Making the Invisible Visible: A Molecular View on Early-Stage Protein Aggregation of α-Synuclein
Gobert Heesink is a PhD student in the department Nanobiophysics. (Co)Promotors are prof.dr. M.M.A.E. Claessens and dr. C. Blum from the faculty of Science & Technology (TNW), University of Twente.
Neurodegenerative diseases (NDs) pose a growing global health challenge, with protein aggregation as a central hallmark. In Parkinson’s disease (PD), the intrinsically disordered protein α-synuclein (αS) aggregates into amyloid fibrils. While fibrils have been extensively studied, the early aggregation mechanisms remain elusive. This thesis focuses on the formation and modulation of αS multimers – transient, pre-amyloid intermediates that may act as regulatory or pathological precursors.
Using single-molecule and bulk fluorescence spectroscopy, combined with computational and biochemical approaches, we assess the full αS aggregation pathway from monomer to fibril, with emphasis on the multimeric stages and their regulation by protein quality control (PQC) systems. Initial method development enabled probing of αS at the single-molecule level, revealing how local sequence features modulate its disordered conformations. We then demonstrated that αS self-assembles into dynamic, liquid-like multimers at sub-micromolar concentrations. These nanoscale assemblies share hallmarks with biomolecular condensates and support a unified phase-transition model of aggregation, where condensation and deposition pathways differ mainly by the length scale of their intermediate states: microscale droplets versus nanoscale multimers.
Physiological modulators, including sequence elements and post-translational modifications, were found to tune multimerization without affecting fibril formation – underscoring its mechanistic distinctness. PQC systems likely target this gateway step. Indeed, the chaperone-like protein 14-3-3τ selectively engages αS multimers to form smaller, off-pathway co-condensates, preventing further aggregation. Phosphorylation at Serine-129 enhances this co-assembly via non-canonical multivalent interactions, highlighting a tuneable mechanism that may preserve protein homeostasis.
Altogether, this thesis advances an integrated view of αS aggregation, from monomer to multimer to fibril, and identifies early multimers as important regulatory nodes. By unveiling these previously “invisible” species, this work reveals novel mechanisms and therapeutic entry points acting at the onset, rather than the aftermath, of pathological protein aggregation in neurodegenerative disease.
