Structural and functional analysis of macromolecular machines involved in the protein quality control system

The protein quality control system is formed by a collection of chaperones and proteases that prevent accumulation of stress-induced protein aggregates in cells by refolding or degrading damaged proteins. These enzymes sequester their active sites in self-compartmentalized structures to prevent unwanted activity on folded proteins. I used electron microscopy complemented with biochemical methods to understand the molecular mechanisms of activation of two protein quality control components: the proteasome that constitutes the main proteolytic enzyme in eukaryotic cells and the bacterial periplasmic chaperone/protease DegP.;Regarding DegP, it has been described that this enzyme rearranges from a hexameric complex to larger oligomers formed by, either 12 or 24 monomers. My research in this area sought to understand the mechanism of substrate recognition as well as the factors determining the oligomeric state adopted by DegP. I found that the PDZ2 domain is essential for the integrity of the hexameric cage whereas PDZ1 recognizes the C-terminal residues of substrates targeted for degradation. This substrate recognition event is the initial step that triggers rearrangement of DegP from a hexamer to the larger active cages allowing the enzyme to enclose substrates of different sizes. DegP works in the context of large cages even at elevated temperatures. Together this work fills important gaps in our understanding of the functional model of these two enzymes and it provides new insights into their regulation.;My research on the proteasome focused on understanding the relative activation of this enzyme by the bovine PA200 and yeast Blm10 homologues to determine whether they are functionally related. I reconstructed a 3D cryo-EM map of Blml0-20S proteasome and found that Blm10 and PA200 proteins are structurally and functionally related. The two proteins activate peptide hydrolysis by the proteasome by inducing opening of the axial channel gates to allow access for substrate or product transport.