結(jié)構(gòu)生物學(xué)精品課件

1、結(jié)構(gòu)生物學(xué)第1頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Recombinant 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 th

2、is protein is not required for normal growth. Hsp104 and ClpB are members of the AAA+ superfamily of ATPasesHsp104第2頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 Uses a Probabilistic Mechanism to Dissolve Disordered Aggregates From this figure, Hsp104DPL has WT ATPase activity, has minimal effect on total ATP

3、ase activity when mixed with WT Hsp104.(DPL, DWA, DWB and DPLDWB are all mutations)第3頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三ClpB Hexamers Are Tuned Differently to Hsp104 HexamersEbuffer dont influence the luciferase reactivationFHsp104DPL and ClpBDPLcaused a roughly linear decline in disaggregase activity第4頁(yè)，

4、共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Different Hsp104 and ClpB mutations have different disaggregase activity第5頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 Hexamers Tolerate Multiple Subunits Defective in ATP Hydrolysis and Substrate BindingDEHsp104DWB and Hsp104DPLDWB have little ATPase activity and incorporate into

5、WT hexamers as predictedKLHsp104DWB has a more severe dominant-negative effect than Hsp104DPLor Hsp104DPLDWB on Hsp104 function in thermotolerance and luciferase disaggregation in vivo第6頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三 It exerts an inhibitory effect if it is adjacent to a mutant subunit Hsp104DPLDWB第7頁(yè)

6、，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 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

7、that necessitated Hsp70 and Hsp40, whereasa-synE46K, Ab42, and Q81 amyloids were generally morerefractory第8頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三CDClpB had limited ability to disaggregate amyloid with or without Hsp70 (DnaK) and Hsp40 (DnaJ)第9頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三EClpB from T. Thermophilus was unable

8、 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

9、effect第10頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三ClpB Reactivates Disordered Aggregates More Effectively Than Hsp104ClpB was more effective than Hsp104 in disordered aggregate dissolution第11頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 Uses a Distinct Mechanism to Resolve Toxic Oligomers and AmyloidsDisassembly of a-syn

10、A30P oligomers, a-synA30P amyloid, and Ure2 prions by Hsp104 was very sensitive to Hsp104DPL (AC), Hsp104DWA (DF), andHsp104DPLDWB (GI)第12頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 Switches Mechanism to Disaggregate Distinct Sup35 Prion StrainsHsp104 subunits might collaborate differently to disaggregate d

11、istinct 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 temperatures第13頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Remodeling each NM prion strain required a different mode of inters

12、ubunit 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 第14頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三Hsp104 Switches Mechanism to Disaggregate Disordered Aggregates versus PrionsAHsp104-catalyzed

13、 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 c

14、an effectively disaggregate luciferase when diverse ATP:ATPgS mixtures populate its NBDs.第15頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三 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 di

15、sordered aggregates versus amyloid. 第16頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三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 aggregate

16、s and could not remodel NM25第17頁(yè)，共20頁(yè)，2022年，5月20日，11點(diǎn)53分，星期三FHsp104D704NDPLDWB 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 Hsp104L4