Curated Optogenetic Publication Database

Abstract: The dynamics of the early steps of protein aggregation remain poorly understood, particularly in the case of α-synuclein (α-syn) aggregation, the hallmark of synucleinopathies. Here, we present a protocol that combines light-inducible protein aggregation (LIPA) with proximity biotinylation using an UltraID construct. We describe the workflow from protein expression to biochemical validation, including the purification of biotinylated proteins prior to liquid chromatography-mass spectrometry (LC-MS) analysis and subsequent validation. This platform provides a powerful strategy to identify proteins interacting with nascent α-syn aggregates. For complete details on the use and execution of this protocol, please refer to Teixeira et al.1.

Abstract: Alpha-synuclein (α-syn) aggregation is a defining feature of Parkinson's disease (PD) and related synucleinopathies. Despite significant research efforts focused on understanding α-syn aggregation mechanisms, the early stages of this process remain elusive, largely due to limitations in experimental tools that lack the temporal resolution to capture these dynamic events. Here, we introduce UltraID-LIPA, an innovative platform that combines the light-inducible protein aggregation (LIPA) system with the UltraID proximity-dependent biotinylation assay to identify α-syn-interacting proteins and uncover key mechanisms driving its oligomerization. UltraID-LIPA successfully identified 38 α-syn-interacting proteins, including both established and previously unreported candidates, highlighting the accuracy and robustness of the approach. Notably, a strong interaction with endolysosomal and membrane-associated proteins was observed, supporting the hypothesis that interactions with membrane-bound organelles are pivotal in the early stages of α-syn aggregation. This powerful platform provides new insights into dynamic protein aggregation events, enhancing our understanding of synucleinopathies and other proteinopathies.