INSITUFOLD · In situ analysis of chaperone mediated protein folding and stability

Molecular chaperones mediate protein folding and conformational maintenance. Failure of these fundamental cellular functions results in a breakdown of protein homeostasis (proteostasis) and facilitates the manifestation of diseases associated with protein aggregation. Much progress has been made in understanding the mechanisms of individual chaperone systems, such as the Hsp70s and the chaperonins, based on analyses in vitro. How these machineries function in the intact cell is not well understood, however. The goal of this proposal is to observe and characterize the dynamic action of the major chaperone systems and their interplay in intact cells in real time.Using live cell single molecule imaging combined with biochemical analyses, proteomic profiling, and genetic screening, we will address the following questions:i) How does the chaperone machinery assist protein chains during and after translation to achieve folding and assembly? We will develop single particle tracking to monitor the chaperone engagement of nascent chains in situ in mammalian cells.ii) How is the conformational stability of chaperone clients assessed, maintained and triaged in healthy cells and in cells burdened with pathological aggregates? We will track the interactions of metastable proteins with chaperones and proteolytic machinery.iii) What is the efficiency of protein folding across the proteome under normal conditions and in chaperone-deficient cells? Pulse proteolysis/mass spectrometry will be developed to analyze the folding of nascent polypeptides and will be scaled to analyze the entire proteome of different cell types. By depleting specific proteostasis components we will explore the capacity of the chaperone network for compensatory rewiring and identify vulnerable proteins with an obligate chaperone dependence.Overall, this work will employ new technology to advance our understanding of the chaperone system in protein folding and proteome maintenance at the cellular level.