Biology subjects

Isas, J. M.

Publications and source records attributed to Isas, J. M..

3 recordsLinked to original sources

bioRxiv2025

Protofibril Binding Peptides Recognize and Inhibit Huntingtin Amyloid Formation in vitro and in vivo

In Huntingtons disease, polyglutamine expansion in huntingtin exon 1 (Httex1) results in stepwise misfolding, amyloid formation, and neuronal death. Here we used mRNA display directed evolution to generate peptide ligands targeting Httex1 protofibrils, an early, toxic misfolding intermediate. Two distinct peptide families bind protofibrils, one tryptophan rich and the other glutamine rich, resulting in two predominant peptides HD1 (W-rich) and HD8 (Q-rich). Both peptides bind with high affinity and specificity to the misfolded polyQ structure present in protofibrils, a toxic component that is not recognized by existing huntingtin-directed antibodies. Homo- and heterodimers of HD1 and HD8 bind protofibrils with antibody-like affinity, and potently inhibit aggregation of recombinant and cellular Httex1. The HD8-1 heterodimer can be used like an antibody for immunocytochemistry to identify Httex1 aggregates in transfected cells and in the retina of a Huntingtons disease mouse model system (R6/1). Peptide binding to both in vitro and in vivo Httex1 validates in vitro generated Httex1 protofibrils share the same structural features as Httex1 amyloid from cellular and mammalian disease model systems. Further, HD8-1 protofibril recognition enables direct detection of a pathogenic form of Httex1 (misfolded polyQ) as a disease biomarker. Finally, HD1, HD8, and HD8-1 binding and aggregation inhibition defines the protofibril sites that mediate fibril growth. Overall, our observations support developing protofibril-directed ligands as novel, selective, diagnostics and therapeutics for Huntingtons disease. Significance StatementHuntingtin protein polyQ misfolding plays a central role in Huntingtons disease pathogenesis. We used mRNA display to discover a new class of huntingtin-directed ligands protofibril-binding peptides. Unlike presently available antibodies, these peptides selectively recognize misfolded polyQ region, enable using toxic huntingtin aggregates as disease biomarkers, and block huntingtin aggregation in vitro and in vivo. Protofibril binding peptides may thus assist analysis of huntingtin pathology and provide a starting point for designing protein misfolding inhibitors as Huntingtons disease therapeutics.

biochemistry↗

bioRxiv2021

Sonicated fibrils of huntingtin exon-1 preferentially seed neurons and produce toxic assemblies

Huntingtons disease (HD) is a genetically inherited neurodegenerative disorder caused by expansion of a polyglutamine (polyQ) repeats in the exon-1 of huntingtin protein (HTT). The expanded polyQ enhances the amyloidogenic propensity of HTT exon 1 (HTTex1), which forms a heterogeneous mixture of assemblies with some being neurotoxic. While predominantly intracellular, monomeric and aggregated mutant HTT species are also present in the cerebrospinal fluids of HD patients, however, their biological properties are not well understood. To explore the role of extracellular mutant HTT in aggregation and toxicity, we investigated the possible uptake and amplification of recombinant HTTex1 assemblies in cell culture models. We found seeding-competent species in the sonicated HTTex1 fibrils, which preferentially entered human neurons and triggered the amplification of neurotoxic assemblies; astrocytes or epithelial cells were not permissive to the HTTex1 seeding. The aggregation of HTTex1 seeds in neurons depleted endogenous HTT protein with non-pathogenic polyQ repeat, activated apoptotic caspase-3 pathway and induced nuclear fragmentation. Using a panel of novel monoclonal antibodies and genetic mutation, we identified epitopes within the N-terminal 17 amino acids and proline-rich domain of HTTex1 mediating neural seeding. Synaptosome preparations from the brains of HD mice also contained similar neurotoxic seeding-competent mutant HTT species. Our findings suggest that amyloidogenic extracellular mutant HTT assemblies may selectively enter neurons, propagate and produce neurotoxic assemblies.

neuroscience↗

bioRxiv2019

Huntingtin fibrils with different toxicity, structure, and seeding potential can be reversibly interconverted

The first exon of the huntingtin protein (HTTex1) important in Huntingtons Disease (HD) can form cross-{beta} fibrils of varying toxicity. We find that the difference between these fibrils is the degree of entanglement and dynamics of the C-terminal proline-rich domain (PRD) in a mechanism analogous to polyproline film formation. In contrast to fibril strains found for other cross-{beta} fibrils, these HTTex1 fibril types can be reversibly interconverted. This is because the structure of their polyQ fibril core remains unchanged. Further, we find that more toxic fibrils of low entanglement have higher affinities for protein interactors and are more effective seeds for recombinant HTTex1 and HTTex1 in cells. Together these data show how the structure of a framing sequence at the surface of a fibril can modulate seeding, protein-protein interactions, and thereby toxicity in neurodegenerative disease.

biophysics↗