Charting the Proteostasis Network as a Therapeutic Target

 

MARC BREHME

PROTEOSTASIS THERAPEUTICS INC. AND CCSB, DANA-FARB

 

Proteins very rarely act in isolation, but are organized in networks and large multiprotein molecular machines whose function relies on its individual components. While each protein constantly faces acute and chronic challenges to its structural and functional integrity, molecular chaperones ensure proper folding of nascent polypeptides into their native three-dimensional conformations and are involved in the prevention of misfolding, aggregation and degradation. Eukaryotic protein homeostasis, or ‘proteostasis’, enables healthy development and aging at a cellular and organismal level thereby protecting against disease. Proteostasis deficiencies lead to metabolic, oncological, neurodegenerative and cardiovascular disorders. A wealth of supporting evidence has recently been gathered that small-molecule or biological ‘proteostasis regulators’ can readjust proteostasis and ameliorate some of the most challenging diseases of our era. The ‘proteostasis network’ represents a set of interacting pathways, including the heat shock and unfolded protein response pathways. To generate a unified view of the human proteostasis network, we are systematically mapping its binary protein-protein interactions using a highly specific, high-throughput yeast two-hybrid (Y2H) system. Initially, we are using ~260 human chaperones and their co-factors, the ‘human chaperome’, to elucidate its central roles in proteostasis, and then we will expand the analysis to the complete proteostasis network. The Y2H screens are done using the hORFeome v5.1 collection consisting of 15,483 ORFs and representing 12,794 non-redundant human genes. We aim at characterizing the resulting networks in disease conditions to facilitate strategies to restore the ‘diseased’ networks back to their ‘healthy’ states using proteostasis regulators. We will also use Y2H to analyze the interactomes of single residue missense mutant proteins known to act through folding defects as compared to their wildtype counterparts in order to identify both edgetic (edge-specific) and more global changes to their interaction profiles as a consequence of folding defects. These analyses will be performed under various conditions such as temperature shifting or treatment with candidate proteostasis regulator compounds. We are aiming to establish experimental conditions for rapid testing of a large array of different disease-associated mutations in order to specifically screen for those that act through folding defects. Towards the discovery of proteostasis regulator compounds and their as therapeutic agents in proteostasis deficiency disease intervention we will carry out proof-of-concept experiments in order to demonstrate alleviating effects on mutant protein folding efficiency, interactome architecture as well as on the systems and phenotypic integrity of the mutant organism.