Academic literature on the topic 'PFN1 Mutations'

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 mutation-induced destabilization can account for the high propensity of ALS-linked 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.

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.

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 ALS-associated 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.

Abstract Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive. Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice. Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients. Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. Disclosures No relevant conflicts of interest to declare.

Introduction Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of immature myeloid progenitor cells in the bone marrow (BM). Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of AML cases, with Internal Tandem Duplications (ITD) being the most common type of mutation. Several FLT3 specific inhibitors (TKI) have been developed such as quizartinib, crenolanib and midostaurin (recently approved for clinical use). Nevertheless FLT3-ITD is associated with unfavorable prognosis and patients develop drug resistance with the underlying mechanisms remaining largely unexplained. Recently, changes within the actin cytoskeleton were associated with drug resistance development in various cancers. FLT3-ITD mutations are associated with RAC1 activation. RAC1 belongs to the family of RHO GTPases and enhances the actin polymerization by inducing the expression of N-WASP or WAVE2 and ARP2/3 complex. Therefore, we investigated actin cytoskeleton rearrangements through RAC1 activation as a potential mechanism contributing to Midostaurin resistance in AML. Material and methods First, we developed two Midostaurin resistant AML cell lines (MID-RES, MV4-11 and MOLM-13). Single cell measurements of Cell Stiffnes, cell adhesion forces between tumor and HS5 stroma cells and Actin filaments were performed by Atomic Force Microscopy (FluidFM®) and SIM microscopy, respectively. RAC1 activation was measured by RAC1 activation kit provided by Cytoskeleton. FLT3 surface and intracellular expression was measured by Flow cytometry and western blot, respectively. Cell death was analyzed by Annexin/PI staining in flow cytometry. Results The MID-RES cell lines MV4-11/MOLM-13 showed higher FLT3 surface and intracellular expression compared to their MID sensitive parental cells. In line with our expectations, we observed RAC1 activation, as well as an up-regulation of actin polymerization positive regulators such as N-WASP, WAVE2, PFN1 and ARP2/3 complex and the inhibition of actin polymerization negative regulator P-ser3 CFL1 in MID-RES cells. FLT3 receptor knock down by siRNAs reversed the MID resistance and reduced RAC1 activation and actin polymerization inducers expression. Likewise, bioinformatic analysis from publicly available microarray expression data (E-MTAB-3444), confirmed positive correlation between actin polymerization inducers and FLT3 signaling expression in 178 FLT3-ITD (r=0,67) and 461 FLT3 WT(r= 0,57) de novoAML patients. RAC1 induced Actin polymerization positively correlates with actin filaments growth and cell stiffness, which was observed in our MID-RES cells, higher load of actin filaments and increased cell stiffness. The combination between RAC1 specific inhibitor, EHT1864 and Midostaurin synergistically induces cell death in MID-RES cells by arresting cell cycle in G0/G1 phase and activating apoptosis. Beside, this combination reduced the adhesion forces to stroma cells, decreased the expression of PFN1/N-WASP/ARP2 and consequently reduced drastically the number of actin filaments and cell stiffness in MID-RES cells. EHT1864 and Midostaurin (alone and in combination) were not toxic in PBMCs obtained from healthy donors. Interestingly, this combination increase >45 % cell death in cells obtained from refractory FLT3-mutated AML patient (this patient was relapsed (≥ 50% residual blasts in the bone marrow)under Chemotherapy+Midostaurin combination).The specific knock down of PFN1/N-WASP/ARP2 with siRNAs equally reversed the resistance to Midostaurin. Of note, RAC1 regulates the anti-apoptotic BCL2. Indeed, EHT1864 in combination with Midostaurin reduced anti-apoptotic family BCL2/MCL1 expression and increases the pro-apoptotic proteins BAX/PUMA. As expected, our MID-RES cells showed higher sensitivity to BCL2 inhibitor Venetoclax, than their parental cells. The combinations EHT1864+venetoclax, venetoclax+midostaurin and venetoclax+Midostaurin+EHT1864 synergistically induced cell death in MID-RES cells. Conclusion Actin polymerization inducers N-WASP, ARP2/3 complex and PFN1 may provide a valuable approach to overcome Midostaurin resistance in AML. Our data further suggest that the addition of BCL2 inhibition through EHT1864 and venetoclax could represent an interesting strategy to potentiate the activity of Midostaurin in FLT3 mutated AML. Disclosures Duell: Regeneron Pharmaceuticals, Inc.: Research Funding. Rosenwald:MorphoSys: Consultancy.

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated (∼45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure–function relationships for lymphocyte perforin.

Amyotrophic lateral sclerosis (ALS) is a progressive adult neurodegenerative disease that causes death of both upper and lower motor neurons. Approximately 90 percent of ALS cases are sporadic (SALS), and 10 percent are inherited (FALS). Mutations in the PFN1 gene have been identified as causative for one percent of FALS. PFN1 is a small actin-binding protein that promotes actin polymerization, but how ALS-linked PFN1 mutations affect its cognate functions or acquire gain-of-function toxicity remains largely unknown. To elucidate the contribution of ALS-linked PFN1 mutations to neurodegeneration, we have characterized these mutants in both mammalian cultured cells and Drosophila models. In mammalian neuronal cells, we demonstrate that ALS-linked PFN1 mutants form ubiquitinated aggregates and alter neuronal morphology. We also show that ALS-linked PFN1 mutants have partial loss-of-function effects on actin polymerization in growth cones of mouse primary motor neurons and larval neuromuscular junctions (NMJ) in Drosophila. In Drosophila, we also observe that PFN1 level influences integrity of adult motor neurons, as demonstrated by locomotion, lifespan, and leg NMJ morphology. In sum, the work presented in this dissertation has shed light on PFN1- linked ALS pathogenesis by demonstrating a loss-of-function mechanism. We have also developed a Drosophila PFN1 model that will serve as a valuable tool to further uncover PFN1-associated cellular pathways that mediate motor neuron functions.

Amyotrophic lateral sclerosis (ALS) is a progressive adult neurodegenerative disease that causes death of both upper and lower motor neurons. Approximately 90 percent of ALS cases are sporadic (SALS), and 10 percent are inherited (FALS). Mutations in the PFN1 gene have been identified as causative for one percent of FALS. PFN1 is a small actin-binding protein that promotes actin polymerization, but how ALS-linked PFN1 mutations affect its cognate functions or acquire gain-of-function toxicity remains largely unknown. To elucidate the contribution of ALS-linked PFN1 mutations to neurodegeneration, we have characterized these mutants in both mammalian cultured cells and Drosophila models. In mammalian neuronal cells, we demonstrate that ALS-linked PFN1 mutants form ubiquitinated aggregates and alter neuronal morphology. We also show that ALS-linked PFN1 mutants have partial loss-of-function effects on actin polymerization in growth cones of mouse primary motor neurons and larval neuromuscular junctions (NMJ) in Drosophila. In Drosophila, we also observe that PFN1 level influences integrity of adult motor neurons, as demonstrated by locomotion, lifespan, and leg NMJ morphology. In sum, the work presented in this dissertation has shed light on PFN1- linked ALS pathogenesis by demonstrating a loss-of-function mechanism. We have also developed a Drosophila PFN1 model that will serve as a valuable tool to further uncover PFN1-associated cellular pathways that mediate motor neuron functions.