A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

Hsp40s play complementary roles in the prevention of tau amyloid formation.

The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer's disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation and compare these to the small heat shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.

Several neurological conditions, such as Alzheimer's and Parkinson's disease, are characterized by the build-up of protein clumps known as aggregates. In the case of Alzheimer's disease, a key protein, called tau, aggregates to form fibers that are harmful to neuronal cells in the brain. One of the ways our cells can prevent this from occurring is through the action of proteins known as molecular chaperones, which can bind to tau proteins and prevent them from sticking together. Tau can take on many forms. For example, a single tau protein on its own, known as a monomer, is unstructured. In patients with Alzheimer's, these monomers join together into small clusters, known as seeds, that rapidly aggregate and accumulate into rigid, structured fibers. One chaperone, HSPB1, is known to bind to tau monomers and prevent them from being incorporated into fibers. Recently, another group of chaperones, called J-domain proteins, was also found to interact with tau. However, it was unclear how these chaperones prevent aggregation and whether they bind to tau in a similar manner as HSPB1. To help answer this question, Irwin, Faust et al. studied the effect of two J-domain proteins, as well as the chaperone HSBP1, on tau aggregation. This revealed that, unlike HSBP1, the two J-domain proteins can bind to multiple forms of tau, including when it has already aggregated in to seeds and fibers. This suggests that these chaperones can stop the accumulation of fibers at several different stages of the aggregation process. Further experiments examining which sections of the J-domain proteins bind to tau, showed that both attach to fibers via the same region. However, the two J-domain proteins are not identical in their interaction with tau. While one of them uses a distinct region to bind to tau monomers, the other does not bind to single tau proteins at all. These results demonstrate how different cellular chaperones can complement one another in order to inhibit harmful protein aggregation. Further studies will be needed to understand the full role of J-domain proteins in preventing tau from accumulating into fibers, as well as their potential as drug targets for developing new treatments.

HSP40 proteins use class-specific regulation to drive HSP70 functional diversity.

The ubiquitous heat shock protein 70 (HSP70) family consists of ATP-dependent molecular chaperones, which perform numerous cellular functions that affect almost all aspects of the protein life cycle from synthesis to degradation1-3. Achieving this broad spectrum of functions requires precise regulation of HSP70 activity. Proteins of the HSP40 family, also known as J-domain proteins (JDPs), have a key role in this process by preselecting substrates for transfer to their HSP70 partners and by stimulating the ATP hydrolysis of HSP70, leading to stable substrate binding3,4. In humans, JDPs constitute a large and diverse family with more than 40 different members2, which vary in their substrate selectivity and in the nature and number of their client-binding domains5. Here we show that JDPs can also differ fundamentally in their interactions with HSP70 chaperones. Using nuclear magnetic resonance spectroscopy6,7 we find that the major class B JDPs are regulated by an autoinhibitory mechanism that is not present in other classes. Although in all JDPs the interaction of the characteristic J-domain is responsible for the activation of HSP70, in DNAJB1 the HSP70-binding sites in this domain are intrinsically blocked by an adjacent glycine-phenylalanine rich region-an inhibition that can be released upon the interaction of a second site on DNAJB1 with the HSP70 C-terminal tail. This regulation, which controls substrate targeting to HSP70, is essential for the disaggregation of amyloid fibres by HSP70-DNAJB1, illustrating why no other class of JDPs can substitute for class B in this function. Moreover, this regulatory layer, which governs the functional specificities of JDP co-chaperones and their interactions with HSP70s, could be key to the wide range of cellular functions of HSP70.

Structural and Biochemical Properties of Hsp40/Hsp70 Chaperone System.

Hsp70s are ubiquitous molecular chaperones that act in a myriad of cellular functions, affecting virtually all aspects in the life of proteins from synthesis to degradation. Hsp70 proteins act in the cell in cooperation with a large set of dedicated co-chaperones consisting of J-domain proteins and nucleotide exchange factors that regulate the Hsp70 chaperone cycle. Recent studies have made significant progress towards obtaining a better understanding of the mechanisms through which Hsp70s and their co-chaperones operate, providing insights into structural, kinetic, and functional features of the various members of this network. In this chapter we describe the emerging working principles of the Hsp70 machine and its co-chaperones, and highlight how mechanistic aspects of this network are tied to distinct protein folding functions.

Protein Regulation by Intrinsically Disordered Regions: A Role for Subdomains in the IDR of the HIV-1 Rev Protein.

Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent-accessible residues. Here we show that the disordered C-terminal domain (CTD) of HIV-1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.

A role of disordered domains in regulating protein oligomerization and stability.

Intrinsically disordered proteins (IDPs) or regions (IDRs) in proteins hold many functions but their biological roles are still not fully understood. Here we describe a new role of such regions. Using the HIV-1 Rev protein, we show that disordered domains have a role in maintaining the correct oligomeric state and the thermodynamic stability of proteins.