Database Construction And Its Application Of Protein Phosphorylation And Ubiquitination Modifications

Post-translational modification(PTM)is one of the most important regulatory mechanisms of biological processes in vivo.The aberrance of PTM system is closely associated with human diseases.As the two most common types of PTMs,protein phosphorylation and ubiquitination have complex crosstalk,and involve in the regulation of almost all biological processes,such as liquid-liquid phase separation(LLPS).With the in-depth studies of PTMs and the rapid development in mass spectrometry-based PTMome,a large number of modification regulators,substrates and sites have been experimentally characterized.The key question is,how to integrate PTM data and systematically analyse the crosstalk among PTM types.Consequently,this article collected,integrated,and annotated the data of protein phosphorylation and ubiquitination.Then,two comprehensive databases for PTMs were constructed,and the regulations of phosphorylation and ubiquitination in LLPS were systematically analyzed.The identification of the modification site in proteins is the fundament of understanding the modification function.Focused on protein phosphorylation,1 451 629 newly identified phosphorylation sites(p-sites)in 195 351 phosphoproteins were collected from highthroughput phosphoproteomic studies by literature curation.Meanwhile,known p-sites from 13 public databases were also integrated.Then,the eukaryotic phosphorylation site database(EPSD),which contained 1 616 804 experimentally identified p-sites in 209 326 phosphoproteins from 68 eukaryotic species,was designed and constructed.Moreover,based on the knowledge of 100 additional resources that covered 15 aspects,EPSD carefully annotated the phosphoproteins and p-sites of eight model organisms.Compared with other databases,the p-sites in EPSD increased by 174.9%,with a total data size of 14.1 GB.Modification regulators directly control the modification state of substrates.The integrated annotations for ubiquitin and ubiquitin-like conjugation database(i UUCD 2.0),which included 136 512 known and computationally detected ubiquitin and ubiquitin-like regulators,was developed for the ubiquitin and ubiquitin-like conjugation.Then,i UUCD2.0 performed the detailed annotations for 7 138 regulators of eight model species,based on 67 public databases in 11 aspects,and provided an online database with interactive retrieval and sharing of data.Furthermore,based on EPSD dataset,the phosphorylation of regulators was analyzed and used to predict their upstream protein kinases,and found that the ubiquitin and ubiquitin-like regulators have complex phosphorylation regulations.PTM is a key regulation mechanism of liquid-liquid phase separation.Based on the PTM data of EPSD and i UUCD 2.0 databases,the phosphorylation and ubiquitination of LLPS-associated proteins from the constructed database named data resource of liquidliquid phase separation(Dr LLPS),were systematically analysed.The results reveal that the phosphorylation may involve in LLPS by the cooperative regulation of multi p-sites or the activity regulation of ubiquitin and ubiquitin-like regulators.In summary,this article focused on the protein phosphorylation and ubiquitination,and developed two integrated databases for PTMs,through the collection,integration and comprehensive annotation of PTM data.The new databases were not only used to systematically analyze the crosstalk between phosphorylation and ubiquitination during LLPS,but also provide important data resources for further analysis of PTMs.