The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70.

J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform. Cryoelectron microscopy and mutational studies reveal that different domains are involved in self-association. Oligomer dissociation into dimers potentiates its interaction with unfolded client proteins. The J-domains are accessible to Hsc70 within the tubular structure. They allow binding of closely spaced Hsc70 molecules that could be transferred to the unfolded substrate for its cooperative remodelling, explaining the efficient recovery of DNAJA2-bound clients. The disordered C-terminal domain, comprising the last 52 residues, regulates its holding activity and productive interaction with Hsc70. These in vitro findings suggest that the association equilibrium of DNAJA2 could regulate its interaction with client proteins and Hsc70.

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species.

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Extraction and Refolding Determinants of Chaperone-Driven Aggregated Protein Reactivation.

Reactivation of protein aggregates plays a fundamental role in numerous situations, including essential cellular processes, hematological and neurological disorders, and biotechnological applications. The molecular details of the chaperone systems involved are known to a great extent but how the overall reactivation process is achieved has remained unclear. Here, we quantified reactivation over time through a predictive mechanistic model and identified the key parameters that control the overall dynamics. We performed new targeted experiments and analyzed classical data, covering multiple types of non-ordered aggregates, chaperone combinations, and experimental conditions. We found that, irrespective of the behavior observed, the balance of surface disaggregation and refolding in solution universally determines the reactivation dynamics, which is broadly described by two characteristic times. This characterization makes it possible to use activity measurements to accurately infer the underlying loss of aggregated protein and to quantify, for the first time, the refolding rates of the soluble intermediates.

Structural and functional insights on the roles of molecular chaperones in the mistargeting and aggregation phenotypes associated with primary hyperoxaluria type I.

To carry out their biological function in cells, proteins must be folded and targeted to the appropriate subcellular location. These processes are controlled by a vast collection of interacting proteins collectively known as the protein homeostasis network, in which molecular chaperones play a prominent role. Protein homeostasis can be impaired by inherited mutations leading to genetic diseases. In this chapter, we focus on a particular disease, primary hyperoxaluria type 1 (PH1), in which disease-associated mutations exacerbate protein aggregation in the cell and mistarget the peroxisomal alanine:glyoxylate aminotransferase (AGT) protein to mitochondria, in part due to native state destabilization and enhanced interaction with Hsp60, 70 and 90 chaperone systems. After a general introduction of molecular chaperones and PH1, we review our current knowledge on the structural and energetic features of PH1-causing mutants that lead to these particular pathogenic mechanisms. From this perspective, and in the context of the key role of molecular chaperones in PH1 pathogenesis, we present and discuss current and future perspectives for pharmacological treatments for this disease.

Regulation of Human Hsc70 ATPase and Chaperone Activities by Apg2: Role of the Acidic Subdomain.

Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.

Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate.

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the ß subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the ß subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.

Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery.

Chaperone-mediated protein aggregate reactivation is a complex reaction that depends on the sequential association of molecular chaperones on their interaction with protein aggregates and on substrate refolding. This process could be modulated by the highly crowded intracellular environment, which is known to affect protein conformational change, enzymatic activity, and protein-protein interactions. Here, we report that molecular crowding shapes the chaperone activity of bacterial disaggregase composed of the DnaK system (DnaK, DnaJ, and GrpE) and the molecular motor ClpB. A combination of biophysical and biochemical methods shows that the excluded volume conditions modify the conformation of DnaK and DnaJ without affecting that of GrpE. These crowding-induced conformational rearrangements activate DnaK, enhance the affinity of DnaK for DnaJ, but not for GrpE, and increase the sensitivity of the chaperone activity to cochaperone concentration, explaining the tight control of their relative intracellular amounts. Furthermore, crowding-mediated disordering of the G/F domain of DnaJ facilitates the reversible formation of intermolecular DnaJ conglomerates. These assemblies could drive the formation of Hsp70 clusters at the aggregate surface with the consequent enhancement of the disaggregation efficiency through their coordinated action via entropic pulling. Finally, crowding helps ClpB to outcompete GrpE for DnaK binding, a key aspect of DnaK/ClpB cooperation given the low affinity of the disaggregase for DnaK. Excluded volume conditions promote the formation of the bichaperone complex that disentangles aggregates, enhancing the efficiency of the disaggregation reaction.

Chaperone-assisted protein aggregate reactivation: Different solutions for the same problem.

The oligomeric AAA+ chaperones Hsp104 in yeast and ClpB in bacteria are responsible for the reactivation of aggregated proteins, an activity essential for cell survival during severe stress. The protein disaggregase activity of these members of the Hsp100 family is linked to the activity of chaperones from the Hsp70 and Hsp40 families. The precise mechanism by which these proteins untangle protein aggregates remains unclear. Strikingly, Hsp100 proteins are not present in metazoans. This does not mean that animal cells do not have a disaggregase activity, but that this activity is performed by the Hsp70 system and a representative of the Hsp110 family instead of a Hsp100 protein. This review describes the actual view of Hsp100-mediated aggregate reactivation, including the ATP-induced conformational changes associated with their disaggregase activity, the dynamics of the oligomeric assembly that is regulated by its ATPase cycle and the DnaK system, and the tight allosteric coupling between the ATPase domains within the hexameric ring complexes. The lack of homologs of these disaggregases in metazoans has suggested that they might be used as potential targets to develop antimicrobials. The current knowledge of the human disaggregase machinery and the role of Hsp110 are also discussed.

ClpB dynamics is driven by its ATPase cycle and regulated by the DnaK system and substrate proteins.

The hexameric AAA+ (ATPase associated with various cellular activities) chaperone ClpB reactivates protein aggregates in collaboration with the DnaK system. An intriguing aspect of ClpB function is that the active hexamer is unstable and therefore questions how this chaperone uses multiple rounds of ATP hydrolysis to translocate substrates through its central channel. In the present paper, we report the use of biochemical and fluorescence tools to explore ClpB dynamics under different experimental conditions. The analysis of the chaperone activity and the kinetics of subunit exchange between protein hexamers labelled at different protein domains indicates, in contrast with the current view, that (i) ATP favours assembly and ADP dissociation of the hexameric assembly, (ii) subunit exchange kinetics is at least one order of magnitude slower than the ATP hydrolysis rate, (iii) ClpB dynamics and activity are related processes, and (iv) DnaK and substrate proteins regulate the ATPase activity and dynamics of ClpB. These data suggest that ClpB hexamers remain associated during several ATP hydrolysis events required to partially or completely translocate substrates through the protein central channel, and that ClpB dynamics is tuned by DnaK and substrate proteins.

Allosteric communication between the nucleotide binding domains of caseinolytic peptidase B.

ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K(d) = 3-7 µm) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the K(d) decreases from ≈160-300 to 50-60 µm) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.

Nucleotide utilization requirements that render ClpB active as a chaperone.

ClpB is a member of the AAA+ superfamily that forms a ring-shaped homohexamer. Each protomer contains two nucleotide binding domains arranged in two rings that hydrolyze ATP. We extend here previous studies on ClpB nucleotide utilization requirements by using an experimental approach that maximizes random incorporation of different subunits into the protein hexamer. Incorporation of one subunit unable to bind or hydrolyze ATP knocks down the chaperone activity, while the wt hexamer can accommodate two mutant subunits that hydrolyze ATP in only one protein ring. Four subunits seem to build the functional cooperative unit, provided that one of the protein rings contains active nucleotide binding sites.