ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization

Abstract

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin–PFN1–polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Profilin Isoforms in Health and Disease – All the Same but Different

“…Such internal voids are structurally more disruptive than peripheral, solvent-exposed cavities, which explains the particularly high instability and aggregation propensity of PFN1 C71G among the biochemically characterized ALS-PFN1 mutants (Eriksson et al, 1992b,a;Del Poggetto et al, 2015a;Xue et al, 2019). In line with the structural and biochemical findings is the particularly high susceptibility of PFN1 C71G to proteasomal degradation in cells (Schmidt et al, 2021). Interestingly, the PFN1 E117G and PFN1 Q139L mutants are less prone to aggregate (Smith et al, 2015).…”

“…Moreover, PFN1 M144T and PFN1 G118V significantly enhance the actin assembly rate of those formins. The augmenting function of both mutations relies on a heightened flexibility in the PFN1 α4 helix, which contacts the actin as well as the poly-proline binding site (Schmidt et al, 2021). In addition, disturbances in nuclear pore complexes and nuclear-cytoplasmic transport occur in ALS PFN1 mutants expressing neurons (Giampetruzzi et al, 2019).…”

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

“…Thus, we sought to assess the effects of each ALS-associated profilin variant on formin-mediated actin polymerization. For these analyses we chose the representative formin mDia1 for three reasons: 1) mDia1 polymerizes actin at the highest rates recorded among all the mammalian formins, thus impairments might be easier to detect; 2) wild-type profilin and mDia1 interact at discrete sites of cellular actin polymerization (Jacquemet et al, 2019); and 3) mDia1 is known to interact with a subset of ALS-associated profilins (G118V and M114T) (Schmidt et al, 2021). First, we compared the average rates of formin-mediated actin assembly in the presence of each ALS-associated profilin in bulk pyrene fluorescence assays (Figure S1B).…”

“…Each of these variants enhanced formin-mediated actin elongation. While both mutations reside far from the PLP binding surface (Figure 1A), they may enhance forminbased elongation by increasing the off-rate of profilin from PLP sequences in the formin FH1 domains (Figure 5A and 5B) (Liu and Henty-Ridilla, 2022; Schmidt et al, 2021). Alternatively, binding differences seen with this two PLP motifcontaining peptide may be accentuated in formins that contain many PLP motifs; for example, mDia1 contains at least fifteen PLP binding sites on each of the two FH1s present in the active dimer (Courtemanche, 2018;Courtemanche and Pollard, 2012;Paul et al, 2008;Zweifel and Courtemanche, 2020).…”

“…Through these combined mechanisms, profilin effectively regulates most of the known mechanisms of cellular actin assembly (Funk et al, 2019;Skruber et al, 2020Skruber et al, , 2018Suarez and Kovar, 2016). ALS-associated mutations in profilin are commonly thought to disrupt actin filament assembly mechanisms, either through direct interactions with actin monomers or indirectly through their association with actin assembly promoting proteins like Ena/VASP and formin (Alkam et al, 2017;Giampetruzzi et al, 2019;Schmidt et al, 2021). In a synthetic lethality screen from yeast, the C71G, M114T, and G118V ALS-related variants fail to complement the loss of wild-type profilin (Figley et al, 2014).…”