The Study On Protein Post-Translational Modifications Of Trypanosoma Brucei And T.Evansi And Their Biological Functions

Trypanosomes are unicellular vector-borne zoonotic protozoan parasites that cause substantial mortality and morbidity in mammalian hosts worldwide,and there is no vaccine available for trypanosomiasis,the search for new drug targets is urgent.Post-translational modifications（PTMs）introduce various chemical groups into specific amino acids of proteins and ultimately affect their functions by altering their physicochemical properties,spatial conformation,and binding capacity.PTMs are very important factors in determining the distribution,structure and function of proteins in cells.Dissecting the regulatory mechanisms of protein PTM processes,identifying the key sites where PTMs occur,and determining the key enzymes that mediate modifications and de-modifications are essential for future drug development.As an early branching organism,the energy metabolism and histone sequences of African trypanosomes are distinctly different from those of traditional eukaryotes;however,we still lack a comprehensive study of the PTM regulatory network of African trypanosomes,and the overall dynamic regulation mechanism of trypanosomal PTMs remains to be elucidated.In this study,we firstly performed the first and most comprehensive study of the protein post-translational modifications group in two African trypanosomes,Trypanosoma brucei and T.evansi,using high-throughput proteomics technology,including in-depth analysis of 10PTMs,and identified a total of about 40,000 modification sites and 150 histone markers,providing the landscape of protein PTMs in African trypanosomes.The PTMs are widely involved in various critical life processes,including gene expression,protein synthesis,and energy metabolism.Since these two African trypanosomes have great biological differences but their genomes are extremely similar,we analyzed the differential expressed and modified proteins of the two trypanosomes to investigate whether the biological differences between these two closely related African trypanosomes are caused by dynamic changes in protein expression and post-translational modifications.The results show that the two trypanosomes exhibit different PTM profiles,regulatory pathways and networks.PTMs involved in the redox system were mainly highly expressed and modified in T.brucei,while motility-related proteins were expressed and modified at higher levels in T.evansi.The establishment of multiple databases of PTMs in both parasites provides new directions in the study of the biological mechanisms involved in the life course of trypanosomes with different life cycles.Many substrates or cofactors of PTMs are intermediate products of cellular metabolism,among which lactate was long considered a metabolic waste product,but recent studies have shown that it can not only be reused for energy but also mediates the occurrence of protein lactation modifications.To investigate the regulation between the level of glycolysis and the level of lactylation in African trypanosomes,we regulated the level of glycolysis by adding inhibitors.Our results showed that with the decrease in glycolysis rate,the level of lactate in the parasites gradually decreased,and the level of lactylation also decreased specifically.The specific change in lactylation level was verified by immunoprecipitation of histone H3.By examining the changes in transcriptional levels of gene regulators which capable of lactylation,we found that this regulation significantly affected the expression of many gene regulators.To find the key enzymes regulating lactylation and thus further understand the regulatory mechanism of lactylation in African trypanosomes,we screened for the silence information regulator 2-related protein 5（SIRT5）protein,an NAD⁺-dependent deacetylation modifying enzyme associated with cellular metabolic regulation,and performed prokaryotic expression,purification and polyclonal antibody preparation of SIR2rp3,the most homologous SIR2rp3 in T.brucei,and demonstrated its natural expression in trypanosomes and localized in mitochondria.Further,we constructed the SIR2rp3 knockout strain using the CRISPR/Cas9system and found that it could regulate a variety of acylations including lactylation;through animal infection experiments,we demonstrated that SIR2rp3 had a significant effect on the pathogenicity of the parasite.The survival rate of mice infected with the SIR2rp3^-/-strain was100%,and all mice survived after being infected with the wild strain,indicating that the SIR2rp3^-/-strain stimulated complete immunity against infection in mice.In conclusion,this study we describe the first comprehensive multiple PTM-omics atlas of African trypanosomes and reveals the specific PTMs among different trypanosomes through the in-depth analysis of modified sites proteins;the level of glucose metabolism in African trypanosomes regulates the level of lactylation,which provides energy and regulates various biological functions of trypanosomes;SIR2rp3 is a key enzyme for lactylation in trypanosomes,its deletion deprives the parasite of its pathogenicity to mice,but stimulates the host to develop immunity against infection.These findings will help us to characterize the systemic biology of trypanosomes and promote the development of new antitrypanosomal drugs and vaccines.