Effects Of Simulated High Altitude Hypoxia On Protein Phosphorylation In Brain Of Neodon Fuscus

The processes of sensing and regulating oxygen in organisms are very complicated.The response mechanism mainly involves genome,transcriptome and proteome,among which proteome and post-translational modification proteome are important tools to study such problems.Currently,the mechanism of biological adaptation to high altitude hypoxia is a research hotspot,but there is a lack of research on the mechanism of proteome and phosphoproteome in the biological response to hypoxic environment.Neodon fuscus(N.fuscus)has lived in the alpine meadow area of the Qinghai-Tibet plateau for generations,while Lasiopodomys brandtii(L.brandtii)is distributed in the grasslands of Inner Mongolia.The two species are close in taxonomic status and have similar habitat types,which lead them to be good species for the study of high altitude hypoxia.In this study,N.fuscus was taken as objects and L.brandtii as contrasts.After screening with personalization and fatness,20 individuals were placed in the laboratory hypoxia chamber which simulated the oxygen environment at the altitude of 100 m,3000m,5000 m and 7000 m,and the brain tissues were collected after 24-hour treatment.Then,the quantitative proteome and phosphoproteome of these two species were obtained by high-throughput mass spectrometry(MS).The expression trends of differential proteins were determined through differential protein identification methods,while the methods of GO and KEGG enrichment,and protein domain prediction were used to analyze the differential proteins of specific expression trend types.Furthermore,GPS 2.1 algorithm and the protein-protein interactions were adopted to predict the site-specific kinase-substrate relations(ss KSRs),and the corresponding kinase-site phosphorylation networks(KSPNs)were constructed to analyze the regulation mechanism of the networks in hypoxia adaptation.The purpose of this study was to reveal the molecular mechanism of proteins and thier phosphorylation level in N.fuscus brain tissue in response to high altitude hypoxia,and to provide theoretical basis and technical support for the research of high altitude hypoxia adaptation.The main results are as follows:1.The personalization scores of N.fuscus and L.brandtii were normally distributed and had significant differences(P < 0.001),wheras the relative fatness did not conform to the normal distribution and had no significant differences.20 individuals of N.fuscus and L.brandtii were scelected for hypoxic experiments from the personalized score range of 4-12 and 12-18 with the relative fatness of 4-6.5 range.2.Through the quality control of MS spectrogram and peptide length distribution,the proteome and phosphoproteome of N.fuscus and L.brandtii accorded with the analysis criteria were obtained.At the level of proteome,5628 and 5683 proteins of N.fuscus and L.brandtii were identified,of which 4368 and 4374 were quantified,respectively.At the phosphorylation level,9840 and 8972 phosphorylation sites(p-sites)on 3262 and 3069 substrates of N.fuscus and L.brandtii were identified,among which 9444 and 8661 p-sites on 3189 and 3001 proteins were quantified,respectively.3.Taking Fc=1 and P<0.05 as the standard,the numbers of significant differential expressed proteins(DEPs)in N.fuscus and L.brandtii were 733 and 469,respectively.1)The protein expression trends of these two species can be divided into 10 categories with the same trend in general,while the DEPs were concentrated in the up-regulated group and down-regulated group.2)N.fuscus up-regulated the proteins related to nucleotide metabolism,oxidative phosphorylation,glycolysis and citric acid cycle,as well as the domains of substance transport and nucleic acid binding function;And the related proteins involved in protein complex decomposition,depolymerization and protein processing,as well as the domains of cytoskeleton and protein interaction were down-regulated in N.fuscus.3)L.brandtii up-regulated the proteins related with lysosomal pathway and NADP binding,and the domains about protein interaction and RNA binding,while it down-regulated proteins involved in energy coupled ion transmembrane transport and oxidative phosphorylation,as well as the domains of cytoskeleton and protein interaction.4)Two species responded to altitude-related changes in oxygen levels through the proteins related with synaptic tissue,ion transmembrane transport,cytoskeletal-dependent cytoplasmic transport,coenzyme binding and ATPase activity.4.Based on Fc=1.5 and P<0.05,there were 682 and 541 proteins with significantly different in phosphorylation levels of N.fuscus and L.brandtii respectively.1)The sequential and structural preferences of p-sites in these two species showed that the proportion of p-Serine was the highest,and p-sites were enriched in the disordered region,protein surface and coli structure(P < 0.001).Different types of p-sites have different sequence characteristics,while most of the phosphorylated proteins were in the nucleus.2)The analysis of DEPs in phosphorylation at different altitudes indicated that N.fuscus responded to high altitude hypoxia by increasing the phosphorylation level of proteins related to neuroendocrine system,digestive system and platelet activation,wheras L.brandtii responded to high altitude hypoxia through enhancing synaptic plasticity and regulating the phosphorylation level of proteins involved in endocrine system and oxytocin signaling pathway.3)The prediction of the kinase and substrate relationship showed that 4984 and 2863 site-specific kinases and substrates relationship were predicted of N.fuscus and L.brandtii respectively.The network analysis indicated that the kinases of N.fuscus involved in m RNA splicing and calcium homeostasis regulation significantly had significant changes in activity,while the kinases of L.brandtii involved in apoptosis induction and autophagy regulation significantly changed significantly in activity.4)TGF-? and MAPK signaling pathways as well as cell cycle-dependent protein kinases may be the common pathway involved in hypoxic regulation of N.fuscus and L.brandtii.Main conclusion:1)N.fuscus and L.brandtii may cope with the changes of oxygen by changing the protein expression of synapse tissue,neurotransmitter production and secretion,energy coupled ion transmembrane transport,and regulating the phosphorylation levels of proteins related with the cell cycle regulation,TGF-?,and MAPK signaling pathways.2)N.fuscus may up-regulate the proteins related to anaerobic respiration,downregulate the proteins involved in protein processing,change the phosphorylation level of protein related to the regulation of neuroendocrine system,blood oxygen and blood glucose,and enhance the activity of calcium homeostasis regulated kinases in response to hypoxia.3)L.brandtii may up-regulate the proteins related to lysosome and NADP binding,down-regulate the proteins involved in energy-coupled ion transmemmembrane transport and oxidative phosphorylation,enhance the phosphorylation level of protein related to synaptic plasticity,the endocrine system regulation and the oxytocin signaling pathway,and reduce the kinases activity that induce apoptosis and autophagy in response to hypoxia.