![]() Beyond this simple anti-aggregation activity, groups of chaperones are able to carry out more sophisticated functions, such as folding or dis-aggregating proteins. Some chaperones, such as Hsp70, accomplish this task by interacting reversibly with exposed hydrophobic regions, limiting aberrant ( i.e., non-native) contacts. In their simplest form, the “job” of the chaperones is to bind clients and protect them from aggregation. The heat shock proteins are abundant and conserved through all kingdoms of life, suggesting that they are an ancient way of protecting proteomes. The largest class of molecular chaperones is the heat shock proteins 1, which are named for their apparent molecular mass (in kDa): Hsp90, Hsp70, Hsp60, Hsp40 and the small heat shock proteins (sHsps). One of the major products of this research field has been the identification of putative PPI drug targets within the chaperone network, which might be used to change chaperone “decisions” and re-balance proteostasis.ġ. Introduction to the Chaperone Network. ![]() In addition, these molecules have provided leads for the potential treatment of protein misfolding diseases. Indeed, chemical biology has played a particularly important role in this effort, as molecules that either promote or inhibit specific PPIs have proven to be invaluable research probes in cells and animals. Guided by these efforts and –omics approaches to measuring PPIs, new advances in high throughput chemical screening, specially designed to account for the challenges of this system, have emerged. Structural biology methods, including X-ray crystallography, NMR and electron microscopy, have all played important roles in visualizing the chaperone PPIs. This Account will discuss the efforts of our group and others to map, measure and chemically perturb the PPIs within the molecular chaperone network. The key to understanding chaperone-mediated proteostasis might be to understand how PPIs are regulated. Somehow, this collection of PPIs draws together chaperone families and creates multi-protein subnetworks that are able to make the “decisions” of protein quality control. Many of these motifs have the same binding surfaces on shared partners, such that members of one chaperone class often compete for the same interactions. The PPIs of the chaperone network have a wide range of affinity values (nanomolar to micromolar) and involve many distinct types of domain modules, such as J domains, zinc fingers and tetratricopeptide repeats. PPIs also link chaperones and their clients to other cellular pathways, such as those that mediate trafficking ( e.g., cytoskeleton) and degradation ( e.g., proteasome). These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the “hand off” of clients between them. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Further, once a chaperone binds a client, how does it “decide” what to do with it? One clue comes from observations that individual chaperones engage in protein-protein interactions (PPIs) – both with each other and with their clients. Remarkably, these activities are carried out by only ~180 dedicated chaperones in humans. ![]() To perform these diverse tasks, chaperones need the malleability to bind nearly any “client” protein and the fidelity to detect when it is misfolded. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.Molecular chaperones play a central role in protein homeostasis (aka proteostasis) by balancing protein folding, quality control and turnover. Conclusion: The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Materials & methods: Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds. Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. We report the structure-guided optimization of the carbazole-based stabilizer PK083. Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. ![]()
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