1. Aim In principle proteins are able to spontaneously fold into their native structure. Protein folding reactions, however, turn out to be rather inefficient as folding often results in a significant fraction of misfolded and aggregated protein. As a result cellular organisms have evolved a sophisticated ATP-dependent machinery of molecular chaperones to assist protein folding. Chaperones play a determining role in all aspects of protein quality control (PQC), including protein synthesis, translocation, disaggregation and degradation. It is known that some chaperones (such as Hsp70s) play a role in almost every aspect of PQC, while others are dedicated to specific pathways such as de novo folding, refolding, disaggregation or degradation and antigen presentation. Although the decisional mechanism of protein translocation by means of signaling peptides is relatively well understood, it is still totally unknown on what basis the PQC determines whether a particular misfolded protein needs to be refolded or degraded. Given the inefficiency of protein folding and the resulting high metabolic cost of protein synthesis (it is estimated that only between 5-30% of synthesized proteins result in native protein in vivo), it is extremely important that the PQC is able to determine the most cost-effective option between recycling or replacement of misfolded protein. The SWITCH laboratory developed the algorithm TANGO to predict protein aggregation and has used it extensively to study the sequence characteristics of aggregation-prone polypeptides in more than 20 entire proteomes. This work showed that there is strong evolutionary pressure against protein aggregation. This finding led to the discovery of so-called 'gatekeeper' residues that specifically oppose aggregation. Typically gatekeeping is performed by flanking aggregation-prone hydrophobic sequences with charged residues. Importantly, our work showed that strongly aggregating sequences are preferentially flanked by positive residues while mildly aggregating sequences are more often flanked by negative residues. Interestingly in vitro studies have shown that chaperones have a preference for binding positively charged gatekeepers but can in some cases (as with the Hsp60 chaperones) increase the refolding rate of negatively charged substrates. The main aim of the SWITCH laboratory, therefore, is to determine whether gatekeepers play a role in executive decisions of the protein quality machinery. The approach of the laboratory is to combine computer modeling with in vitro and in vivo experimentation. The present research proposal focuses on the exploration of chaperone ligand recognition by structural modeling. Although some experimental studies have already been devoted to chaperone specificity, most of these have only extracted general properties of chaperone ligands such as hydrophobicity and preference of positive charges. However, in order to analyse whether chaperone recognition can provide a signal that induces PQC routing, a position specific recognition profile of chaperones needs to be established. Here we will determine the position-specific recognition mode of several chaperone families including Hsp60, Hsp70 and Hsp90. 2. Objectives * Study the substrate specificity of Hsp70 homologues in an array of species using a combinatorial library of in silico peptide scans. * Create a predictor tool for predicting affinity of a peptide for a specific chaperone based on peptide sequence profiles. * Validate the predictor tool by comparing predicted chaperone-substrate stability results with the results of binding affinity experiments. * Create a website that allows public use of the predicting tool. * See if aggregation tendency of peptides is linked to the binding affinity and recognition ability of chaperones. Do chaperones exploit the existence of "gatekeepers" to recognize aggregation-prone sequences? * Is the chaperone machinery within a species trained to recognize species-specific aggregation-prone sequences? If yes, what are the sequence determinants? * Study the effect of mutations in chaperone binding sequences or mutations in chaperones that can lead to a loss of chaperone-substrate binding, eventually promoting misfolding or aggregation. * Explore whether chaperones distinguish between amyloid precursors and aggregate (TANGO) precursors.
|Effective start/end date||1/10/07 → 30/09/13|
Flemish discipline codes
- Biological sciences