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The role of charges in protein folding, aggregation and homeostasis

Boek - Dissertatie

Most proteins are only functional upon the acquisition of a specific three-dimensional structure, their native fold. Upon their ribosomal birth however, proteins are elongated, linear concatenations of amino acids. This necessitates an intricate folding reaction, by which the unfolded molecule is shaped into its functional conformation. A major driver in this process is hydrophobicity: in the aqueous cytosol, it is energetically favorable to bury hydrophobic amino acids in the protein core, while leaving polar residues exposed. Furthermore, the protein backbone engages in specific hydrogen-bonding patterns yielding secondary structure elements such as a-helices and b-sheets. Unfortunately, these prerequisites for globular protein folding, i.e. hydrophobicity and a propensity for b-sheet formation, are also key drivers of protein aggregation. In this process, short stretches of mostly hydrophobic amino acids self-assemble through b-strand interactions and interdigitation of their sidechains, forming highly structured and highly stable ensembles commonly known as cross-b structures. Since the protein segments driving aggregation are mostly buried in the native state, aggregation usually precludes proper protein folding, and thereby function. Moreover, it is becoming increasingly clear that the presence of aggregated structures can be toxic to cells. Given that the same physicochemical properties that drive protein folding also drive aggregation, the latter is not a rare phenomenon. Indeed, it has been suggested that most proteins, under the right conditions and given enough time, have an inherent tendency to form cross-b structured aggregates. Both the ubiquity of aggregation and its detrimental consequences are reflected in the increasing number of human disorders that are being linked to the aggregation of specific proteins, with around 50 identified up to date. The study of protein aggregation has allowed for a determination of the physicochemical properties that drive it, which in turn made possible the development of algorithms that predict segments of proteins with an inherent tendency to engage in cross-b interactions. An analysis with one of these algorithms, called TANGO, across almost 30 different proteomes revealed that most proteins contain at least one, and usually several Aggregation-Prone Regions (APRs). The same study also showed that there is a strong enrichment of charged residues and Proline in the residues directly flanking the APRs. All of these residue types reduce the aggregation propensity of the APR they flank, and are therefore referred to as GateKeeper residues (GKs). As such, the GKs form a protein-intrinsic defense mechanism against protein aggregation. On top of such protein-intrinsic measures to reduce aggregation propensity, cells dispose of an intricate network of components which maintain protein homeostasis by shielding APRs, unfolding and refolding misfolded species, dislodging monomers from aggregated states, degrading terminally misfolded proteins and generally guiding proteins towards their native fold. This network is known as the Protein Quality Control (PQC) system. It is essential for cellular life, has been conserved throughout evolution and is one of the major energy consumers in the cell.The primary objective of this PhD was to understand whether the charged GKs - Arg, Lys, Asp and Glu - differ in terms of their aggregation-breaking potential as, although these residues have the same absolute charge, they are structurally very different. Indeed, we were able to show that basic residues Arg and Lys are intrinsically worse GKs than the acidic Asp and Glu, in large part because of their longer, more flexible sidechains. These allow them to move their charged moieties farther apart when incorporated into a tightly packed cross-b structure, reducing charge repulsion. Moreover, their longer aliphatic sidechains impart increased hydrophobicity, further favoring burial into an aggregate core. Although reducing gatekeeping potential, that same long aliphatic sidechain does make basic residues more compatible with the globular protein fold in that the backbone can be buried deep into the structure, while keeping polar atoms at the hydrophilic surface. Indeed, we were able to show that basic residues are less disruptive to protein structure than their acidic counterparts, and are more readily used as protective GKs for APRs buried deep in the protein core. Taken together, this means that some APRs are buried too far into the hydrophobic core to allow the use of the superior acidic GKs, and are therefore potentially inadequately protected from engaging in aggregation. In the final part of this work, we sought to understand how these potentially dangerous APRs are dealt with. Work from other groups over the last few decades has found that many elements of the PQC machinery in fact preferentially interact with basic residues (GKs) in a hydrophobic context (APRs), whilst avoiding acidic residues. An adequate explanation for this preference this however, has hitherto been lacking. We posited that through this charge preference, the PQC triages inadequately protected APRs, thereby homing in on regions most likely to disrupt the folding process. Indeed, we were able to show for one of these PQC elements, bacterial DnaK, that by binding basic residues over acidic ones it effectively compensates for the inferior aggregation-breaking potential of the inferior GKs. In summary, this work provides an explanation for why the PQC seems to be geared towards basic residues, and offers evidence of a co-evolution between molecular chaperones and aggregation gatekeepers.
Jaar van publicatie:2020