Effect of chaperones with intrinsically disordered regions (IDRs) on the fibrillogenesis of A? amyloid peptide

Molecular chaperones play essential and several roles in many cellular processes, including protein folding, targeting, transport, and are essential in fighting the consequences of protein misfolding and aggregation or enhancing disaggregation of toxic aggregates by clearance mechanisms [1]. There are several mechanisms by which chaperones exert their protective action, many of which involve protein disordered regions. These segments can assume multiple distinct conformations upon binding to different partner proteins. Examples are provided by chaperones Hsp70 (E. coli DnaK), Hsp60 (E. coli GroEL), and Hsp90 (E. coli HtpG) that undergo (ATPase)-driven conformational changes in their interaction with client proteins [2]. In the case of Hsp60 and its homolog GroEL, both the C-terminal and N-terminal disordered segments project into the central, substrate-binding cavity and contribute to chaperone activity [3,4]. GroEL apparently acts on folding proteins by preventing them from ending up in a thermodynamically unfavourable conformation, rather than by actively accelerating the rate of folding (entropic pulling). Local motion in GroEL is frozen by formation of a binary complex with partially folded proteins [5]. Moreover the C termini that are disordered in the ATP-free apo-GroEL result to be more ordered in the ATP-bound form [6]. Nothing in this regard has been demonstrated for the human chaperonin Hsp60, neither it is known whether, like GroEL, Hsp60 belongs to the class of ATP-dependent chaperones [2]. There are some differences involving the disordered regions of the two proteins. In particular, two of the four residues R452, E461, S463 and V464 that reside in one of the protein unordered segments and are essential for the GroEL double ring formation, are different in the corresponding Hsp60 positions [7]. Moreover, the "two-stroke engine" mechanism, necessary for GroES release from GroEL/GroES complex, is not obligatory for Hsp60, due to the very lower affinity to its co-chaperonin Hsp10, hence it is able to function as an efficient "one stroke engine"[7]. In this study we explored the functional consequences of Hsp60 and GroEL specific disorder features by investigating the effect of the chaperones on the fibrillogenesis of A? amyloid peptide involved in Alzheimer Disease. These analyses have been performed by Thioflavin T fluorescence assay, by Circular Dichroism and we are planning a SAXS experiment on 5.2 Elettra Beamline (Trieste, Italy) in the next future. Moreover, binding parameters of A?/GroEL and A?/Hsp60 have be quantified by isothermal titration calorimetry. The experiments have been performed in the presence and in the absence of ATP and at different concentration values. This allowed us to hypothesize a mechanism of action of the chaperones among the most likely in this context (refolding by "entropic pulling" or holding processes) [8] and to clear the functional role for disorder, also offering insight in the field of prevention and therapy in Alzheimer Disease, and, in general, of amyloid pathologies. 1. Muchowski PJ et al. (2005) Modulation of neurodegeneration by molecular chaperones, Nature Reviews Neuroscience 6, 11-22. 2. Bardwell JCA and Jacob U (2012) Conditional disorder in chaperone action, Cell 37(12), 517-5. 3. Tompa P and Csermely P (2004) The role of structural disorder in the function of RNA and protein chaperones, FASEB J. 18(11), 1169-75. 4. Machida K et al. (2008) Hydrophilic residues 526 KNDAAD 531 in the flexible C-terminal region of the chaperonin GroEL are critical forsubstrate protein folding within the central cavity, J Biol Chem. 283(11), 6886-96. 5. Gorovits BM and Horowitz PM (1995) The molecular chaperonin cpn60 displays local flexibility that is reduced after binding with an unfolded protein, J Biol Chem. 270(22), 13057-62. 6. Clare DK et al. (2012) ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin, Cell 149, 113-123. 7. Nielsen KL and Cowan NJ (1998) A single ring is sufficient for productive chaperonin-mediated folding in vivo, Mol Cell. 2, 93-100. 8. Evans CG et al. (2006) Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro, J Biol Chem. 281(44), 33182-91.