| dc.description.abstracteng | Molecular chaperones are diligent helpers of the cell that ensure a well-balanced proteome by regulating protein production, protein folding, refolding and holding as well as protein degradation. In doing so, molecular chaperones assist proteins throughout their lifetime. Within the proteome, i.e. the entirety of all proteins, a special feature is reserved for the group of intrinsically disordered proteins (IDPs). They inherently lack a distinct three-dimensional structure so that, when unbound, their side chains are unprotected. Thereby, IDPs are able to participate in a wide range of interactions and react sensitively to external changes. At the same time this feature bears a particularly high risk for unfavorable contacts that may in the worst case engender protein aggregation. The importance of molecular chaperones to shelter IDPs from such undesired interactions manifests in yet incurable neurodegenerations such as Alzheimer's or Parkinson's disease. In each of these disorders, there are disease specific IDPs that attach to each other, clump together, and deposit as huge protein aggregates.
With regard to IDP chaperoning, a special role is assigned to the heat shock protein (Hsp) family, as they possess the unique capability of protein holding to protect proteins from undesired interactions. Thus, the binding of Hsps to IDPs gained increasing research interest. So far, mainly the interaction of IDPs, including the Alzheimer’s disease related, IDP prime example protein Tau, with individual Hsps has been studied. However, for foldable substrates, it is known that, in addition to their discrete function, Hsps can team up to drive protein turnover. And so also Hsp70 and Hsp90, which together can form the Hsp70/Hsp90 chaperone machinery to particularly prevent protein misfolding. Hence, being on the one side equipped with the marvelous capacity to counteract aggregation via the Hsp70/Hsp90 chaperone machinery and observing on the other side the deposition of insoluble Tau aggregates, poses the central question on whether the cellular defense system of the Hsp70/Hsp90 chaperone machinery isn’t likewise involved in the chaperoning of Tau.
The Hsp70/Hsp90 chaperone machinery comprises the minimal system of the five proteins Hsp70, Hsp40, Hsp90, Hop and p23. In order to understand the role of each of these proteins in the Hsp70/Hsp90 chaperone machinery mediated Tau chaperoning, its stepwise assembly with distinct intermediate states was reconstituted in vitro. The analysis showed that (i) one Hop molecule stabilizes the Hsp902 dimer in a V-shaped conformation, (ii) Hsp70, Hop and Hsp90 together form the Hsp70/Hsp90 chaperone machinery comprising the Hsp702:Hop1:Hsp902 complex, (iii) Tau associates with the preassembled Hsp70/Hsp90 chaperone machinery creating a 710 kDa large (Hsp701:Hop1:Hsp902:Tau1)2 dimeric complex and that (iv) the addition of p23 stabilizes the Hsp70/Hsp90 chaperone machinery:Tau interaction generating a stable 750 kDa large (Hsp701:Hop1:Hsp902:Tau1:p231)2 complex. (v) By means of the co-chaperone CHIP, which marks proteins for proteasomal degradation, it was further shown that the (Hsp701:Hop1:Hsp902:Tau1:p231)2 complex is no irreversible deadlock but part of a co-chaperone-mediated dynamic equilibrium between protein holding via Hop and p23, and protein degradation induced by CHIP. (vi) The Hsp70/Hsp90 chaperone machinery was revealed to control normal as well as abnormal Tau, as pathologically modified, hyperphosphorylated Tau (PTau) equally associated with the Hsp70/Hsp90 chaperone machinery forming Hsp70:Hop:Hsp90:PTau and Hsp70:Hop:Hsp90:PTau:p23 complexes.
Altogether, the Hsp70/Hsp90 chaperone machinery was revealed as interaction partner and thus potential chaperone system for the intrinsically disordered protein Tau. An integrated approach of biochemical and biophysical methods including native page, tryptophane fluorescence, isothermal titration calorimetry, nuclear magnetic resonance, dynamic light scattering, sucrose density gradient centrifugation, chromatography and chemical cross-linking coupled to mass spectrometry was used to describe a detailed model of stepwise machinery assembly accompanied with Tau binding. The knowledge gained about binding sites, conformational changes, stoichiometries and affinities presents novel structural and biochemical insights into the IDP chaperoning by the Hsp70/Hsp90 chaperone machinery. The obtained data thus may serve as fundamental basis for high-resolution structure determination and future research directions deciphering why the Hsp70/Hsp90 chaperone machinery fails to protect against Tau aggregation during disease. | de |