Data Integration And Bioinformatic Studies Of Hypoxia-associated Proteins And Protein Cysteine Modifications

Oxygen is an essential element for the survival of aerobic organisms.Decreased oxygen concentration(hypoxia)may threaten the survival of cells,tissues and individuals.Eukaryotes have evolved sophisticated oxygen-sensing systems and response mechanisms to adapt to low-oxygen conditions.In addition,hypoxia also exists in many physiological and pathological processes,becoming one of the research hotspots in evolution and biomedicine.At present,the research on hypoxia mainly focused on transcriptomics and proteomics.Post-translational modifications(PTMs)are chemical modifications of protein after transcription and translation.PTMs can affect the structure and function of protein by adding functional groups to amino acid residues.Among them,cysteine has high nucleophilicity,could be intensively modified by various PTMs,and participate in a wide range of biological processes such as redox homeostasis,signal transduction,autophagy and others.It has been presented that protein cysteine modifications(PCMs)play crucial roles in regulating hypoxia response.The rapid development of high-throughput technology promoted the accumulation of hypoxia-related proteins and substrates with sites for PCMs.However,these data are scattered in different literature,and how to collect and integrate these data to provide useful help for related research has become a challenge.Therefore,data integration and bioinformatic studies around hypoxia-related proteins and cysteine modifications were carried out in this work.We first developed an integrative database for hypoxia-associated proteins in animals(iHypoxia).Through literature curation and public databases integration,we collected 1,215 hypoxia-associated proteins validated by low-throughput experiments(LTE proteins)and 36,194 potential hypoxia-associated proteins identified by highthroughput experiments(HTE proteins)from 5 mammals.The detailed experimental information,including samples,hypoxic concentration and treatment time,expression patterns,and other data were carefully collected and integrated.In addition,we further expanded our data to 618,171 proteins in 50 animals by orthologous search.Compared with other databases,the iHypoxia contained more data,covered a wider range of animal species,and had more comprehensive annotation information.Enrichment analysis for human LTE proteins showed that hypoxia-associated proteins were significantly enriched in certain drug targets and oncogenes.By annotating known PTM sites to LTE proteins,it was found that most hypoxia-associated proteins can undergo extensive PTMs,especially phosphorylation,ubiquitination and acetylation.Secondly,an integrative database for protein cysteine modifications in eukaryotes named iCysMod was constructed.Through mining the experimentally identified PCM substrates and sites from published literature and databases,we totally obtained108,030 PCM events for 85,747 sites on 31,483 proteins from 48 eukaryotes for 8 types of PCMs,including oxidation,S-nitrosylation,S-glutathionylation,disulfide formation,S-sulfhydration,S-sulfenylation,S-sulfinylation and S-palmitoylation.With human dataset in iCysMod,the sequence features around the cysteine modification sites for each PCM type were analyzed,and the results indicated that various types of PCMs presented distinct sequence recognition preferences.Moreover,it was found that15,458 cysteine sites can be modified in various PCMs,indicating that different PCMs can crosstalk with each other to synergistically orchestrate specific biological processes.Based on the data of iHypoxia and iCysMod,a series of bioinformatic studies were carried out to analyze the regulation of PCMs in hypoxia-associated proteins.The results showed that hypoxia-associated proteins could be regulated by a variety of cysteine modifications,while hypoxia-associated proteins which were prone to PCM were significantly related to signaling pathways and human diseases.Through mapping the cancer mutations to the PCM sites of human hypoxia-associated proteins,it was found that somatic mutations more preferably occured in PCM regions than in nonmodified regions.In summary,through the collection,integration and annotation of the animal hypoxia-related proteins and eukaryotic PCM substrates with sites,the databases of iHypoxia and iCysMod were constructed,respectively.Based on that,the regulation of PCMs in hypoxia response was further analyzed.Our study would provide data resources and new strategies for further exploring the molecular mechanisms and the complicated regulatory networks of hypoxia response and cysteine modifications.