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The Dynamic Composition of the Protein Chaperone Network: Unraveling Human Protein Disaggregation via NMR Spectroscopy

Periodic Reporting for period 2 - NMR-DisAgg (The Dynamic Composition of the Protein Chaperone Network: Unraveling Human Protein Disaggregation via NMR Spectroscopy)

Reporting period: 2020-03-01 to 2021-08-31

Molecular chaperones are a diverse group of proteins critical to maintaining cellular homeostasis. Aside from protein refolding, it has recently been discovered that certain combinations of human chaperones can break apart toxic protein aggregates and even amyloids that have been linked to a host of neurodegenerative diseases. The first chaperones in this disaggregation reaction, which are responsible for recognizing and performing initial remodeling of aggregates, are members of the Hsp40 (DnaJ) and small heat shock protein (sHSP) families. Very little, though, is known regarding how these chaperones perform their functions. Moreover, characterization of sHsp- and DnaJ-substrate complexes by most structural techniques has proven extremely challenging, as most chaperones are dynamic in nature and typically operate through a series of transient interactions with both their clients and other chaperones.

The advanced NMR techniques used in our lab, however, are ideally suited for the study of these exact types of dynamic systems, to monitor the transient and low populated protein states typical of chaperone-chaperone and chaperone-client interactions, and to study the structure of these typically very large protein complexes. By further combining advanced NMR with biophysical and functional assays, we intend to identify the specific sets of chaperones that function with the Hsp70 system, and the manner in which they operate to prevent or reverse the aggregation process.

Our overall objectives are:
1) Determine how the DnaJ and sHsp chaperone families recognize and remodel their client proteins.
2) Elucidate how chaperone-chaperone complexes enhance client-binding properties and cellular functions.
3) Identify the specific chaperone combinations required to break apart cytotoxic aggregates, such as those associated with Alzheimer's, Parkinson's, and Huntington's diseases, and the mechanisms through which the chaperones operate.

The successful implementation of this proposal will have a profound impact on our understanding of the human aggregation prevention and disaggregation mechanisms and the molecular chaperones that help protect our cells from the devastating effects of protein aggregate- and amyloid-associated diseases.

In addition to our goals for these specific systems, we believe the obtained knowledge and new methodologies stemming from our work will also pave the way for the study of many additional, chaperone-mediated cellular functions, as no such description is currently available for any other cellular pathway of similar complexity.
During this period we have identified how Hsp40 (DnaJ) and sHsp chaperones interact and remodel several physiologically relevant client proteins, such as a-synuclein and tau, that form disease-associated amyloid fibers. We found that different chaperones interact with α-synuclein at different positions and obtained structural information for each of these chaperones bound to monomeric α-synuclein.

In the case of tau aggregates, we observed that all chaperones tested show aggregation prevention activity, however class A Hsp40s, class B Hsp40s, and sHSPs perform this function via very different modes. sHsps bind to soluble monomeric tau in solution, preventing it from forming the cross-beta amyloid structures, while class B Hsp40s interact with formed fibers to both slow down their growth, and, with the help of Hsp70 chaperones, even break the fibers apart. Class A Hsp40s, on the other hand, interact with both the monomers and preformed fibers slowing their growth, but are unable to dissolve the fibers once they have formed. We also found that these functional differences are closely related to the binding sites of the chaperones on tau and the nature of the binding grooves on the chaperones themselves.

In addition, in this period we studied the interaction mode of class A DnaJs with misfolded proteins, mapping the interaction to the C-terminal binding domains of DnaJA1 and DnaJA2, as well as to their zinc finger-like regions. We also established that the interaction with these chaperones keeps the proteins in a soluble, folded state, utilizing this physical interaction to sequester the hydrophobic residues normally exposed during misfolding.
Investigations of different combinations of molecular chaperones that are functional in the disaggregation of amyloid fibers have systematically shown that only class B Hsp40 chaperones (and not class A) can work together with Hsp70 chaperones to accomplish this task. Through our research, we have uncovered a regulatory mechanism that is present only in class B Hsp40s, which, when removed (by mutations), abolishes the disaggregation activity of the Hsp70 machinery.

Furthermore, we found that this regulation is both important for proper recruitment of Hsp70 chaperones to the amyloid fibers, and absent in class A Hsp40s - explaining why only class B chaperones can solubilize amyloids. In addition, we have identified that this regulatory element is present in all cytosolic members of the class B Hsp40 family. We next plan to determine whether this regulation is likewise vital for additional functionalities of class B Hsp40 chaperones in the cell.
Amyloid disaggregation