Varkey, J. | Search bioRxiv

bioRxiv2026

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

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.

neuroscience↗

bioRxiv2020

Structural model of the proline-rich domain of huntingtin exon-1 fibrils

Huntingtons disease (HD) is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine (polyQ) domain that increases the propensity of huntingtin exon-1 (HTTex1) to form cross-{beta} fibrils. While the polyQ domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of HTTex1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800 ns long molecular dynamics (MD) simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired EPR DEER distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline II (PPII) helical conformation. The two polyproline (polyP) regions within the PRD stay in a PPII helix for most of the simulation, whereas occasional kinks in the proline rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD. Statement of SignificanceHD is caused by a polyQ expansion in the exon-1 of huntingtin, which results in the formation of fibrillar huntingtin aggregates. Although the polyQ domain is the site of the disease-causing mutation, the PRD domain of HTTex1 is important for fibril toxicity and contains many epitopes of fibril-specific HTTex1 antibodies. Here, we present a structural and dynamic model of the highly dynamic PRD domain using a combination of EPR, solid-state NMR, and MD simulations. This model paves the way for studying known HTTex1 fibril specific binders and designing new ones.

biophysics↗