结构生物学.pptx

Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Nonamyloid ClientsMorgan E.DeSantisCell 151,778793,November 9,2012Recombinant HSP104 containing 908 amino acids and having a molecular mass of 102 kDa HSP104 is a molecular chaperone required for stress toleranceHsp104 can protect cells against hightemperature and high concen-tration of ethanol but mutation studies have shown this protein is not required for normal growth.Hsp104 and ClpB are members of the AAA+superfamily of ATPasesHsp104Hsp104 Uses a Probabilistic Mechanism to Dissolve Disordered Aggregates From this figure,Hsp104DPL has WT ATPase activity,has minimal effect on total ATPase activity when mixed with WT Hsp104.(DPL,DWA,DWB and DPLDWB are all mutations)ClpB Hexamers Are Tuned Differently to Hsp104 HexamersEbuffer dont influence the luciferase reactivationFHsp104DPL and ClpBDPLcaused a roughly linear decline in disaggregase activityDifferent Hsp104 and ClpB mutations have different disaggregase activityHsp104 Hexamers Tolerate Multiple Subunits Defective in ATP Hydrolysis and Substrate BindingDEHsp104DWB and Hsp104DPLDWB have little ATPase activity and incorporate into WT hexamers as predictedKLHsp104DWB has a more severe dominant-negative effect than Hsp104DPLor Hsp104DPLDWB on Hsp104 function in thermotolerance and luciferase disaggregation in vivo It exerts an inhibitory effect if it is adjacent to a mutant subunit Hsp104DPLDWBHsp104 Remodels Diverse Amyloids,Whereas ClpB Has Limited Activity Hsp104DWA was inactive,but Hsp104 remodeled the majority of these amyloids in a manner that wasslightly enhanced by Hsp70(Ssa1)and Hsp40(Sis1),whichwere inactive alone.Rnq1 prions were an exception that necessitated Hsp70 and Hsp40,whereasa-synE46K,Ab42,and Q81 amyloids were generally morerefractoryCDClpB had limited ability to disaggregate amyloid with or without Hsp70(DnaK)and Hsp40(DnaJ)EClpB from T.Thermophilus was unable to disaggregate amyloid,whereas the A.Thaliana homolog,Hsp101,remodeled various amyloidsFamyloids inhibited ClpB ATPase activity by 30%,whereas disordered aggregates stimulated by 20%Hsp104 ATPase activity was stimulated by disordered aggregates and several amyloids,but some amyloids had no effectClpB Reactivates Disordered Aggregates More Effectively Than Hsp104ClpB was more effective than Hsp104 in disordered aggregate dissolutionHsp104 Uses a Distinct Mechanism to Resolve Toxic Oligomers and AmyloidsDisassembly of a-synA30P oligomers,a-synA30P amyloid,and Ure2 prions by Hsp104 was very sensitive to Hsp104DPL(AC),Hsp104DWA(DF),andHsp104DPLDWB(GI)Hsp104 Switches Mechanism to Disaggregate Distinct Sup35 Prion StrainsHsp104 subunits might collaborate differently to disaggregate distinct amyloid strains formed by the same protein.To examine this possibility,we exploited Sup35s prion domain,termed NM,which spontaneously forms different prion strains at different temperaturesRemodeling each NM prion strain required a different mode of intersubunit collaboration by Hsp104.Thus,NM4 remodeling was less sensitive than NM25 or NM37 to Hsp104DPL(Figures A,D,G),Hsp104DWA(B,E,H),Hsp104DPLDWB(C,F,I),or Hsp104DWB Hsp104 Switches Mechanism to Disaggregate Disordered Aggregates versus PrionsAHsp104-catalyzed luciferase reactivation was insensitive to a 20-fold excess of p370,whereas NM4 remodeling was inhibited and NM37 remodeling was abolishedBeffect of various ratios of ATP and ATPgS,a slowly hydrolyzable ATP analog.These activity profiles illustrate the adaptability of the Hsp104 hexamer,which can effectively disaggregate luciferase when diverse ATP:ATPgS mixtures populate its NBDs.Hsp104-catalyzed remodeling of NM4 was sharply inhibited by low fractions of ATPgS,and NM37 was even more sensitive,Thus,WT Hsp104 uses a distinct mechanism to disaggregate disordered aggregates versus amyloid.Key Middle Domain and NBD2 Residues Enable Hsp104 to Switch Mechanism Hsp104D704N had reduced ATPase activity,whereas Hsp104L462R had WT levels of ATPase activity.Both mutants had reduced ability to reactivate luciferase aggregates and could not remodel NM25FHsp104D704NDPLDWB subunits elicited an approximately linear decline in Hsp104D704N luciferase reactivation activity rather than the stimulation observed with WT Hsp104 or sharp inhibition observed with ClpBGDoping Hsp104L462RDPLDWB elicited a roughly linear decline in Hsp104L462R luciferase reactivation activity rather than the stimulation observed with WT Hsp104 Mechanisms of Intersubunit Collaboration for Hsp104 and ClpBThank you