ClpB3 from Arabidopsis thaliana displays chaperone activity per se

CECCARELLI, EDUARDO; ROSANO, GERMÁN; PARCERISA, IVANA

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentina de Bioquímica y Biología Molecular (SAIB) y 9 Sociedades más

All cells have developed a protein network involved in maintaining the proteome homeostasis, particularly after periods of stress. When massive protein aggregation occurs due to heat exposure, cell survival relies on the ClpB/Hsp100 subfamily of molecular chaperones which promote the solubilization and reactivation of protein aggregates. The bacterial chaperone ClpB and its homologue in yeast, Hsp104 are the most extensively studied disaggregases. Like many members of the AAA+ superfamily, ClpB/Hsp104 protomers form ring-like homohexameric complexes.The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer. ClpB and Hsp104 collaborate with the DnaK or Hsp70 chaperone system, respectively, to dissolve protein aggregates both in vivo and in vitro, with DnaK/Hsp70 playing a main function in the regulation of ClpB/Hsp104 activity. In the present study, we have explored the substrate recognition by ClpB from A. thaliana chloroplasts (AtClpB3) and the role of its cpHsp70 chaperones in this recognition. We demonstrated that AtClpB3 ATPase activity is stimulated by caseins, poly-lysine and glucose-6-phosphate dehydrogenase (G6PDH) aggregates, but not by luciferase aggregates. In addition, cpHsp70s did not enhance AtClpB3 activity, albeit the presence of caseins. Likewise, AtClpB3 could mediate G6PDH disaggregation independently from cpHsp70 chaperones, recovering the activity of the denatured enzyme up to a 65 %. Besides, it has distinct properties not previously reported for Hsp104or ClpB, as it interacts more efficiently with the aggregated G6PDH in the presence of the non-hydrolysable ATP analog AMP-PNP rather than ATPγS. This is the first evidence that a ClpB disaggregating activity can be exerted without the participation of other proteins and establishes a first approach to understanding AtClpB3 substrate preferences.