Biology subjects

Perez, V. A.

Publications and source records attributed to Perez, V. A..

5 recordsLinked to original sources

bioRxiv2024

Functional classification of tauopathy strains reveals the role of protofilament core residues.

Distinct tau amyloid assemblies underlie diverse tauopathies but defy rapid classification. Cell and animal experiments indicate tau functions as a prion, as different strains propagated in cells cause unique, transmissible neuropathology after inoculation. Strain amplification requires compatibility of the monomer and amyloid template. We used cryo-EM to study one cell-based YFP-tagged strain, resolving its amyloid nature. We then used sequential alanine (Ala) substitution (scan) within tau repeat domain (RD) to measure incorporation to pre-existing tau RD-YFP aggregates. This robustly discriminated strains, defining sequences critical for monomer incorporation. We then created 3R/4R or 4R WT RD (aa 246-408) biosensors. Ala scan of recombinant tau seeds with the Alzheimers Disease fold matched that of AD homogenate. We scanned 22 brain lysates comprising 4 tauopathies. This clustered cases by neuropathological syndrome, revealed the role of amino acids in protofilament folds, and allowed strain discrimination based on amino acid requirements for prion replication. TeaserDiscrimination of tau strains based on the relative contribution of each amino acid to templated propagation of the amyloid.

neuroscience↗

bioRxiv2023

Ensemble-based design of tau to inhibit aggregation while preserving biological activity.

The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. However, the structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to engineer tau sequences that modulate its structural ensemble. Our findings show that substitutions near the conserved PGGG {beta}-turn motif informed by tau isoform context reduce tau aggregation in vitro and cells and can even counteract aggregation induced by turn destabilizing disease-associated proline-to-serine mutations. Engineered tau sequences maintain microtubule binding and explain why 3R isoforms exhibit reduced pathogenesis compared to 4R. We propose a simple mechanism to reduce the formation of pathogenic species while preserving biological function, thus offering insights for therapeutic strategies aimed at reducing tau protein misfolding in neurodegenerative diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/571598v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@178a79org.highwire.dtl.DTLVardef@1842dc7org.highwire.dtl.DTLVardef@d9a32borg.highwire.dtl.DTLVardef@676dec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

DnaJC7 specifically regulates tau seeding

Neurodegenerative tauopathies are caused by accumulation of toxic tau protein assemblies. This appears to involve template-based seeding events, whereby tau monomer changes conformation and is recruited to a growing aggregate. Several large families of chaperone proteins, including Hsp70s and J domain proteins (JDPs) cooperate to regulate the folding of intracellular proteins such as tau, but the factors that coordinate this activity are not well known. The JDP DnaJC7 binds tau and reduces its intracellular aggregation. However, it is unknown whether this is specific to DnaJC7 or if other JDPs might be similarly involved. We used proteomics within a cell model to determine that DnaJC7 co-purified with insoluble tau and colocalized with intracellular aggregates. We individually knocked out every possible JDP and tested the effect on intracellular aggregation and seeding. DnaJC7 knockout decreased aggregate clearance and increased intracellular tau seeding. This depended on the ability of the J domain (JD) of DnaJC7 to bind to Hsp70, as JD mutations that block binding to Hsp70 abrogated the protective activity. Disease-associated mutations in the JD and substrate binding site of DnaJC7 also abrogated its protective activity. DnaJC7 thus specifically regulates tau aggregation in cooperation with Hsp70.

molecular biology↗

bioRxiv2022

Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only [~]1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and "biosensor" cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.

cell biology↗

bioRxiv2020

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation

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.

biophysics↗