Mechanism Involved In The Functional Diversity Of Multiple Hsp70s In Myxococcus Xanthus

The 70-kDa heat shock proteins(Hsp70s)are highly conserved molecular chaperones that are widely distributed in all kingdom of life and act in a large variety of cellular process.Hsp70s have a remarkable capacity to maintain proteostasis by assisting in protein folding、assembly、degradation,and are induced to help proteins resisting various environmental stresses.Thus,Hsp70 plays an import role in protein quality control.Across all eukaryotes,cells encode multiple Hsp70 family members.The differential expression,abundance and subcellular localization for distinct Hsp70 isoforms all contribute to diversification of the cellular functions of Hsp70s.Similar,prokaryotic cells also encode multiple Hsp70s.However,many previous studies have focused on the heat-shock inducible Hsp70,while the functions and significances of other isoforms remain unclear.Owing to the universality and importance of Hsp70s,the study of functional diversity and mechanisms of multiple Hsp70s can further understand the protein quality control of prokaryotes.Myxobacteria are a group of Gram-negative bacteria that widely distributed and are characterized by the complex social behavior and the huge genome.The model strain of myxobacteria,Myxococcus xanthus DK1622 possesses a 9.14-Mb genome,which contains many duplicate genes,including different hsp70s.Here,we investigated the functional diversity and its mechanisms of multiple Hsp70s in DK1622,and got the following results:1.Performed conserved domain analysis in representation prokaryotic genomes.Profiling the existance of multiple Hsp70s in prokaryotes.We screened the existence of Hsp70s in 5551 representative prokaryotic genomes and retrieved 9292 Hsp70 superfamily proteins,which can be divided into several types(PRK00290、cd10170 and CbnR proteins)according to the conserved domain.The number of hsp70 genes in a genome range from zero to 23 and 99.1%of these genomes contain one or more hsp70 genes.the PRK00290 protein was the most widely distributed Hsp70 types,present in 98.6%of the prokaryotic genomes,while the cd10170 and CbnR proteins were only present in 36.7%and 4.4%.Moreover,2295 x genomes possessed two or more hsp70 genes,accouting for 41.3%of the total prokaryotic genomes.Among them,38 sequenced myxobacterial genomes all encoded multiple Hsp70s from different isoforms.Thus,multiple Hsp70s are widely distributed in prokaryotes,especially in myxobacteria.2.Explored the cellular functions of multiple Hsp70s in DK1622,Characterized the functional diversity of highly conserved PRK00290 isoforms.In M.xanthus DK1622,there are 15 genes encoding the Hsp70 superfamily proteins.Based on phylogenetic analysis,we selected six hsp70 genes,two encoding the PRK00290 proteins and four encoding the proteins containing the c135085 or cd10170 domain,to investigate their cellular functions.Our results demonstrated that the highly conserved PRK00290 proteins play important roles in complex cellular life cycle.Among them,MxDnaK1 is undeletable,which not result of its extremely high transcriptional level.Moreover,MxDnaK1 is significantly induced by heat shock,which are always regarded as the properties of heat shock proteins.The other PRK00290 protein MxDnaK2 take part in several social behaviors:deletion of mxdnaK2 affects multicellular fruiting body development、sporulation and S-motility.Moreover,MxDnaK2 play an important role in oxidative resistance.In the contrast,deletions of each of the other four hsp70 genes decreased sporulation and oxidativeresistance at lower extents.Thus,MxDnaK1 and MxDnaK2 have significant functional diversification.3.Identification and comparision of MxDnaKl and MxDnaK2 substrates.To explore the mechanisms in functional diversity of MxDnaKs,we identificated and compared the substrates of them.Although their substrates have the same preference in structure、molecular weight and isoelectric point,they are largely nonoverlapping for substrate recognition and each have a lot of specific substrates.MxDnaK1 preferentially associates with proteins without hydrophobic core which are prone to be influenced by heat shock,that reflects the stress-induced function of MxDnaK1.In the contrast,MxDnaK2-specific substrates and MxDnaKl/2 substrates have more membrane proteins,that are consistent with their COG function prediction.And the MxDnaK1-specific substrates have a similar subcellular located distribution with the heat-induced Hsp70 of E.coli.Moreover,MxDnaKl and MxDnaK2 associate with different cochaperones.These results demonstrated that,MxDnaKl exhibits similar transcriptional regulation and substrate preference as canonical Hsp70s,while MxDnaK2 has evolved for unique cellular processes of myxobacteria.4.Explored the effects of functional region on MxDnaKs,confirmed that the NBD drives the functional diversity of MxDnaKl and MxDnaK2.According to the results of protein modeling and sequence analysis,the NBD and SBDβ are highly conserved in MxDnaK1 and MxDnaK2,the most differences lie in SBDa and CTT.However,exchange individual NBD of them badly destroy the cellular functions of MxDnaK,which lead to almost same phenotypes between region swapping mutants and deletion mutants.Moreover,exchange of NBD significantly decreased the substrate numbers and influent the recognization of cochaperone.Comparatively,exchange of SBDβ have a weaker influence on phenotypes and substrate recognization,and exchange of SBDα or CTT have almost no effect.The in vitro experiments demonstate that exchange of NBD retain the cochaperone-free holdase activity,but reverse the oligomeric states of MxDnaK1 and MxDnaK2,which might be the reason of importance of NBD in functional diversity.Thus,the highly conserved NBD drives the functional diversity of MxDnaKl and MxDnaK2.Here,by bioinformatic analysis and several experiments,we analyze the function diversity of Hsp70 proteins and its mechanisms.We found that multiple Hsp70s are widespread in prokaryotes;MxDnaK1 and MxDnaK2 have significant functional diversification.And explored the mechanisms of functional diversity from substrates recognization and structure differences.These results might have important implications for the protein quality control,functional diversity of duplicate genes,and the stress resistance of bacteria.It also provides a possible reference for the study of functionally specialized Hsp70s.