ATTR amyloidosis is a fatal disease associated with the accumulation of transthyretin (ATTR) fibrils that lead to organ failure and death. Mutations in the TTR gene or aging may accelerate the deposition of variant (ATTRv) or wild-type (ATTRwt) transthyretin, respectively. Although ATTR amyloidosis patients accumulate ATTR fibrils in virtually every organ, the clinical presentation is often unpredictable and variable. Recent studies in cryo-electron microscopy (cryo-EM) have revealed that in tauopathies and synucleinopathies, diseases involving amyloidosis of tau and α-synuclein, respectively, all the patients of the same disease display the same fibril fold, or polymorph. In this study, we use cryo-EM to explore whether fibrils from heart tissue of different patients with cardiac ATTR amyloidosis share a common fold. We determined the molecular structures of fibrils extracted from the hearts of seven patients, including both ATTRv and ATTRwt carriers, at resolutions of 3.0 to 3.7 Å. We found that ATTRv mutations perturb the fibril conformation whereas ATTRwt fibrils share a common structure fold. Our findings suggest that unlike in tauopathies and synucleinopathies, ATTRv fibrils display structural polymorphism driven by each individual and their genotypes. ATTR polymorphism challenges the current paradigm of ″one disease equals one fibril polymorph,″ and questions whether similarly novel conformations occur in other amyloidoses.

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 275VQIINK280 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.

The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) genome is evolving as the viral pandemic continues its active phase around the world. The Papain-like protease (PLpro) is a domain of Nsp3 – a large multi-domain protein that is an essential component of the replication-transcription complex, making it a good therapeutic target. PLpro is a multi-functional protein encoded in coronaviruses that can cleave viral polyproteins, poly-ubiquitin and protective Interferon Stimulated Gene 15 product, ISG15, which mimics a head-to-tail linked ubiquitin (Ub) dimer. PLpro across coronavirus families showed divergent selectivity for recognition and cleavage of these protein substrates despite sequence conservation. However, it is not clear how sequence changes in SARS-CoV-2 PLpro alter its selectivity for substrates and what outcome this has on the pathogenesis of the virus. We show that SARS-CoV-2 PLpro preferentially binds ISG15 over Ub and K48-linked Ub2. We determined crystal structures of PLpro in complex with human K48-Ub2 and ISG15 revealing that dual domain recognition of ISG15 drives substrate selectivity over Ub and Ub2. We also characterized the PLpro substrate interactions using solution NMR, cross-linking mass spectrometry to support that ISG15 is recognized via two domains while Ub2 binds primarily through one Ub domain. Finally, energetic analysis of the binding interfaces between PLpro from SARS-CoV-1 and SARS-CoV-2 with ISG15 and Ub2 define the sequence determinants for how PLpros from different coronaviruses recognize two topologically distinct substrates and how evolution of the protease altered its substrate selectivity. Our work reveals how PLpro substrate selectivity may evolve in PLpro coronaviruses variants enabling design of more effective therapeutics.

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau converts into β-sheet-rich amyloid conformations, which underlie pathology in over 25 related tauopathies. Structural studies of tau amyloid fibrils isolated from human tauopathy tissues have revealed that tau adopts diverse structural polymorphs, each linked to a different disease. Molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. New data supports the model that chaperones selectively recognize different conformations of tau to limit the accumulation of proteotoxic species. The challenge now is to understand how chaperones influence disease processes across different tauopathies, which will help guide the development of novel conformationspecific diagnostic and therapeutic strategies. Molecular chaperone function in tauopathiesA class of more than 25 different neurodegenerative diseases, collectively called tauopathies, are associated with brain deposition of fibrillar aggregates of the protein microtubule-associated protein tau (MAPT) [1]. New research in the field has shown that monomeric tau can be converted from a soluble state into a pathogenic state that self-propagates the distinct β-sheet aggregates observed in diseases in a prion (see Glossary) -like manner [2][3][4][5][6][7]. These different states that are observed in vivo are stable, unique conformations that 'seed' or induce the native, inert monomer to assemble into amyloid fibrils or their intermediates [8][9][10][11]. New work on the structures of tau fibrils derived from patient samples have indicated that each tau strain (and/or resulting tauopathy) can be classified by a unique structural polymorph [12,13], implying that there is a direct link between disease and tau conformation. Molecular chaperones bind to tau and modify its conformation to prevent tau assembly and also to disaggregate fibrils. This review will discuss new concepts in tau fibril conformation and the role of molecular chaperones to influence tau strains and their capacity to regulate formation of pathogenic species in disease. We first describe tau function and its aggregation mechanism followed by interactions with molecular chaperones in a hierarchy determined by tau binding, refolding, and disaggregation: J-domain proteins (JDPs), heat shock protein 70 (Hsp70), heat shock protein 90 (Hsp90), and small heat shock protein (sHSP). Importantly, insights into chaperone function in the initial formation and subsequent propagation of distinct tau conformations could guide the design of diagnostic and therapeutic strategies. The MAPTThe human MAPT gene encodes 16 exons, of which exons 2, 3, and 10 are alternatively spliced. In the human brain, tau exists as six isoforms that are defined by the presence or absence of the two N terminal domains (NTDs) and four repeat domains (RDs) (i.e., 2N4R; two NTDs and four RDs; detailed in Figure 1) and are highly expressed in n...

Molecular chaperones, including Hsp70/Hsp40 families, play central roles in binding substrates to prevent their aggregation. How Hsp40s select different conformations of substrates remains poorly understood. Here, we report a novel interaction between the Hsp40 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 beta-turn element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau beta-turn fragments, but not mutant fragments, 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. This identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

SUMMARYJ-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerizationin vitroand in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregationin vitroand in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.

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