ENAH Binding to the Huntingtin Proline-Rich Domain Modulates HTT1a Assembly

Abstract

The proline-rich domain (PRD) of huntingtin (HTT), located C-terminal to the polyglutamine tract within the exon-1 region, plays a critical role in modulating the aggregation and toxicity of mutant HTT1a. To identify PRD-interacting proteins, we performed mass spectrometry analysis of PRD peptide pulldowns from human neurons. ENAH, a member of the Ena/VASP family of actin regulatory proteins, emerged as the top enriched interactor from neuronal membrane fractions. In neurons, ENAH colocalized with HTT1a species, suggesting a biologically relevant interaction. Because the EVH1 domain of ENAH binds polyproline motifs with high affinity, we examined its interaction with HTT1a using recombinant proteins. EVH1 directly bound HTT1a and unexpectedly formed condensate-like assemblies capable of recruiting HTT1a monomers. Remarkably, EVH1 robustly suppressed HTT1a oligomerization and promoted remodeling of preformed HTT1a fibrils in vitro. These findings demonstrate that ENAH-PRD interactions modulate HTT1a aggregation dynamics and identify EVH1 assembly as a potential proteostatic mechanism regulating mutant HTT1a states. More broadly, this work establishes a framework for investigating how EVH1-containing proteins may influence HTT1a proteostasis and neurotoxicity in Huntington's disease.