intense manual curation with machine learning approach . although e3miner contains many useful e3-centered information , obtaining high accuracy in the text - mining method is difficult . ubiquitination is a conserved biological process from yeasts to humans ( 1 , 912 ) . understanding of the human ubiquitination system has gradually become clearer as more enzymes and ubiquitinated proteins have been discovered ( 1315 ) . however , no database demonstrating those of the human ubiquitination system exists . hubiquitome was constructed based on published papers in pubmed , and all the ubiquitination proteins and cascades were experimentally validated . hubiquitome provides a user - friendly interface through which information can be easily retrieved , including e1 , e2 and e3 substrates , deubiquitination enzymes ( dubs ) and the relationship among these elements . ubiquitin lysine sites or sequences are provided if they were identified in the reference papers . if some e3 functions are in the form of complexes , a complex name would be provided . hubiquitome aims to provide as many precise information as confirmed by experiments in the ubiquitination cascades . the ubiquitination cascade data documented in the current version , hubiquitome 1.1.1 , were collected manually by searching the pubmed database for primary research articles published or e - published up to the time of the present study with a list of keywords ( e1 ubiquitin or e2 ubiquitin - conjugating enzyme or e3 ubiquitin ligases limits : humans ) . studies that described experimentally identified relationships between e2 and e3 and e3 and substrate were included . experimental evidence includes in vitro reaction of e3 and substrate , immunoblot with ubiquitin antibody , autoradiogram of isotope - labeled ubiquitin , tag - labeled ubiquitin detection , substrate degradation by proteome , substrate stability detection and so on . additional information , such as e3 complex components and ubiquitinated sites and sequences , are important features of hubiquitome . seventeen terms of dubs with identified substrates were collected and added after e3-substrate cascade terms . hubiquitome 1.1.1 currently contains 1 e1 enzyme , 12 e2 enzymes , 138 e3 ligases or e3 ligases complexes , 279 different substrate proteins and 17 dub terms . all the proteins and the related papers in the website are hyperlinked to uniprot and pubmed . search : hubiquitome can be used to search for an enzyme or substrate using the uniprotkb protein accession number or the uniprotkb protein entry name . for example , input ' mdm2 ' inside the searchbox and search . however , users can deduce that the same e2 could be used with other e3-substrates reactions that share the same e3 . for example , a paper ( pmid:18784257 ) reported that mdm2 ubiquitinate ut2 uses ub2d1 as e2 . thus , ub2d1 might also be used as e2 in mdm2-mediated ubiquitination of other substrates , such as runx3 , pde4d and aqp2 . in some cases , e3 functions in the form of a complex . for example , ku70 and mdm2 function together to ubiquitinate ccne1 . a complex name is given if e3 functions as a complex . some experiments identified the ubiquitinated lysine sites and sequence motifs , such as mdm2 ubiquitinate runx3 at lysines 94 and 148 . the experimentally identified ubiquitinated lysine sites and sequence motifs are given as soon as they are available . the experimental evidence ( mass spectrometry , mutation or both ) identifying the ubiquitinated lysine sites are also included . although the number of dubs is small than that of e3s , we include them in the cascades , for example , ubp7 have been reported to be a dub for daxx in a paper ( pmid : 20153724 ) . mdm2 and ubp7 arranged in one row has the only meaning of sharing the same substrates . the pubmed i d is hyperlinked to its source page at ncbi , and each protein is ultimately hyperlinked to major biological databases like uniprot and entrez gene . 
 figure 1.general view using  mdm2 as an example . blast : the hubiquitome blast reports lysine sites in submitted sequences that match with ubiquitinated lysine sites in the hubiquitome database . thirty - five unique lysine sites were found to be ubiquitinated by known e3s ( excluding those identified by mass spectrometry without e3 information ) . thus , the biological context of the blast results should be evaluated by the user . submit and download : users can submit their own proteins or even entire ubiquination cascades to hubiquitome . contributors just need to fill out the excel form provided at the submission page and then send it back to the database administrator . all data in hubiquitome are freely available for download as tab - delimited text files without password protection for academic users . functional analysis of e3 substrates : e3 can be divided into two main classes ( ring : really interesting new gene and hect : homologous to e6-ap c - terminus e3 ) according to the domain structure they contain ( 13 , 15 ) . other kinds of e3s contain few numbers , such as u - box and phd e3s ( 20 , 21 ) . whether different e3 classes prefer to catalyze functionally different substrates has never been reported . in this article , the biological functions of substrates catalyzed by ring or hect and other e3s are analyzed using the online david tool ( 22 ) . ring substrates ( ring ) or hect and other substrates ( hect ) are submitted to the david website with the background of the whole human proteome . the functional annotation tool , gene ontology biological process ( goterm_bp ) , was selected to analyze the functional enrichment of the two kinds of substrates under default parameters . the enrichment was defined by benjamini - adjusted p - value designed to control false discovery rates . from the enriched terms in both the ring and hect group results , nine terms were selected according to the different enrichment and biological meanings in the two groups . the findings show that apoptosis - associated processes are enriched in ring e3s ( figure 2a ) , whereas gene transcription regulation - associated processes are enriched in hect and in other e3s ( figure 2b ) . ( a ) biological processes enriched by ring e3s ; ( b ) biological processed enriched by hect and other e3s . a , regulation of apoptosis ; b , apoptosis ; c , cellular response to stress ; d , cell cycle ; e : response to dna damage stimulus ; f : positive regulation of macromolecule metabolic process ; g : positive regulation of transcription ; h : positive regulation of rna metabolic process ; i : positive regulation of gene expression . functional analysis of ring e3s , hect and other e3s . ( a ) biological processes enriched by ring e3s ; ( b ) biological processed enriched by hect and other e3s . a , regulation of apoptosis ; b , apoptosis ; c , cellular response to stress ; d , cell cycle ; e : response to dna damage stimulus ; f : positive regulation of macromolecule metabolic process ; g : positive regulation of transcription ; h : positive regulation of rna metabolic process ; i : positive regulation of gene expression . database implementation : hubiquitome consists of a relational sqlite ( http://www.sqlite.org/ ) database and a jqueryui ( http://jquery.com/web interface ) , constructed in python ( http://www.python.org/ ) with django ( http://www.djangoproject.com/ ) and run via an apache server ( http://www.apache.org/ ) . hubiquitome , a database that focuses on the human ubiquitination system , collects exact ubiquitination information which have been experimentally validated . for every ubiquitinated substrate , hubiquitome provides the other ubiquitination cascades information in one row which includes e2 , e3 , ubiquitinated sites and sequences if possible . based on the collection , the biological process preferences of ring e3s and other e3s are found , indicating biological differences among various kinds of e3s . hubiquitome was designed to cover all experimentally validated ubiquitination associated proteins ( enzymes and substrates ) and cascades in humans . more ubiquitinated proteins will soon be discovered , although the current version of hubiquitome includes hundreds of them . these newly demonstrated ubiquitinated proteins will be added to hubiquitome when the database is updated . hubiquitome does not collect ubiquitinated proteins without e3 information produced by large - scale mass spectrometry methods ( 2325 ) . some of these proteins are even hyperlinked to the original papers to provide further information . second , based on the characteristics of ubiquitinated peptide sequences , the blast function provides researchers a tool to estimate possible ubiquitinations of unknown peptides . third , using the entire data which can be freely downloaded from the website , researchers can conduct further analysis . other interesting analyses , such as e2 and e3 substrate network construction , are expected . national basic research program of china ( 2011cba01104 ) ; national natural science foundation of china ( 60905014 and 31030041 ) . the funding agencies played no role in the study design , data collection , analysis , decision to publish or preparation of the manuscript . funding for open access charge : national natural science foundation of china ( 60905014 ) .
protein ubiquitination is an evolutionarily conserved and functionally diverse post - translational modification achieved through the sequential action of e1-activating enzymes , e2-conjugating enzymes and e3 ligases . 
 a summary of validated ubiquitination substrates have been presented and a prediction of new substrates have been conducted in yeast . however , a systematic summary of human ubiquitination substrates containing experimental evidence and the enzymatic cascade of each substrate is not available . in the present study , 
 hubiquitome web resource is introduced , a public resource for the retrieval of experimentally verified human ubiquitination enzymes and substrates . 
 hubiquitome is the first comprehensive database of human ubiquitination cascades . 
 currently , hubiquitome has in its repertoire curated data comprising 1 e1 enzyme , 12 e2 enzymes , 138 e3 ligases or complexes , 279 different substrate proteins and 17 deubiquitination enzyme terms . 
 the biological functions of substrates from different kinds of e3s were analyzed using the collected data . 
 the findings show that substrates ubiquitinated by ring ( really interesting new gene ) e3s are enriched most in apoptosis - related processes , whereas substrates ubiquitinated by other e3s are enriched in gene expression - associated processes . 
 an analysis of the data demonstrates the biological process preferences of the different kinds of e3s . 
 hubiquitome is the first database to systematically collect experimentally validated ubiquitinated proteins and related ubiquitination cascade enzymes which might be helpful in the field of ubiquitination - modification research.database url : http://202.38.126.151/hmdd/hubi/