Characteristics Of Protein Post-translational Modifications During The Intra-erythrocyte Developmental Cycle Of Plasmodium Falciparum

Malaria is still one of the deadliest diseases in the world.Plasmodium falciparum is the most pathogenic and the most harmful human malaria parasite,and the 48 h erythrocytic cycle is the main pathogenic period of Plasmodium falciparum.Although artemisinin-based combination therapy(ACT)is currently the most effective anti-malarial method,the emergence and worldwide spread of anti-artemisinin strains makes the mining of malaria vaccines and new antimalarial drugs imminent.However,there are still many important scientific problems to be revealed in the pathogeny biology and pathogenesis of malaria parasites so that many difficulties in malaria control cannot be solved.The regulation mechanism of malaria development and reproduction has always been an important direction of malaria research,and post-translational modifications(PTMs)seem to provide valuable ideas for the analysis of these scientific problems,and molecules involved in the addition and removal of protein modification have become important targets for the mining of biopharmaceuticals.At present,there have been many researches on the protein modification of P.falciparum,for example,histone acetylation,methylation,palmitoylation,phosphorylation,small ubiquitin(SUMO);and phosphorylation,acetylation,ubiquitination of non-histone proteins.The PTM research of P.falciparum explains the regulatory mechanism of the development and pathogenicity of the parasite to some degree.However,the current understanding of PTM on the regulatory mechanism of parasite development and pathogenesis is still partial.Current PTM analysis on P.falciparum has been limited to certain modification types and there are few studies on dynamic changes during the 48 h erythrocytic development stage,except for histone modifications.What's more,the knowledge on the posttranslational modification of the P.falciparum-infected red blood cells(p RBCs)of human is almost blank.Herein,ten kinds of PTM pan-antibodies(Tyrosine phosphorylation,Lysine acetylation,crotonylation,2-hydroxyisobutyrylation,ubiquitination,trimethylation,propionylation,malonylation,glutarylation and succinylation)were used as primary antibodies of Western blotting to identify the dynamic modification levels of P.falciparum-infected erythrocytes during the intra-erythrocyte development cycle.Tandem mass tag(TMT)labeling,mass spectrometry(MS)-based proteomics and PTM-omics were employed to identify samples of P.falciparum and its host erythrocytes at six time points(8,16,24,32,40,48 h after invasion),with healthy erythrocytes as controls.Dynamic modification levels of six PTM-omics(phosphorylation,acetylation,crotonylation,2-hydroxyisobutyrylation,N-glycosylation,and ubiquitination)were comprehensively analyzed during the 48 h intra-erythrocyte stage;and bioinformatics analysis was carried out from four aspects: the function,dynamic subcellular localization,and correlation of each modification.We drew schematic diagrams for the three key metabolic pathways,including glycolysis,pentose phosphate pathway,and purine salvage synthesis,and labeled the PTMs of the related regulatory enzymes.Then we screened and tested the antimalarial activity of regulatory enzyme inhibitors related to the six PTMs.In this study,we drew the most comprehensive dynamic maps of the six post-translational modifications of P.falciparum and the host proteins during the intra-erythrocyte developmental cycle for the first time,revealing the developmental changes of P.falciparum during the intraerythrocyte developmental cycle(IDC)from the perspective of PTMs.Among them,crotonylation,2-hydroxyisobutyrylation,and N-glycosylation are novel modifications in the field of P.falciparum epigenetics,and the dynamic protein modifications of the host red blood cells of P.falciparum is unprecedented.We found that PTMs were widely involved in important biological processes such as merozoite invasion,erythrocyte remodeling,immune evasion,gene expression,nutritional metabolism,and malaria pathogenicity.Throughout the intraerythrocyte cycle,the six modifications performed rapid and immediate dynamic regulation of proteins derived from P.falciparum and its host cells.Compared with healthy red blood cells,the phosphorylation and ubiquitination of P.falciparum-infected erythrocytes underwent significant changes,and the abundance of these two modifications decreased significantly during the first invasion.The N-glycosylation level of erythrocyte proteins changed extremely during the release and invasion stages of P.falciparum and many non-sialic acid-dependent erythrocyte receptors were modified by N-glycosylation,such as CR1,Basigin,etc.This research drew the most comprehensive modification map of histones and its variants of P.falciparum and marked the modification sites identified in previous studies,laying an important foundation for the analysis of the "histone code" and the study of histone-mediated gene expression regulation.Moreover,we screened proteins related to gene expression,and drew an interaction network based on the known protein interaction relationships and the abundance levels of the modification sites identified in this study.This research drew the most comprehensive modification map of histones and its variants of P.falciparum,and marked the modification sites identified in previous studies.Moreover,we screened proteins related to gene expression,and drew an interaction network based on the known protein interaction relationships and the abundance levels of the modification sites identified in this study.The results showed that in the process of gene expression,the structural changes of nucleosomes containing histones are most closely related to acetylation;the transcription process and translation initiation are mainly regulated by phosphorylation,while translation elongation and ribosome processing are modified by multiple modifications.The PTM analysis of the regulatory enzymes in the metabolic pathway of P.falciparum showed that the rate-limiting enzyme pyruvate kinase(PK)of the glycolytic pathway was acetylated and 2-hydroxyisobutyrylated in the early-trophozoite stage,and was crotonylated,2-hydroxyisobutylated and phosphorylated in the late stage.Hypoxanthine-guanine phosphoribosyltransferase(HPRT),the key enzyme of the purine salvage synthesis pathway,was 2-hydroxyisobutylated,acetylated and crotonylated.Glucose 6-phosphate dehydrogenase(G6PD,the key enzyme in the pentose phosphate pathway)in human was phosphorylated and 2-hydroxyisobutyrylated.The antimalarial activity test showed that the PTM-related enzyme inhibitors had different degrees of antimalarial activities,suggesting that the modified enzyme has a non-negligible potential for the mining of antimalarial drugs.Overall,we provided a comprehensive and in-depth view of the dynamic epigenetic networks governing the development of the deadliest malaria parasite P.falciparum and the resulted pathogenesis throughout the IDC.And our research involved the biology of malaria parasites from the overall PTM-omics level,laying an important foundation for pathogenic biology,cell biology,and drug mining of new antimalarial drugs.