FUS Protein Research Articles

HOTAIR regulates SIRT3-mediated cardiomyocyte survival after myocardial ischemia/reperfusion by interacting with FUS

BackgroundMyocardial ischemia/reperfusion (I/R) contributes to serious myocardial injury and even death. Therefore, prevention and mitigation of myocardial I/R is particularly important. LncRNA HOTAIR has been reported to be implicated in myocardial I/R progression. However, the detailed molecular mechanism of HOTAIR in cardiomyocyte was explored in myocardial I/R.MethodsFirstly, cell model of myocardial I/R was established through hypoxia/reoxygenation (H/R). Apoptosis and cell cycle were evaluated utilizing flow cytometry. The corresponding test kits were conducted to monitor the levels of LDH, Caspase3 and Caspase9. The gene expression and protein levels were detected by qPCR and western blot, respectively. RNA pull-down and RIP were performed to verify the interaction between FUS and lncRNA HOTAIR.ResultsIn AC16 cardiomyocytes treated with H/R, lncRNA HOTAIR and SIRT3 expression were obviously decreased. Overexpression of HOTAIR or SIRT3 could ameliorate H/R-induced cardiomyocyte injury by promoting cell viability, lowering LDH levels, and suppressing cell apoptosis. Further, lncRNA HOTAIR upregulated the expression of SIRT3 via interacting with FUS, thereby promoting the survival of H/R-injured cardiomyocytes.ConclusionLncRNA HOTAIR can improve myocardial I/R by affecting cardiomyocyte survival through regulation of SIRT3 by binding to the RNA binding protein FUS.

Hypoxia-responsive circular RNA circAAGAB reduces breast cancer malignancy by activating p38 MAPK and sponging miR-378 h

BackgroundBreast cancer is a prevalent disease in women, with high prevalence worldwide. The hypoxic microenvironment of solid tumors develops during the progress of carcinogenesis and leads to greater malignancy and treatment resistance. Recently, accumulating evidence indicates that non-coding RNAs, such as circular RNAs (circRNAs), play a pivotal role in altering cellular functions. However, the underlying mechanisms of circRNAs in breast cancer are still unclear. Therefore, the purpose of this study was to investigate the role of a tumor-suppressive circRNA, circAAGAB, in breast cancer by assuming down-regulation of circAAGAB under hypoxia and the properties of a tumor suppressor.MethodsFirstly, circAAGAB was identified from expression profiling by next generation sequencing. Next, the stability of circAAGAB increased by interacting with the RNA binding protein FUS. Moreover, cellular and nuclear fractionation showed that most circAAGAB resided in the cytoplasm and that it up-regulated KIAA1522, NKX3-1, and JADE3 by sponging miR-378 h. Lastly, the functions of circAAGAB were explored by identifying its down-stream genes using Affymetrix microarrays and validated by in vitro assays.ResultsThe results showed that circAAGAB reduced cell colony formation, cell migration, and signaling through p38 MAPK pathway, as well as increased radiosensitivity.ConclusionThese findings suggest that the oxygen-responsive circAAGAB acts as a tumor suppressor in breast cancer, and may contribute to the development of a more specific therapeutic regimen for breast cancer.

Comprehensive fluorescence lifetime fluctuation spectroscopy to investigate protein mobility during aggregation processes in living cells.

How proteins move and interact in the complicated, crowded cellular environment is still unknown. The formation of membrane-less condensates, caused by interacting proteins and RNA molecules, in the so-called liquid-to-liquid phase transition has recently raised scientific interest. Primarily related to protein mutations, the condensates can undergo a liquid-to-solid transition often associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Here, we introduce a novel method based on fluorescence fluctuation spectroscopy (FFS) as a tool to study proteins interactions, movement, and aggregate formation comprehensively. FFS is a family of analytical methods that allow quantifying molecular processes by studying the fluorescence intensity fluctuations generated by a population of fluorescent molecules entering and leaving a confined microscope focal volume. We enhance conventional FFS methods by employing a novel single-photon-avalanche-diode array detector in an ISM confocal microscope, which allows us to spatially and temporally tag single photons. Within the same system, we can systematically, and with high throughput, access a wide range of spatial and temporal scales, leading to a comprehensive knowledge of the process. In particular, we can follow slow changes in the overall cell organization by performing time-lapse super-resolved image scanning microscopy, fast-slow molecular movements on the level of single proteins, e.g., time-resolved FFS, and interaction with dual-color cross-correlation methods. Moreover, we introduce fluorescence lifetime fluctuation spectroscopy, a novel paradigm where the molecule's mobility and diffusion mode are simultaneously investigated with fluorescence lifetime, allowing us to directly correlate changes in diffusion mode/mobility with structural changes in the molecule's environment. We validated our methods by measuring the time evolution of stress granules formation, focusing on G3BP1 and FUS proteins, in an in vitro cellular system that recapitulates several features of the ALS pathology.

Experimental investigations of coupled polymer and membrane phase transitions.

Polymers can separate into polymer dense and polymer dilute liquid phases in solution while multicomponent lipid membranes can separate into distinct phases within the two-dimensional plane of the membrane. Recent theoretical and simulation work predicts the presence of a polymer rich surface phase when these two-phase transitions are coupled by including polymer conjugated lipids in membranes, and that the location of this phase boundary is tuned by both polymer and lipid interactions1. Here, we experimentally observe the predicted domains of polymer rich surface phase when a small subset of polymers are conjugated to membranes. These surface phases appear at concentrations where polymers do not condense in the bulk, and the concentration needed to form surface phase depends on membrane composition. Experiments have been conducted both with a simple polyelectrolyte and with FUS proteins in separate measurements, demonstrating the robustness of this effect. 1. Rouches, M., Veatch, S. & Machta, B. Surface Densities Prewet a Near-Critical Membrane. bioRxiv2021.02.17.431700 (2021).

Complex coacervation contributes to the joint phase behaviors of mixtures of charge-deficient prion-like low-complexity domains.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of various cellular biomolecular condensates, including stress granules and transcriptional condensates, which form via phase separation coupled to percolation. Our recent work has uncovered how evolutionarily conserved sequence features drive PLCD phase behavior through homotypic interactions. Since many condensates encompass a diverse mixture of proteins with PLCDs, we asked if mixtures of PLCDs have emergent phase behaviors that go beyond the intrinsic phase behaviors that have been well-documented through recent, in-depth biophysical studies. Specifically, we combined coarse-grained, single-bead-per-residue stickers-and-spacers simulations with experimental phase separation assays to study mixtures of PLCDs from the proteins hnRNPA1 and FUS. We refer to these domains as A1-LCD and FUS-LCD, respectively. Surprisingly, we find that a 1:1 mixture of the two species phase separates more readily than either sequence on its own. We demonstrate that the enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD comes from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic sticker residues. Our findings are striking because we find evidence for an enhancement mediated by complex coacervation even though only 12% and 2.8% of the residues in the A1- and the FUS-LCD are charged. Importantly, the condensates formed by mixtures of A1- and FUS-LCD show inhomogeneous spatial organization whereby molecules are spatially proximal to species with which they have the strongest interactions. This deviates from a random mixture model and instead reflects a fine interplay between homotypic and heterotypic associations. Our findings support the emerging view that multicomponent condensates involve complex networks of homotypic and heterotypic interactions that drive a coupling between associative and segregative phase transitions.

Molecular insights into the effect of alkanediols on FUS liquid-liquid phase separation.

Numerous cell biology studies have used high concentrations of 1,6-hexanediol to dissolve membraneless organelles and disordered protein biomolecular condensates. Yet, little is known about how alkanediols effect liquid-liquid phase separation (LLPS), and why certain alkanediol isomers are more effective. Here, we evaluate the effect of various alkanediols on the archetypal phase separating protein FUS. Low-complexity domain and full-length FUS LLPS is decreased varyingly, while LLPS of FUS RGG-RNA condensates is even enhanced by some alkanediols. NMR experiments show that all diols act similarly, correlating atomistic changes with LLPS-preventing effects. Furthermore, we find no evidence for specific residue interactions - the largest perturbations are seen at backbone and glutamine side-chain hydrogen bonding sites, not hydrophobic/aromatic residues. Furthermore, 1,6 hexanediol favors formation of protein-solvent hydrogen bonds and increases FUS local motions. These findings show how alkanediols affect water-disordered protein interactions, underscoring the difficulty in using alkanediol-derivatives to target dissolution of specific membraneless organelles.

RNA structure controls assembly of multi-layered paraspeckles.

Biomolecular condensates mediate key cellular functions from gene expression to signaling. Condensates assemble via multivalent interactions amongst proteins and nucleic acids and often behave as liquid-like droplets. An essential requirement for condensate function is the establishment of a distinct compositional identity. How cells maintain coexisting populations of different condensates, despite the propensity of liquids to fuse, is poorly understood. Multi-layered condensates, comprising layers of distinct biomolecular composition, are useful for understanding the establishment of molecular identity and the opposition to mixing. We set out to uncover molecular cues that encode multi-layered condensates by studying nuclear paraspeckles, composed of a core and shell. Paraspeckles are scaffolded by a long noncoding RNA called NEAT1 that bridges the core and shell and recruits different proteins to each layer. The molecular features of NEAT1 that encode for each layer are unknown. We found that isolated core or shell fragments of NEAT1 can assemble condensates with the paraspeckle core-associated protein FUS. When both RNAs are present, condensates do not fuse but instead form a core and shell reminiscent of paraspeckles. Quantitative imaging showed that condensates formed with shell RNA display a higher protein/RNA ratio compared to core RNA. Probing RNA structure uncovered that shell RNA was more well-structured compared to core RNA, suggesting that RNA structure contributes to differential protein recruitment to paraspeckle layers. Consistent with this idea, disrupting the native structures of each RNA via melting and rapid cooling drove dramatic changes in protein and RNA recruitment. Thus, the structural features along an RNA strand likely control the spatial arrangement of the RNA within paraspeckles and tune protein recruitment to each layer. More broadly, our findings suggest that RNA structure is a powerful control knob that determines condensate identity and function in diverse contexts.

Circular RNA circGlis3 protects against islet β-cell dysfunction and apoptosis in obesity

Pancreatic β-cell compensation is a major mechanism in delaying T2DM progression. Here we report the abnormal high expression of circGlis3 in islets of male mice with obesity and serum of people with obesity. Increasing circGlis3 is regulated by Quaking (QKI)-mediated splicing circularization. circGlis3 overexpression enhances insulin secretion and inhibits obesity-induced apoptosis in vitro and in vivo. Mechanistically, circGlis3 promotes insulin secretion by up-regulating NeuroD1 and Creb1 via sponging miR-124-3p and decreases apoptosis via interacting with the pro-apoptotic factor SCOTIN. The RNA binding protein FUS recruits circGlis3 and collectively assemble abnormal stable cytoplasmic stress granules (SG) in response to cellular stress. These findings highlight a physiological role for circRNAs in β-cell compensation and indicate that modulation of circGlis3 expression may represent a potential strategy to prevent β-cell dysfunction and apoptosis after obesity.

Impaired RNA Binding Does Not Prevent Histone Modification Changes in a FUS ALS/FTD Yeast Model.

Mutations in the RNA-binding protein FUS are linked to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). FUS mutants mislocalize and aggregate in dying neurons. We previously established that FUS proteinopathy is linked to changes in the histone modification landscape in a yeast ALS/FTD model. Here, we examine whether FUS' RNA binding is necessary for this connection. We find that overexpression of a FUS mutant unable to bind RNA is still associated with reduced levels of H3S10ph, H3K14ac and H3K56ac. Hence, FUS' ability to bind RNA is not required in the mechanism connecting FUS proteinopathy to altered histone post-translational modifications.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix.

Disentangling selective vulnerability underlying sleep disfunction in neurodegenerative diseases: differential gene expression in neuromodulatory subcortical system in Alzheimer’s disease vs. Progressive supranuclear palsy vs. controls

AbstractBackgroundSleep‐wake disturbance precedes the prodromal stage of Alzheimer’s disease (AD) and is characterized by excessive daytime sleepiness and sundowning. In contrast, hyper‐insomnia is seen in progressive supranuclear palsy (PSP), a pure 4‐repeat tauopathy. The wake‐promoting histaminergic (HA) neurons of the tuberomamillary nucleus (TMN) together with the orexinergic (OX) neurons of the lateral hypothalamic area (LHA) modulate the sleep‐wake cycle. Previously, we reported wake‐promoting nucleus (WPNs) are selectively vulnerable to AD‐tau toxicity than PSP, and neuronal loss in TMN is specific to AD. In the present study, we investigated changes in RNA expression in the TMN and LHA to understand the selective vulnerability of WPNs in AD at the molecular level.MethodLHA and TMN were dissected from serial histological sections from the postmortem brains of AD, PSP, and healthy control (HC) subjects. 50ng total RNA was extracted using RNeasy Micro Kit, and RNA integrity was accessed using Nanodrop. Cases with RIN > 5 were included for RNA sequencing. The nCounter Neuropathology panel and nSolver software (NanoString Technologies) was employed to obtain and analyze the data.ResultMolecular profiling of TMN in AD demonstrated 13 upregulated genes associated with angiogenesis, apoptosis, chromatin modification, disease‐association, and transcription factor, and 12 genes in PSP. The proto‐oncogene RELA, RNA binding protein FUS and oligodendrocyte specific NKX6‐2 genes were upregulated in AD and PSP. Further, in the LHA, we identified 17 differentially expressed genes associated with circadian rhythm, inflammatory cytokines, apoptosis, autophagy, and angiogenesis in AD over HC and 72 genes in PSP over HC. Both IL4 and CRY2 genes demonstrated downregulation in the AD and PSP.ConclusionDifferential gene expression in the WPNs revealed a significant upregulation of genes associated with pro‐inflammatory cytokines, activated microglia, apoptosis, oxidative stress, and autophagy in AD over PSP. The neuromodulatory subcortical system shows different vulnerability patterns in AD vs. PSP making it an excellent candidate to interrogate the molecular bases of vulnerability.

Conformational Heterogeneity of RNA Stem-Loop Hairpins Bound to FUS-RNA Recognition Motif with Disordered RGG Tail Revealed by Unbiased Molecular Dynamics Simulations.

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here, we used an extended set of microsecond-scale MD trajectories (400 μs in total) to study two FUS-RNA constructs previously characterized by nuclear magnetic resonance (NMR) spectroscopy. The FUS protein contains a well-structured RNA recognition motif domain followed by a presumably disordered RGG tail that binds RNA stem-loop hairpins. Our simulations not only provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data. We propose that the dynamics is so rich that its converged description is not achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops, which can explain the NMR data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate dynamic RNA-protein complexes.

Stress-induced perturbations in intracellular amino acids reprogram mRNA translation in osmoadaptation independently of the ISR.

SUMMARYThe integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury–associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury–specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury–induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury–induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

Intrinsically Disordered Protein Condensate-Modified Surface for Mitigation of Biofouling and Foreign Body Response.

Mitigation of biofouling and the host's foreign body response (FBR) is a critical challenge with biomedical implants. The surface coating with various anti-fouling materials provides a solution to overcome it, but limited options in clinic and their potential immunogenicity drive the development of more alternative coating materials. Herein, inspired by liquid-liquid phase separation of intrinsically disordered proteins (IDPs) to form separated condensates in physiological conditions, we develop a new type of low-fouling biomaterial based on flexible IDP of FUS protein containing rich hydrophilic residues. A chemical structure-defined FUS IDP sequence tagged with a tetra-cysteine motif (IDPFUS) was engineered and applied for covalent immobilization on various surfaces to form a uniform layer of protein tangles, which boosted strong hydration on surfaces, as revealed by molecular dynamics simulation. The IDPFUS-coated surfaces displayed excellent performance in resisting adsorption of various proteins and adhesion of different cells, platelets, and bacteria. Moreover, the IDPFUS-coated implants largely mitigated the host's FBR compared with bare implants and particularly outperformed PEG-coated implants in reducing collagen encapsulation. Thus, this novel low-fouling and anti-FBR strategy provides a potential surface coating material for biomedical implants, which will also shed light on exploring similar applications of other IDP proteins.

Upregulation of β-catenin due to loss of miR-139 contributes to motor neuron death in amyotrophic lateral sclerosis.

SummaryAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons (MNs). There are no effective treatments and patients usually die within 2–5 years of diagnosis. Emerging commonalities between familial and sporadic cases of this complex multifactorial disorder include disruption to RNA processing and cytoplasmic inclusion bodies containing TDP-43 and/or FUS protein aggregates. Both TDP-43 and FUS have been implicated in RNA processing functions, including microRNA biogenesis, transcription, and splicing. In this study, we explore the misexpression of microRNAs in an iPSC-based disease model of FUS ALS. We identify the downregulation of miR-139, an MN-enriched microRNA, in FUS and sporadic ALS MN. We discover that miR-139 downregulation leads to the activation of canonical WNT signaling and demonstrate that the WNT transcriptional mediator β-catenin is a major driver of MN degeneration in ALS. Our results highlight the importance of homeostatic RNA networks in ALS.

Circ-0002814 participates in proliferation and migration through miR-210 and FUS/VEGF pathway of preeclampsia.

Circular RNA plays a critical role in cell proliferation, differentiation, and autophagy. However, the role and mechanism of circRNA in preeclampsia is yet to be clarified. The present study investigated the function and mechanism of circ-0002814 in preeclampsia. The expression level of circ-0002814, microRNA 210 (miR210), Notch receptor 1 (Notch1), or cytoplasmic polyadenylation element-binding protein 2 (CPEB2) was investigated by Reverse transcription Real-time Quantitative PCR (RT-qPCR). The level of Notch1, CPEB2, and FUS (FUS RNA-binding protein) proteins was detected by western blot. The localization of circ-0002814 and miR-210 was determined by FISH assay. Cell proliferation and invasion were detected by EdU and transwell assays, respectively. The interaction between circ-0002814 and FUS protein was detected by RNA pulldown assay, while that between circ-0002814 and miR-210 was detected by dual-luciferase reporter assay. The expression of circ-0002814 was decreased in the placenta and plasma of patients with preeclampsia. The ectopic expression of circ-0002814 promoted the invasion and proliferation of HTR8 cells compared to the control group (p < 0.05). Silencing circ-0002814 suppressed the invasion and proliferation of JEG3 cells compared to the control group (p < 0.05). circ_0002814-regulated Notch1 and CPEB2 by interaction with miR-210 and also regulated FUS/VEGF axis in HTR8 and JEG3 cells. A low expression of circ-0002814 was detected in the plasma and placental tissue in preeclampsia patients. circ-0002814 participated in the proliferation and invasion of HTR8 and JEG3 cells. Thus, circ-0002814 may be considered as a potential diagnostic marker and therapeutic target for preeclampsia.

Advances in Treatment of Frontotemporal Dementia.

In this review, the authors explored the clinical features of frontotemporal dementia (FTD), focusing on treatment. The clinical features of FTD are unique, with disinhibition, apathy, loss of empathy, and compulsions common. Motor changes occur later in the illness. The two major proteins that aggregate in the brain with FTD are tau and TDP-43, whereas a minority of patients aggregate FET proteins, primarily the FUS protein. Genetic causes include mutations in MAPT, GRN, and C9orf72. There are no medications that can slow FTD progression, although new therapies for the genetic forms of FTD are moving into clinical trials. Once a diagnosis is made, therapies should begin, focusing on the family and the patient. In the setting of FTD, families experience a severe burden associated with caregiving, and the clinician should focus on alleviating this burden. Advice around legal and financial issues is usually helpful. Careful consideration of environmental changes to cope with abnormal behaviors is essential. Most compounds that have been used to treat dementia of the Alzheimer's disease type are not effective in FTD, and cholinesterase inhibitors and memantine should be avoided. Although the data are scant, there is some evidence that antidepressants and second-generation antipsychotics may help individual patients.

A molecular view of amyotrophic lateral sclerosis through the lens of interaction network modules.

BackgroundDespite the discovery of familial cases with mutations in Cu/Zn-superoxide dismutase (SOD1), Guanine nucleotide exchange C9orf72, TAR DNA-binding protein 43 (TARDBP) and RNA-binding protein FUS as well as a number of other genes linked to Amyotrophic Lateral Sclerosis (ALS), the etiology and molecular pathogenesis of this devastating disease is still not understood. As proteins do not act alone, conducting an analysis of ALS at the system level may provide new insights into the molecular biology of ALS and put it into relationship to other neurological diseases.MethodsA set of ALS-associated genes/proteins were collected from publicly available databases and text mining of scientific literature. We used these as seed proteins to build protein-protein interaction (PPI) networks serving as a scaffold for further analyses. From the collection of networks, a set of core modules enriched in seed proteins were identified. The molecular biology of the core modules was investigated, as were their associations to other diseases. To assess the core modules’ ability to describe unknown or less well-studied ALS biology, they were queried for proteins more recently associated to ALS and not involved in the primary analysis.ResultsWe describe a set of 26 ALS core modules enriched in ALS-associated proteins. We show that these ALS core modules not only capture most of the current knowledge about ALS, but they also allow us to suggest biological interdependencies. In addition, new associations of ALS networks with other neurodegenerative diseases, e.g. Alzheimer’s, Huntington’s and Parkinson’s disease were found. A follow-up analysis of 140 ALS-associated proteins identified since 2014 reveals a significant overrepresentation of new ALS proteins in these 26 disease modules.ConclusionsUsing protein-protein interaction networks offers a relevant approach for broadening the understanding of the biological context of known ALS-associated genes. Using a bottom-up approach for the analysis of protein-protein interaction networks is a useful method to avoid bias caused by over-connected proteins. Our ALS-enriched modules cover most known biological functions associated with ALS. The presence of recently identified ALS-associated proteins in the core modules highlights the potential for using these as a scaffold for identification of novel ALS disease mechanisms.

Engineered Nuclear Import Receptor Karyopherin‐β2 Chaperones Aberrant Phase Transitions of Disease‐Associated Cargo

Karyopherin‐β2 (Kapβ2) is a nuclear import receptor (NIR) that localizes proteins bearing a proline‐tyrosine nuclear localization signal (PY‐NLS) to the nucleus, including the RNA‐binding protein (RBP) FUS. Recent work has demonstrated that NIRs can chaperone RBPs in vitro and in cells, preventing and reversing their self‐assembly and aggregation. However, when the PY‐NLS is mutated, Kapβ2 no longer efficiently chaperones its cargo. Here, we focus on the case of FUSP525L, an RBP NLS mutant associated with a highly aggressive form of juvenile ALS. Using a structural approach, we have engineered Kapβ2 to recognize mutant FUS and demonstrate improved chaperone activity in vitro, in human cells, and in mice.