Modulation of clpb activity by stabilizing ligands and crowders

Resumen

Molecular chaperones are proteins that help other polypeptides to reach their native conformation, and are not part of their final structure. They are important constituents of the protein quality control system. When severe stress conditions are maintained a prolonged period of time, the protein quality control system cannot avoid protein aggregation. Toxic protein aggregates can be either proteolyzed and recycled or reactivated by the combined action of chaperones of the Hsp70 and Hsp100 families. This Thesis deals with the study of ClpB, the bacterial representative of the Hsp100 family. First, we wanted to consider the effect that the environment in which ClpB operates might have on its biological properties. In particular, the consequences that the high concentration of macromolecules in almost all physiological media can have on the association equilibrium and on its chaperone activity have been characterized. The results presented in chapter 4 show that macromolecular crowding conditions shift the association equilibrium of ClpB towards its active, hexameric form, and activates its ATPase activity. Excluded-volume conditions also promote hetero-association of ClpB with the DnaK system, therefore enhancing the reactivation of protein aggregates. Second, we have also considered the possibility of using ClpB as a target to develop bactericidal compounds. The rationale for this consideration is that ClpB is a virulence factor required for the intracellular replication of many pathogens. To this aim, the interaction of 10.000 compounds with ClpB was analyzed by high-throughput screening method. Those that interacted with several conformation of ClpB (the apo and at least one of its nucleotide-bound conformations) were selected to further examine their effect on the biochemical properties of the chaperone, and on bacterial growth. Interestingly, two compounds displayed a temperature-dependent bactericide activity that relied on ClpB, since deletion is due to the inhibition of a biological process essential to face the thermal stress, in which ClpB is involved. The moderate toxicity of these compounds against human cell lines points to their possible use as leads for the development of new bactericides.