Profilin 1 Associates with Stress Granules and ALS-Linked Mutations Alter Stress Granule Dynamics

110

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...

Section: Discussionmentioning

confidence: 74%

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

Boopathy

Silvas

Tischbein

et al. 2015

110

“…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). Both of these possibilities may result in the aggregation of supersaturated proteins, such as TDP-43, but not do not require the aggregation of profilin1.…”

Section: Protein Supersaturation In the Als Network Underlies The Promentioning

confidence: 90%

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

Ciryam

Lambert-Smith

Bean

et al. 2017

Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that diseaseaffected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.protein aggregation | protein misfolding | protein homeostasis | supersaturation | motor neuron disease

“…Mutant PFN1 binds less efficiently to actin than the wild-type protein, suggesting that the mutations compromise PFN1 function (3). The severest mutations also are incapable of compensating for loss-of-function PFN1 mutation in yeast (6). Other evidence supports a gain of function.…”

mentioning

confidence: 99%

“…Expression of mutant, but not wild-type, PFN1 inhibits filamentous actin formation and impairs growth cone function and neurite growth (3). Furthermore, PFN1 mutants have been shown to alter stress granule dynamics in cultured mammalian cells and form cellular aggregates that may contain other proteins contributing to pathogenesis (6).…”

mentioning

confidence: 99%

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

Yang

Danielson

Qiao

et al. 2016

100

Mutations in the profilin 1 (PFN1) gene cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease caused by the loss of motor neurons leading to paralysis and eventually death. PFN1 is a small actin-binding protein that promotes formin-based actin polymerization and regulates numerous cellular functions, but how the mutations in PFN1 cause ALS is unclear. To investigate this problem, we have generated transgenic mice expressing either the ALSassociated mutant (C71G) or wild-type protein. Here, we report that mice expressing the mutant, but not the wild-type, protein had relentless progression of motor neuron loss with concomitant progressive muscle weakness ending in paralysis and death. Furthermore, mutant, but not wild-type, PFN1 forms insoluble aggregates, disrupts cytoskeletal structure, and elevates ubiquitin and p62/ SQSTM levels in motor neurons. Unexpectedly, the acceleration of motor neuron degeneration precedes the accumulation of mutant PFN1 aggregates. These results suggest that although mutant PFN1 aggregation may contribute to neurodegeneration, it does not trigger its onset. Importantly, these experiments establish a progressive disease model that can contribute toward identifying the mechanisms of ALS pathogenesis and the development of therapeutic treatments.A LS is a neurodegenerative disease that causes a relentless progressive loss of motor neurons, leading to progressive weakness that ends in paralysis and death (1). Mutations in the PFN1 gene have been identified as a genetic cause for ALS (2, 3). PFN1 is a major regulator of actin polymerization through its ability to bind actin-ADP monomers and promote the conversion of actin-ADP to actin-ATP, through transporting the actin-ATP monomers and through its interactions within formins present at the growing end of actin filaments. Additionally PFN1 binds to phosphoinositides and a large network of proteins with poly-Lproline stretches. Through these binding interactions, PFN1 regulates several cellular functions including actin dynamics, membrane trafficking, neuronal synaptic structure and activity, small GTPase signaling, and others (4). Despite our understanding of its function, how PFN1 mutations cause motor neuron degeneration remains elusive. 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). The severest mutations also are incapable of compensating for loss-of-function PFN1 mutation in yeast (6). Other evidence supports a gain of function. Expression of mutant, but not wild-type, PFN1 inhibits filamentous actin formation and impairs growth cone function and neurite growth (3). Furthermore, PFN1 mutants have been shown to alter stress granule dynamics in cultured mammalian cells and form cellular aggregates that may contain other proteins contributing to path...