Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Abstract

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin-mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constants of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-PLP interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

Current insights in the molecular genetic pathogenesis of amyotrophic lateral sclerosis

“…The profilin 1 (PFN1) variants (A20T, C71G, T109M, M114T, E117G, G118V, R136W, and Q139L) cause the autosomal dominant form of ALS by interfering with the actin dynamics in two opposite pathways. Firstly, PFN1 directly binds to the actin monomers, blocking the sites for new monomer addition, and thus preventing the actin filament assembly (Pimm et al, 2020;Liu et al, 2022). Secondly, PFN1 enhances the actin filament assembly by interacting with PFN, a profilamentous actin-binding protein (Schmidt et al, 2021).…”