Database, 2016, 1–14 doi: 10.1093/database/baw054 Original article Original article UbiNet: an online resource for exploring the functional associations and regulatory networks of protein ubiquitylation Van-Nui Nguyen1,2,†, Kai-Yao Huang1,†, Julia Tzu-Ya Weng1,3, K. Robert Lai1,3,* and Tzong-Yi Lee1,3,* 1Department of Computer Science and Engineering, Yuan Ze University, Taoyuan, 320, Taiwan, 2University of Information and Communication Technology, Thai Nguyen University, Vietnam and 3Innovation Center for Big Data and Digital Convergence, Yuan Ze University, 320, Taiwan *Corresponding author: Tel: +886928560313, Email: [email protected] †These authors contributed equally to this work. Correspondence may also be addressed to or K. Robert Lai. Email: [email protected] Citation details: Nguyen,V.-N., Huang,K.-Y., Weng,J.T.-Y., et al. UbiNet: an online resource for exploring the functional as- sociations and regulatory networks of protein ubiquitylation. Database (2016) Vol. 2016: article ID baw054; doi:10.1093/ database/baw054 Received 18 August 2015; Revised 30 September 2015; Accepted 20 March 2016 Abstract Protein ubiquitylation catalyzed by E3 ubiquitin ligases are crucial in the regulation of many cellular processes. Owing to the high throughput of mass spectrometry-based proteomics, a number of methods have been developed for the experimental determin- ation of ubiquitylation sites, leading to a large collection of ubiquitylation data. However, there exist no resources for the exploration of E3-ligase-associated regulatory networks of for ubiquitylated proteins in humans. Therefore, the UbiNet database was developed to provide a full investigation of protein ubiquitylation networks by incorporating experi- mentally verified E3 ligases, ubiquitylated substrates and protein–protein interactions (PPIs). To date, UbiNet has accumulated 43 948 experimentally verified ubiquitylation sites from 14 692 ubiquitylated proteins of humans. Additionally, we have manually cura- ted 499 E3 ligases as well as two E1 activating and 46 E2 conjugating enzymes. To delin- eate the regulatory networks among E3 ligases and ubiquitylated proteins, a total of 430 530 PPIs were integrated into UbiNet for the exploration of ubiquitylation networks with an interactive network viewer. A case study demonstrated that UbiNet was able to de- cipher a scheme for the ubiquitylation of tumor proteins p63 and p73 that is consistent with their functions. Although the essential role of Mdm2 in p53 regulation is well studied, UbiNet revealed that Mdm2 and additional E3 ligases might be implicated in the regulation of other tumor proteins by protein ubiquitylation. Moreover, UbiNet could identify potential substrates for a specific E3 ligase based on PPIs and substrate motifs. With limited knowledge about the mechanisms through which ubiquitylated proteins are VC The Author(s) 2016. Published by Oxford University Press. Page 1 of 14 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. (page number not for citation purposes) Page 2 of 14 Database, Vol. 2016, Article ID baw054 regulated by E3 ligases, UbiNet offers users an effective means for conducting prelimin- ary analyses of protein ubiquitylation. The UbiNet database is now freely accessible via http://csb.cse.yzu.edu.tw/UbiNet/. The content is regularly updated with the literature and newly released data. Database URL: http://csb.cse.yzu.edu.tw/UbiNet/. Introduction Apoptosis/ or Apoptosis inhibitor (IAPs) and Skp, Cullin, F- Protein ubiquitylation, involving ubiquitin conjugation at a box containing complex (SCF), are over-expressed in many target lysine residue, is carried out through a series of enzym- human cancers, and because evidence suggests that the in- atic activities such as E1 activation, E2 conjugation and E3 hibition of these enzymes may lead to growth suppression ligation (1), as illustrated in Supplementary Figure S1.To or apoptosis (19). Therefore, understanding of the mechan- date, high-throughput mass spectrometry (MS)-based prote- isms and interaction networks of E3 ligases may facilitate omics approaches have facilitated the characterization of the development of more efficient cancer treatments. many substrate proteins with ubiquitylation sites (2–5). In The biological significance of E3 ligases in cellular proc- particular, ubiquitylation of lysine residues was found to esses has spurred efforts in the bioinformatics domain. For function as a crucial modulator in transcriptional regulation, instance, E3Net (10) has presented a collection of 1671 signal transduction, development, apoptosis, endocytosis, E3-substrate relations between 493 E3s and 1277 sub- cell proliferation and cancers (6–8). In protein ubiquityla- strates in 42 organisms. Additionally, Sakiyama et al. (20) tion, E3 ligases are responsible for recognizing substrate sites built a database of proteins involved in the ubiquitin sig- for ubiquitin-mediated protein degradation (9). An E3 ligase naling cascade, and analysed their sequence similarities, could regulate and recognize multiple substrates in various domains and distributions across different species. Despite functional networks; alternatively, each substrate may be an increasing interest in ubiquitylated proteins and E3 lig- regulated by multiple E3s (10). These relationships could be ases, there is a lack of resources dedicated to mapping the organized into a network of E3-specific regulatory activities regulatory networks of E3 ligases for large-scale ubiquity- against multiple cellular pathways. As a result, complex cel- lation data. Therefore, the UbiNet database was developed lular processes could be characterized comprehensively with to provide an interactive network viewer for discovering the integration of an E3-specific functional regulatory net- protein ubiquitylation networks. To enable the compre- work by investigating the functional specificity of E3-specific hensive investigation of regulatory networks among E3 lig- substrates along with their networks (11). ases and ubiquitylated proteins, metabolic pathways and As E3 ubiquitin ligases are important in protein regula- protein–protein interactions (PPIs) were incorporated to tion, many studies attempted to characterize E3 structures, explore protein ubiquitylation networks. Moreover, in examine E3-mediated regulatory networks and investi- order to facilitate the study of protein ubiquitylation and gated E3-related diseases (12–15, 17). E3 ligases can be their functions, a web interface was developed for users to categorized based on their catalytic mechanisms. In gen- search for their proteins of interest. Published literature in- eral, E3 ligases involved in ubiquitylation can be classified formation related to E3 ligases, ubiquitylated proteins, into three classes, including the HECT homologous to E6- ubiquitylation sites and PPIs are also provided in this on- AP C-terminus (HECT), really interesting new gene line resource. Finally, case studies demonstrated that (RING) and RINGbetween-RING (RBR) types (16). UbiNet could help users identify E3 ligase-mediated ubiq- RING-type (really interesting new gene) E3s facilitate the uitylation networks and their biological roles. Within the transfer of ubiquitin from an E2 enzyme to the substrate, scope of our current knowledge about the relationships establishing binding between the E2Ub thioester and the among E3 ligases and ubiquitylated proteins, UbiNet pro- substrate; while E3s belonging to the HECT-type (homolo- vides potential E3 ligases for ubiquitylated proteins based gous to E6-AP C-terminus) and RBR-type (RING-be- on PPI information and substrate site specificities. tween-RING) function by transferring ubiquitin from an E2 to the cysteine containing active site in an E3 enzyme, Materials and Methods and subsequently to the substrate. The significance of E3-mediated ubiquitylation is further Database construction of UbiNet underscored by their association with diseases (18). Indeed, Figure 1 presents the flowchart for the construction of several E3 ligases, such as Mdm2/Hdm2, Inhibitor of UbiNet, including the database construction of E3 ligases Database, Vol. 2016, Article ID baw054 Page 3 of 14 and ubiquitylated proteins, functional and structural ana- a means of rationalizing the complementary efforts of the lyses, as well as the building of regulatory networks and PROSITE (31), PRINTS (32), Pfam (33) and ProDom (34) identification of ubiquitylation sites with substrate motifs. databases, is an integrated resource that provides essential In this study, experimentally verified data of human E1 information such as protein families, domains and func- activating enzymes, E2 conjugating enzymes and E3 ubi- tional sites. It has been reported that ubiquitylation regu- quitin ligases were manually curated from various data- lates the degradation of proteins, coordinates protein bases. In total, two distinct proteins for E1 activating cellular localizations and modulates PPIs. (35–37). Thus, enzymes were extracted from UUCD-Version 1.0 (21). The functional domain information could be utilized to infer number of distinct E3 and E2 enzymes, as well as the sour- the functional roles of ubiquitylation sites located in a spe- ces from which they were obtained, are listed in cific protein domain. Recently, the importance