Structural basis for mutation-induced destabilization of profilin 1 in ALS

Abstract

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutationinduced destabilization can account for the high propensity of ALSlinked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.amyotrophic lateral sclerosis | profilin 1 | protein stability | X-ray crystallography | protein misfolding M utations in the profilin 1 gene (PFN1) were recently associated with both familial and sporadic forms of amyotrophic lateral sclerosis (ALS) (1, 2), an incurable and fatal neurodegenerative disease that primarily targets motor neurons (3). The etiology of sporadic ALS is poorly understood, whereas familial ALS is caused by inheritable genetic defects in defined genes such as PFN1 (3). PFN1 is a 15-kDa protein that is best known for its role in actin dynamics in the context of endocytosis, membrane trafficking, cell motility, and neuronal growth and differentiation (4). In addition to binding monomeric or G-actin, PFN1 also binds to a host of different proteins through their poly-L-proline motifs and to lipids such as phosphatidylinositol 4,5-bisphosphate (4, 5). However, little is known about the mechanism(s) associated with PFN1-mediated ALS pathogenesis. The observation that most ALS-linked PFN1 variants are highly prone to aggregation in mammalian cultured cells suggests that diseasecausing mutations induce an altered, or misfolded, conformation within PFN1 (2). Protein misfolding is a hallmark feature of most neurodegenerative diseases, including ALS (3), and can contribute to disease through both gain-of-toxic-function and loss-of-normalfunction mechanisms (6). Although mutations in PFN1 cause ALS through a dominant inheritance mode (2), there is some evidence supporting a loss-of-function mechanism for mutant PFN1. For example, ALS-linked mutations were shown to abrogate the binding of PFN1 to actin (2) and to impair the incorporation of PFN1 into cytoplasmic stress granules during arsenite-induced stress (7) in cultured cells. Moreover, ectopic expression of these variants in murine motor neurons led to a reduction in both axon outgrowth and growth cone size, consistent with a loss of function through a dominant-negative mechanism (2).Although ALS-linked mutations were shown to induce PFN1 aggregation, the ef...

Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS

“…For example, profilin1 has so far not been found in human inclusions (27). When overexpressed, the mutant variant aggregates in cell culture (27), and structural studies show that mutations cause protein instability leading to a shorter half-life and loss of function (55). Our model predicts that destabilization of profilin1 might result in aggregateindependent proteostasis distraction by (i) production of a destabilized profilin1 that requires increased interaction from chaperones and degradation machinery and/or (ii) disruption of its function associated with cytoskeleton stability (56).…”

Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity

“…Some evidence implicates a loss of function in the mutants. The ALS-associated mutations cause structural instability and accumulate in cells at lower levels than the wild-type protein (5). Mutant PFN1 binds less efficiently to actin than the wild-type protein, suggesting that the mutations compromise PFN1 function (3).…”

Delineating the relationship between amyotrophic lateral sclerosis and frontotemporal dementia: Sequence and structure-based predictions

“…G118V is located within a solvent-exposed flexible loop and is less destabilizing. FTD-ALS linked mutation, E117G has only a modest effect on the stability supporting the view that E117G is a risk factor for the occurrence of disease rather than being pathogenic [63].…”

“…The 3D crystal structure of WT PFN1, E117G and M114T proteins is known (PDB ID: codes 4X1L, 4X1M, and 4 × 25) [63].…”

“…It is the most common cause of dementia after Alzheimer's disease (AD). The prevalence and incidence rate of FTD among the people belonging to [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] year age group have been estimated to be 10-20 per 100,000 and 3.5-4.1 per 100,000 per year, respectively [1][2][3]. ALS is a devastating neurodegenerative condition affecting the motor neurons from the brain and spinal cord and is usually fatal due to respiratory paralysis which develops within 1-5 years of symptom onset [4].…”