Protein-Protein Interactions in the Molecular Chaperone Network.

Molecular chaperones play a central role in protein homeostasis (a.k.a. proteostasis) by balancing protein folding, quality control, and turnover. To perform these diverse tasks, chaperones need the malleability to bind nearly any "client" protein and the fidelity to detect when it is misfolded. Remarkably, these activities are carried out by only ∼180 dedicated chaperones in humans. How do a relatively small number of chaperones maintain cellular and organismal proteostasis for an entire proteome? Furthermore, 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. These physical links coordinate multiple chaperones into organized, functional complexes and facilitate the "handoff" of clients between them. 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). 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. 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. Somehow, this collection of PPIs draws together chaperone families and creates multiprotein subnetworks that are able to make the "decisions" of protein quality control. The key to understanding chaperone-mediated proteostasis might be to understand how PPIs are regulated. This Account will discuss the efforts of our group and others to map, measure, and chemically perturb the PPIs within the molecular chaperone network. Structural biology methods, including X-ray crystallography, NMR spectroscopy, and electron microscopy, have all played important roles in visualizing the chaperone PPIs. Guided by these efforts and -omics approaches to measure PPIs, new advances in high-throughput chemical screening that are specially designed to account for the challenges of this system have emerged. 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. In addition, these molecules have provided leads for the potential treatment of protein misfolding diseases. 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 rebalance proteostasis.

Pathogenic Tau Impairs Axon Initial Segment Plasticity and Excitability Homeostasis.

Dysregulation of neuronal excitability underlies the pathogenesis of tauopathies, including frontotemporal dementia (FTD) with tau inclusions. A majority of FTD-causing tau mutations are located in the microtubule-binding domain, but how these mutations alter neuronal excitability is largely unknown. Here, using CRISPR/Cas9-based gene editing in human pluripotent stem cell (iPSC)-derived neurons and isogenic controls, we show that the FTD-causing V337M tau mutation impairs activity-dependent plasticity of the cytoskeleton in the axon initial segment (AIS). Extracellular recordings by multi-electrode arrays (MEAs) revealed that the V337M tau mutation in human neurons leads to an abnormal increase in neuronal activity in response to chronic depolarization. Stochastic optical reconstruction microscopy of human neurons with this mutation showed that AIS plasticity is impaired by the abnormal accumulation of end-binding protein 3 (EB3) in the AIS submembrane region. These findings expand our understanding of how FTD-causing tau mutations dysregulate components of the neuronal cytoskeleton, leading to network dysfunction.

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved ß4-ß8 cleft of its alpha-crystallin domain. The ß4-ß8 region is also the site of Hsp27-Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the ß4-ß8 groove acts as a tunable sensor for clients.

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.

Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks.

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.

BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps.