Many devastating neurodegenerative diseases are associated with the transformation of proteins from their normal soluble forms into amyloid fibrils. During amyloidogenesis, these proteins accumulate in the brain and contribute to disorders such as Parkinson’s disease, Alzheimer’s disease, type II diabetes, and Huntington’s disease. α-Synuclein (αS) is a “natively unfolded” 14 kDa protein of unknown physiological function that plays a central role in the pathogenesis of Parkinson’s disease. Numerous studies have established that α-synuclein can convert in vitro from an unfolded, soluble monomer into filamentous β-sheet aggregates. Despite exhaustive research efforts, the mechanism by which α-synuclein transforms, from a soluble unfolded protein to an insoluble aggregate, remain unclear. It is therefore critically important to elucidate the aggregation pathway of α-synuclein, characterize the structural and kinetic features of this process, and develop strategies to inhibit fibril formation. To achieve this, our laboratory uses several techniques like NMR, fluorescence, electron microscopy, atomic force microscopy, circular dichroism, and computational methods.
Schematic of α-synuclein–driven disease progression:
In affected neurons, α-synuclein (αS) monomers misfold and assemble into higher-order aggregates that form Lewy bodies. Fibrillar aggregates are then released and transmitted to neighboring healthy neurons, where they catalyze further aggregation through elongation and secondary nucleation, driving amplification in a prion-like fashion.
Cracking the Code of Secondary Nucleation:
We study how αS fibril surfaces catalyze secondary nucleation to understand how misfolded aggregates amplify and spread in disease. The encounter between the fibril and the intrinsically disordered αS monomer is a complex process involving transient, dynamic binding events. Work from our group and others suggests that monomer recruitment begins through interactions with the disordered fibril C-terminus and that seeding efficiency is closely linked to the conformational flexibility of the monomer at the fibril surface.
To test this, we probe whether monomer–fibril interactions are conformation-specific, sequence-specific, or mediated by multiple weak contacts versus stronger anchoring interactions. Using complementary approaches, including solution-state NMR to capture transient interactions, AFM to visualize fibril surfaces, ion mobility spectrometry to probe monomer conformations, and quenched HDX-NMR to track solvent accessibility, we aim to define how the C-terminal domain (CTD) recruits monomers and promotes new seed formation. By integrating these approaches, we establish how secondary nucleation is catalyzed at fibril surfaces and open new avenues for therapeutic targeting.
Monomer–fibril interface in secondary nucleation:
αS monomers are recruited to the fibril surface through C-terminal contacts, positioning the N-terminal residues 1–12 against the fibril. These interactions promote secondary nucleation and the generation of new seeds.
From Mechanism to Medicine: Engineering Targeted Inhibitors:
Building on these insights, our strategy is to block the CTD interface by engineering binding modules that reduce secondary nucleation and limit seed propagation, directly targeting the amplification step rather than bulk aggregate clearance. PDZ domains, which naturally recognize C-terminal motifs, provide a versatile scaffold; inspired by the multidomain architecture of HtrA1, we test PDZ constructs both alone and in combination with partner domains. In collaboration with Dr. Sagar Khare, we integrate computational design with NMR and biophysical feedback to refine PDZ and PDZ-fusion constructs, iteratively optimizing affinity, avidity, and specificity. This biophysics-guided design loop allows us to outcompete weak monomer–fibril contacts that drive secondary nucleation. Clinical experience with C-terminal–binding antibodies such as prasinezumab (Phase III) highlights the viability of this therapeutic approach, while our own preliminary data suggest that engineered PDZ variants and HtrA1-inspired multidomain proteins can surpass natural scaffolds in potency. By coupling structural insight into fibril-surface catalysis with protein engineering, we aim to develop mechanism-guided therapeutics that not only reduce aggregate amplification but also establish a framework for next-generation protein-based inhibitors of synucleinopathies.
Iterative protein design strategy:
Schematic illustrating the feedback loop between mechanistic insights, biophysical studies, and protein engineering. This cycle integrates structural and functional understanding with experimental validation to guide the design and refinement of protein-based inhibitors.
Aggregation Propensity Changes in Synuclein Evolutionary History:
Ancestral sequence reconstruction (ASR) is a tool that allows us to construct sequences of ancient synuclein to probe whether ancestral sequences differ from the modern extant sequences that aggregate to form amyloid fibrils. The aims of this study are to reconstruct the sequences of ancestral synucleins and resurrect them in the lab to analyze its aggregation propensity and biophysical properties. In order to do so, we construct the largest and most complete synuclein phylogenetic tree, trace the ancestors of the synuclein family, and identify key synuclein domains or mutations involved in the synuclein aggregation by applying various biochemical techniques.
