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Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene ( MAPT ) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau’s repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306 VQIVYK 311 . We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306 VQIVYK 311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis. The biophysical mechanisms of how disease-associated tau mutations drive amyloid formation are not well understood. Here the authors use biophysical approaches, cell models and MD simulations and find that the intrinsically disordered repeat domain of tau encodes a metastable local structure and perturbations through mutations and proline isomerization cause an aggregation phenotype in vitro and in cells.

Pathogenesis of tauopathies involves conversion of tau monomer into pathological tau conformers that serve as templates to recruit native tau into growing assemblies. Small soluble tau seeds have been proposed to drive pathological tau assembly in vitro, in cells and in vivo. We have previously described the isolation of monomeric pathogenic tau seeds derived from recombinant samples and tauopathy tissues but in-depth biophysical characterization of these species has not been done. Here we describe a chromatographic method to isolate recombinant soluble tau seeds derived from heparin treatment. We used biochemical and biophysical approaches to show that the seeds are predominantly monomeric and have the capacity to nucleate aggregation of inert forms of tau in vitro and in cells. Finally, we used crosslinking mass spectrometry to identify the topological changes in tau as it converts from an inert state to a pathogenic seed. Future studies will reveal the relationship between soluble seeds and structural polymorphs derived from tauopathies to help diagnose and develop therapeutics targeting specific tauopathies.

Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a β-turn structural element in tau that contains the 275 VQIINK 280 amyloid motif. Wild-type tau, but not mutant, β-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention. Protein binding by the Hsp70/J-domain protein (JDP) chaperones prevents aggregation of the client protein. Here, the authors show that DnaJC7 binds preferentially to natively folded wild-type tau, via a β-turn element in tau that contains the known amyloid motif, while aggregation-prone tau mutants are recognized with reduced affinity.

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformations of β-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We evaluate our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures. The authors developed a computational approach to probe the stability of amyloid fibrils and discover networks of hotspot interactions. Understanding the mechanisms of amyloid folding will help identify novel methods to treat protein (mis)folding diseases.

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network. The Hsp70/Hsp40 system plays an important role in maintaining cellular proteostasis but so far it is not well understood how Hsp70 proteins are recruited to specific Hsp40 co-chaperones. Here, the authors combine biochemical and biophysical approaches to characterise the oligomeric mammalian Hsp40 DnaJB8. They identify an intra-oligomer DnaJB8 interaction between the N-terminal J-Domain and the C-terminal domain that occludes the J-Domain surface that binds Hsp70 and propose a model for DnaJB8-Hsp70 recruitment.

When inserted onto the C-terminus of archaeal proteasome activator PA26, an optimized activator peptide derived from human 19S (-NLSYYT-OH) has been shown to efficiently degrade tau in vitro. Structure-activity relationship experiments show how small steric forces generated from the leucine in position 5 (P5) account for full 20S proteasome gate opening and efficient degradation of tau. Proteasomal activity assays using short 6-amino acids peptides derived from (-NLSYYT) show activity dependency on the presence of leucine. After cloning select mutants P5F, P5Y, P5W, P5V, and P5I onto the C-terminus of aPA26, fluorescence polarization and OpenSPR show these mutations impact PA26-20S affinity as well as activity. Cryo-EM structures of each aPA26 mutant reveal a series of intermediate states of the 20S gate, and how steric interactions between leucine and a conserved arginine on the 20S drive allosteric interactions to fully open the 20S gate. A combination of in vitro assays and cell-based assays were used to track total tau and p-tau degradation in a dose dependent manner. Mutations to the P5 leucine in the optimized NLSYYT proteasome activator peptide led to a significant loss of proteasomal activity. Even biochemically similar amino acids showed significant loss of activity (40%-90%). When this sequence was cloned onto PA26 for multivalent sequence activation of the 20S proteasome, we found that in addition to loss in activity, these mutations also weakened affinity to the 20S proteasome. Cryo-EM structures revealed that steric interactions lift PSMA5 R20 and trigger a network of allosteric interactions to turn and open the 20S gate. For P5W or P5Y, intermediate structures show specific pockets where these mutations break the allosteric network, resulting in partial gate opening. Tau degradation experiments validated that these partial open states yield less efficient degradation compared to full gate opening. The sensitivity of small hydrophobic amino acid leucine in the P5 position of NLSYYT significantly impacts the stability of the 20S gate. Remaining protein-protein interactions within a partially closed 20S gate clash with tau entry. By leveraging this information, proteasome activators designed for tau degradation strategies can be designed around controlling the rate of tau degradation.

Background When inserted onto the C‐terminus of archaeal proteasome activator PA26, an optimized activator peptide derived from human 19S (‐NLSYYT‐OH) has been shown to efficiently degrade tau in vitro. Structure‐activity relationship experiments show how small steric forces generated from the leucine in position 5 (P5) account for full 20S proteasome gate opening and efficient degradation of tau. Method Proteasomal activity assays using short 6‐amino acids peptides derived from (‐NLSYYT) show activity dependency on the presence of leucine. After cloning select mutants P5F, P5Y, P5W, P5V, and P5I onto the C‐terminus of aPA26, fluorescence polarization and OpenSPR show these mutations impact PA26‐20S affinity as well as activity. Cryo‐EM structures of each aPA26 mutant reveal a series of intermediate states of the 20S gate, and how steric interactions between leucine and a conserved arginine on the 20S drive allosteric interactions to fully open the 20S gate. A combination of in vitro assays and cell‐based assays were used to track total tau and p‐tau degradation in a dose dependent manner. Result Mutations to the P5 leucine in the optimized NLSYYT proteasome activator peptide led to a significant loss of proteasomal activity. Even biochemically similar amino acids showed significant loss of activity (40%‐90%). When this sequence was cloned onto PA26 for multivalent sequence activation of the 20S proteasome, we found that in addition to loss in activity, these mutations also weakened affinity to the 20S proteasome. Cryo‐EM structures revealed that steric interactions lift PSMA5 R20 and trigger a network of allosteric interactions to turn and open the 20S gate. For P5W or P5Y, intermediate structures show specific pockets where these mutations break the allosteric network, resulting in partial gate opening. Tau degradation experiments validated that these partial open states yield less efficient degradation compared to full gate opening. Conclusion The sensitivity of small hydrophobic amino acid leucine in the P5 position of NLSYYT significantly impacts the stability of the 20S gate. Remaining protein‐protein interactions within a partially closed 20S gate clash with tau entry. By leveraging this information, proteasome activators designed for tau degradation strategies can be designed around controlling the rate of tau degradation.

Background An optimized 6 amino acid peptide (NLSYYT; herein YΦ) derived from the C‐terminus of h19S proteasome activator Rpt5 has been shown to activate the 20S proteasome and promote tau degradation. Further analysis of this peptide has identified the highly conserved leucine in position 5 (P5) as a key part of the 20S activation mechanism to drive degradation of tau monomers in the absence of proteasome activator complexes. Method Recombinant peptides were used to identify key amino acids required for binding and activating the h20S proteasome. Degradation activity from YΦ‐mediated activation was measured with small peptide markers. Using archaeal activator complex aPA26 with YΦ and P5 mutants cloned onto its tails, P5 was shown to use hydrophobic interactions to stabilize YΦ interactions with the h20S α‐subunit ring. Finally, YΦ activation of the h20S proteasome increased tau degradation in vitro, in cell lysates, and in cells. Result In vitro tau degradation experiments show that simultaneous binding of linked YΦ peptides is sufficient to promote degradation of tau monomer in an ATP‐independent and ubiquitin‐independent system. Mutations to the P5 leucine result in a 3‐fold reduction in h20S proteasome activity for aromatic substitutions, and complete loss of activity for all other natural amino acid substitutions. However, P5 substitutions do not have as dramatic of an effect on binding, demonstrating that the role of P5 leucine uncouples activity and binding, unlike the other amino acids. In the context of aPA26, YΦ mediated activation clears 80% of native tau monomer over the course of 30 minutes, with similar results observed in degrading tau derived from cell culture. Conclusion Structure‐activity relationship data of YΦ activation of the 20S proteasome provides insight into future designs of proteasome activators. With an improved understanding of the role of P5 leucine, a more refined interaction network can be considered when designing h20S proteasome activators for clearing disease‐associated tau in cells.

Centrosomes organize microtubules for mitotic spindle assembly and positioning. Forces mediated by these microtubules create tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. How PCM resists these stresses is unclear at the molecular level. Here, we use cross-linking mass spectrometry (XL-MS) to map interactions underlying multimerization of SPD-5, an essential PCM scaffold component in C. elegans . We identified an interaction hotspot in an alpha helical hairpin motif in SPD-5 (a.a. 541-677). XL-MS data, ab initio structural predictions, and mass photometry suggest that this region dimerizes to form a tetrameric coiled-coil. Mutating a helical section (a.a. 610-640) or a single residue (R592) inhibited PCM assembly in embryos. This phenotype was rescued by eliminating microtubule pulling forces, revealing that PCM assembly and material strength are interrelated. We propose that interactions mediated by the helical hairpin strongly bond SPD-5 molecules to each other, thus enabling PCM to assemble fully and withstand stresses generated by microtubules.Centrosomes organize microtubules for mitotic spindle assembly and positioning. Forces mediated by these microtubules create tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. How PCM resists these stresses is unclear at the molecular level. Here, we use cross-linking mass spectrometry (XL-MS) to map interactions underlying multimerization of SPD-5, an essential PCM scaffold component in C. elegans . We identified an interaction hotspot in an alpha helical hairpin motif in SPD-5 (a.a. 541-677). XL-MS data, ab initio structural predictions, and mass photometry suggest that this region dimerizes to form a tetrameric coiled-coil. Mutating a helical section (a.a. 610-640) or a single residue (R592) inhibited PCM assembly in embryos. This phenotype was rescued by eliminating microtubule pulling forces, revealing that PCM assembly and material strength are interrelated. We propose that interactions mediated by the helical hairpin strongly bond SPD-5 molecules to each other, thus enabling PCM to assemble fully and withstand stresses generated by microtubules.

During mitotic spindle assembly, microtubules generate tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. The molecular interactions that enable PCM to assemble rapidly and resist external forces are unknown. Here we use cross-linking mass spectrometry to identify interactions underlying supramolecular assembly of SPD-5, the main PCM scaffold protein in . Crosslinks map primarily to alpha helices within the phospho-regulated region (PReM), a long C-terminal coiled-coil, and a series of four N-terminal coiled-coils. PLK-1 phosphorylation of SPD-5 creates new homotypic contacts, including two between PReM and the CM2-like domain, and eliminates numerous contacts in disordered linker regions, thus favoring coiled-coil-specific interactions. Mutations within these interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces. Thus, PCM assembly and strength are interdependent. , self-assembly of SPD-5 scales with coiled-coil content, although there is a defined hierarchy of association. We propose that multivalent interactions among coiled-coil regions of SPD-5 build the PCM scaffold and contribute sufficient strength to resist microtubule-mediated forces.