| Innate immunocytes play vital roles in the process of recognizing PAMPs and DAMPs,and mediating the clearance of pathogens and abnormal cells,thereby maintaining immune homeostasis.With the process of anti-viral immune responses in innate immunocytes such as macrophages and dendritic cells(DC),the overall metabolome and synthesis level of a series of metabolic-small molecules were altered,including lactate,itaconic acid,arginine,and succinic acid etc.,and these changes can influence ubiquitous protein post-translational modifications(PTM),such as acetylation,succinylation and other types of lysine acylation modifications.Several research have confirmed that the changes of metabolic molecules and PTMs in innate immunocytes benefit on immune responses,however,these metabolic changes and PTM changes may also be utilized by pathogens,therefore benefiting on pathogens’ immune escape.In 2019,one study reported that the existence of histone lactylation in macrophages,and this type of histone PTM mediate the function of immune negative regulation.However,the expression profiles and functions of lactylation in RNA virus-stimulated macrophages are still not clear.With the progress on the sensitivity of mass spectrometry(MS)and the development of proteomic technologies,analyzing proteomes from different types of pathological tissues and cells by MS and then discover critical physiological or pathological mechanisms from multiple proteomic information have become an important research strategy,such as identifying PTM sites which have important regulation functions,or combining with clinical data to screen disease progress/prognosis-related proteins/PTM.Based on research backgrounds as above,this study conducts the research as following:1.Found that the level of proteome lactylation in peritoneal macrophages was up-regulated under VSV stimulation.To investigate the role of protein lactylation in anti-viral innate immune response,first,we test the variation of Pan-lactylation in mice RNA virus-stimulated peritoneal macrophages,and we found the overall modification level of lactylation was upgraded on VSV-stimulated conditions.Utilizing lactate dehydrogenase(LDH)inhibitors GNE-140/Oxamate,the synthesis of lactate and the modification level of lactylation were undermined,meanwhile the level of innate immune response was enhanced.2.Found that the level of lactylation in peritoneal macrophages is negatively correlated with mice’s age.Based on the difference in the level of glycolysis and the level of lactic acid synthesis between young and old mice,we tested the level of Pan-lactylation in peritoneal macrophages from young mice and old mice.We found that the total level of lactylation in young mice is markedly higher than the level in old mice,and the response of innate immune signaling pathways in old mice are more powerful during the same conditions of VSV infection.In addition,adding lactylation inhibitors in peritoneal macrophages can further enhance the response of innate immune signals.To sum up,these results show that in the process of viral-infection of macrophages,the response level of innate immune signals are negatively correlated with the overall protein lactylation level.3.Identification of lactylation modified proteins in viral stimulated macrophages.Next,we focus on the identification of lactylation site and their potential functions in viral-stimulated macrophages.Utilizing proteomic technologies,we test the proteomes of non-infected and VSV-infected mice peritoneal macrophages by mass spectrometry,and then analyze lysine lactylation sites by two different algorithm——Maxquant and p Find.Based on the basic of software filtering,we further screening by the manual comparison of MS spectra,and we finally keep 26 lactylation sites from 25 different proteins.Then,we construct the Flag-target protein recombinant plasmid and overexpressed in HEK-293 T cells.Utilizing immunoprecipitation,we finally determine 8 credible lactylation sites in vitro.Furthermore,we construct lysine cites’ mutation plasmid of these 8 proteins,and results shows 5 proteins’ overall lactylation modification levels were significantly down-regulated after their lysine sites were mutated to arginine,these proteins are OXCT1,PPME1,STRAP,MAPK3,and GAPDH.4.Screening of regulation mechanisms of innate immune mediated by lactylation proteins.Furthermore,we constructed a dual-luciferase reporter gene system to study the response and function of these five proteins’ lactylation sites to the four innate immune genes including NF-κB,ISRE,IFNα and IFNβ through point mutations at the lactylation sites.As a result,we found that these lactylation modifications have certain regulatory effects on innate immune signals.Among them,the most significant is the mutation of lysine to threonine at position 277 of MAPK3(ERK1)which can significantly up-regulate the expression of luciferase transcribed by IFNβ promoter,and the mutation of lysine to threonine at position 64 of GAPDH can significantly up-regulate the expression of luciferase transcribed by ISRE,IFNα and IFNβ promoters.Therefore,we focus on these two sites as the next research target.Next,we further identified the actual presence of lactylation in 277 K of MAPK3 by mass spectrometry that MAPK3 proteins were overexpressed in 293 T cells and then purified by immunoprecipitation.In addition,we found the mutation of lysine to threonine in MAPK3 277 can significantly upregulate the luciferase transcription by the IFNβ promoter,thus this result further illustrates the regulatory effect of MAPK3 lactylation on the expression of IFNβ.5.Bioinformatics analysis of modification enzymes of lysine lactylation.What’s more,to explore the possible lactyl-transferase and delactyl-transferase,we utilized tumor proteome and acetylome MS data from clinical proteomic tumor analysis consortium(CPTAC)database to analyze the lactylation regulation potential of acyltransferases and deacylation enzymes.The prediction result shows that EP300 and KAT6 A have a higher probability of lactyl-transferase catalytic activity,HDAC3,HDAC4,and HDAC5 have a higher probability of delactyl-transferase catalytic activity.In summary,we first found that overall lactylation of proteins in peritoneal macrophages was up-regulated under VSV stimulation,and the level of lactylation of mouse peritoneal macrophages was negatively correlated with age,meanwhile time was negatively correlated with the response level of innate immune signaling pathways.Next,we identified six lactylation sites by proteomic mass spectrometry and molecular biology experiments.Then,we screened two lactylation proteins—GAPDH and MAPK3 that have regulatory functions on innate immune responses by experiments of dual luciferase reporter gene tests.These phenomena and results indicated that non-histone lysine lactylations play important regulatory roles in antiviral innate immune response of macrophages,and indeed,there are several lactylation proteins that have important functions to regulate the level of innate immune response.All in all,this study for the first time confirmed that macrophages have an overall up-regulation of lactylation under the infection of VSV,and the level of lactylation is negatively correlated with the response of innate immune signaling pathways.In addition,two lactylation proteins including GAPDH and MAPK3,were identified that have the function of regulating the level of innate immune response for the first time.These results of this article will provide important experimental basis for further clarifying and perfecting the regulatory function of lactylation in immunocytes and perfecting molecular regulation mechanisms of innate immune response,meanwhile providing new intervention ideas for clinical anti-infective therapy. |
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