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

Periodic Reporting for period 1 - NMR-DisAgg (The Dynamic Composition of the Protein Chaperone Network: Unraveling Human Protein Disaggregation via NMR Spectroscopy)

Reporting period: 2018-09-01 to 2020-02-29

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, as well as to study the structure of these potentially 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 established robust purification protocols for seven Hsp40s (DnaJs) and four sHsps, obtained complete assignments for five chaperones, and are in the process of assigning the additional ones.

We have also identified how these chaperones interact and remodel several physiologically relevant client proteins such as a-synuclein and Tau that form disease-associated amyloid fibers. We have found that different chaperones interact with α-synuclein at different positions and obtained structural information of each of these chaperones bound to monomeric α-synuclein.
In case of Tau aggregates, we have found that all chaperones tested showed aggregation prevention activity, however class A DnaJs, class B DnaJs, and sHSPs, performed this function via very different modes. sHsps bind to the soluble monomeric Tau in solution, preventing it from forming the cross-beta amyloid structures, while class B DnaJs interact with the formed fibers and slow down their growth as well as break the fibers apart with the help of Hsp70 chaperones. Class A DnaJs, on the other hand, interact with both the monomers and preformed fibers, yet are unable to disaggregate the latter. We have 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 have 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 binding domains. We have also found that the interaction with these chaperones keeps the proteins in a soluble, folded state by protecting the misfolding-exposed hydrophobic residues through a physical interaction.

We have also begun working on determining the interactions between hetero-oligomers of chaperones of the same family. We have mapped the binding interfaces between two class A and 3 class B chaperones, as well as between all four sHsps.

In order to obtain the structures of the complexes, however, the structures of the individual components must first be characterized. Currently no solved structures of DnaJs contain the N-terminal J-domain regions, yet these play a vital part in class A - class B DnaJ-DnaJ interactions. We have therefore been using paramagnetic relaxation enhancements (PREs) and pseudocontact shifts (PCSs) NMR approaches to measure distance and orientation restraints, thereby allowing us to determine the position of the J-domains in these proteins.
Similarly, in the case of sHSps, the structure and the location on the largely disordered N-terminal domain is not known for any of these chaperones, yet is vital for sHSp-sHsp interactions. We are currently in the process of obtaining the structural and dynamics information of these N-terminal regions, through NMR chemical shifts and CPMG relaxation dispersion measurements.

Once the structure calculation has been completed, distance restraints and chemical shift mapping will be used to solve the structures of the complexes. This will then serve to uncover how hetero-oligomerization of chaperones from the same family expands both their range of substrates and their functions.
Investigation of different combinations of molecular chaperones that are functional in the disaggregation of amyloid fibers have systematically shown that only class B DnaJ chaperones (and not class A) can work together with Hsp70 chaperones to accomplish this task. Over the course of the last 18 months we have uncovered a new regulatory mechanism that is present only in class B DnaJs, 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 DnaJs - 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 DnaJ family. We next plan to determine whether this regulation is likewise vital for additional functionalities of class B DnaJ chaperones in the cell.