| Under high temperature stress,proteins are more likely to suffer damage,and then lose their function.The timely removal of these damaged proteins is essential for cells to maintain normal structure and function.Lon protease,as a kind of ATP-dependent protease widely existing in organisms,mainly initiates the degradation of damaged proteins by identifying the exposed hydrophobic core region of the protein,thereby preventing their aggregation from causing irreversible damage to cells,and promoting the turnover of amino acids.At present,the structure and function of Lon in E.coli,Bacillus subtilis and some pathogenic bacteria such as Brucella,Salmonella and Vibrio cholerae have been extensively studied,but there are few researches about the Lon of extreme environment microorganism.Deinococcus radiodurans R1 contains two Lon protease homologues(DR?1974 and DR?0349),but there is no specific research on their function.Therefore,this study investigated the functions of Lon1 and Lon2 proteases under high temperature stress by conducting bioinformatics analysis,mutant construction,transcript expression analysis and proteomic analysis of Lon1 and Lon2.1.The homologous analysis showed that there were two Lon protein homologs in D.radiodurans R1,encoded by dr?1974 and dr?0349 on chromosome 1,respectively;the identities of amino acid sequence alignment were 49.0% with the Bacillus subtilis Lon protease.The typical N-terminal domain,ATPase domain(AAA+)and proteolytic domain of Lon A-type proteases were found in Lon1 and Lon2.Evolutionary analysis showed that Lon1 and Lon2 are most closely related to Thermus.Therefore,we hypothesized that both DR?1974 and DR?0349 are Lon A proteases and named DR?1974 as Lon1,DR?0349 as Lon2.2.The results of q RT-PCR showed that the transcript levels of both lon1 and lon2 were significantly up-regulated under 48?,with lon1 and lon2 being up-regulated 13.6-fold and 10.5-fold at48°C for 2 h,respectively;while there was no significant change in transcript levels under hydrogen peroxide,Na Cl and UV stress conditions.The deletion of lon1 and lon2 did not affect the biomass of the strain under normal culture conditions,but resulted in the strain being sensitive to high temperature and Na Cl.In addition,the electron microscopic observation showed that the deletion of lon1 caused the abnormal cell division of D.radiodurans,forming giant cells and damage cell membrane,the comlon1 could make the strain completely restore its normal morphology.The above results suggest that lon1 is involved in the morphogenesis of cells,lon1 and lon2 may be directly involved in high temperature stress.3.The proteomics of the D.radiodurans wild type,?lon1 and ?lon2 under normal condition and 48? for 2 h were analyzed by Label-free mass spectrometry,the results showed that both Lon1 and Lon2 were involved in DNA repair,molecular chaperones and proteases,metabolism of proteins related to energy metabolism in response to high temperature stress.The Fold Change of differentially expressed protein(DEPs)of ?lon1 was significantly lower than?lon2,and the expression of Lon2 was up-regulated 4.52-fold in the absence of Lon1,but the deletion of Lon2 did not cause the up-regulated expression of Lon1.However,compared to the 10 unique differentially expressed proteins associated with Lon1,33 unique proteins were associated with Lon2 were differential expression in response to high temperature stress.Among them,12 amino acid synthesis-related proteins,several proteins involved in TCA cycle and glycolysis with significantly up-regulated expression,and the above results suggest that Lon2 plays a major function in response to high temperature stress.4.To analyze the substrate characteristics of Lon1 and Lon2,11 potential substrates shared by Lon1 and Lon2 were initially screened based on the Fold Change according to the proteome results,including the molecular chaperone proteins DR?1172,DR?0607,DR?0146 and DR?0128;the transcriptional regulatory protein DR?2548;the DNA repair proteins DR?2263,DR?0326,membrane-associated protein DR?2508,and metabolism-related proteins DR?1720,DR?A0147,and DR?1131.In addition,Gro EL,a kind of chaperone,and DR?1172,a class of molecular chaperone proteins that plays an important role in abiotic stresses of D.radiodurans,which were validated as a substrate for of Lon1 and Lon2 by Western Blot.In summary,lon1 and lon2 of D.radiodurans are specifically induced by high temperature stress are associated with high temperature stress resistance.However,Lon1 is mainly involved in the morphogenesis of cells,while Lon2 responds to high temperature stress by participating in several biological processes. |
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