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. A list of cascaded contexts for Mdm2 appears (). E1 is common to all the cascades, so it is omitted. Papers reporting E3-substrates reactions do not always inculde E2 information. To be precise, only E2 information in the cascades reported in previous papers are shown. 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 ID is hyperlinked to its source page at NCBI, and each protein is ultimately hyperlinked to major biological databases like Uniprot and Entrez Gene.
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). The hUbiquitome Blast is useful for retrieving possible ubiquitinated lysine sites of interesting proteins. However, Blast does not give meaningful significant values. 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
). Other kinds of E3s contain few numbers, such as U-box and PHD E3s (20
). 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. Detailed information can be found in supplement materials
. The findings show that apoptosis-associated processes are enriched in RING E3s (A), whereas gene transcription regulation-associated processes are enriched in HECT and in other E3s (B). This result indicates that different classes of E3s may prefer different biological processes.
Figure 2. 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 (more ...)