N-Terminal Fragments of TDP-43-In Vitro Analysis and Implication in the Pathophysiology of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration.

Abnormal cytoplasmic aggregates containing the TDP-43 protein and its fragments are present in the central nervous system of the majority of patients with amyotrophic lateral sclerosis (ALS) and in patients with frontotemporal lobar degeneration (FTLD). Many studies have focused on the C-terminal cleavage products of TDP-43 (CTFs), but few have focused on the N-terminal products (NTFs), yet several works and their protein domain composition support the involvement of NTFs in pathophysiology. In the present study, we expressed six NTFs of TDP-43, normally generated in vivo by proteases or following the presence of pathogenic genetic truncating variants, in HEK-293T cells. The N-terminal domain (NTD) alone was not sufficient to produce aggregates. Fragments containing the NTD and all or part of the RRM1 domain produced nuclear aggregates without affecting cell viability. Only large fragments also containing the RRM2 domain, with or without the glycine-rich domain, produced cytoplasmic aggregates. Of these, only NTFs containing even a very short portion of the glycine-rich domain caused a reduction in cell viability. Our results provide insights into the involvement of different TDP-43 domains in the formation of nuclear or cytoplasmic aggregates and support the idea that work on the development of therapeutic molecules targeting TDP-43 must also take into account NTFs and, in particular, those containing even a small part of the glycine-rich domain.

Exploring the effects of 4-chloro-o-phenylenediamine on human fibrinogen: A comprehensive investigation via biochemical, biophysical and computational approaches.

Fibrinogen (Fg), an essential plasma glycoprotein involved in the coagulation cascade, undergoes structural alterations upon exposure to various chemicals, impacting its functionality and contributing to pathological conditions. This research article explored the effects of 4-Chloro-o-phenylenediamine (4-Cl-o-PD), a common hair dye component (IUPAC = 1-Chloro-3,4-diaminobenzene), on human fibrinogen through comprehensive computational, biophysical, and biochemical approaches. The formation of a stable ligand-protein complex is confirmed through molecular docking and molecular dynamics simulations, revealing possible interaction having a favorable -4.8 kcal/mol binding energy. Biophysical results, including UV-vis and fluorescence spectroscopies, corroborated with the computational findings, whereas Fourier transform infrared spectroscopy (FT-IR) and circular dichroism spectroscopy (CD) provide insights into the alterations of secondary structures upon interaction with 4-Cl-o-PD. Anilinonaphthalene-sulfonic acid (ANS) fluorescence showed a partially unfolded protein, with enhanced α to ß-sheet transition as evidenced by thioflavin T (ThT) spectroscopy and microscopy. Moreover, biochemical assays confirmed the formation of carbonyl compounds that may be responsible for the oxidation of methionine residues in fibrinogen. Electrophoresis and electron microscopy confirmed the formation of aggregates. Our findings elucidate the interaction pattern of 4-Cl-o-PD with Fg, leading to structural perturbation, which may have potential implications for fibrinogen misfolding or its aggregation. Protein aggregation or its misfolded products affect peripheral tissues and the central nervous system. Many chronic progressive diseases, like type II diabetes mellitus, Alzheimer's disease, Parkison's disease, and Creutzfeldt-Jakob disease are associated with intrinsically aberrant disordered proteins. Understanding these interactions may offer new perspectives on the safety and biocompatibility of dye compounds, which may contribute to developing improved strategies for acquired amyloidogenesis.

Aggrescan4D: A comprehensive tool for pH-dependent analysis and engineering of protein aggregation propensity.

Aggrescan4D (A4D) is an advanced computational tool designed for predicting protein aggregation, leveraging structural information and the influence of pH. Building upon its predecessor, Aggrescan3D (A3D), A4D has undergone numerous enhancements aimed at assisting the improvement of protein solubility. This manuscript reviews A4D's updated functionalities and explains the fundamental principles behind its pH-dependent calculations. Additionally, it presents an antibody case study to evaluate its performance in comparison with other structure-based predictors. Notably, A4D integrates advanced protein engineering protocols with pH-dependent calculations, enhancing its utility in advising solubility-enhancing mutations. A4D considers the impact of structural flexibility on aggregation propensities, and includes a large set of precalculated predictions. These capabilities should help to open new avenues for both understanding and managing protein aggregation. A4D is accessible through a dedicated web server at https://biocomp.chem.uw.edu.pl/a4d/.

An updated AL-Base reveals ranked enrichment of immunoglobulin light chain variable genes in AL amyloidosis.

Background: Each monoclonal antibody light chain associated with AL amyloidosis has a unique sequence. Defining how these sequences lead to amyloid deposition could facilitate faster diagnosis and lead to new treatments. Methods: Light chain sequences are collected in the Boston University AL-Base repository. Monoclonal sequences from AL amyloidosis, multiple myeloma and the healthy polyclonal immune repertoire were compared to identify differences in precursor gene use, mutation frequency and physicochemical properties. Results: AL-Base now contains 2,193 monoclonal light chain sequences from plasma cell dyscrasias. Sixteen germline precursor genes were enriched in AL amyloidosis, relative to multiple myeloma and the polyclonal repertoire. Two genes, IGKV1-16 and IGLV1-36, were infrequently observed but highly enriched in AL amyloidosis. The number of mutations varied widely between light chains. AL-associated κ light chains harbored significantly more mutations compared to multiple myeloma and polyclonal sequences, whereas AL-associated λ light chains had fewer mutations. Machine learning tools designed to predict amyloid propensity were less accurate for new sequences than their original training data. Conclusions: Rarely-observed light chain variable genes may carry a high risk of AL amyloidosis. New approaches are needed to define sequence-associated risk factors for AL amyloidosis. AL-Base is a foundational resource for such studies.

Study of Insulin Aggregation and Fibril Structure under Different Environmental Conditions.

Protein amyloid aggregation is linked with widespread and fatal neurodegenerative disorders as well as several amyloidoses. Insulin, a small polypeptide hormone, is associated with injection-site amyloidosis and is a popular model protein for in vitro studies of amyloid aggregation processes as well as in the search for potential anti-amyloid compounds. Despite hundreds of studies conducted with this specific protein, the procedures used have employed a vast array of different means of achieving fibril formation. These conditions include the use of different solution components, pH values, ionic strengths, and other additives. In turn, this variety of conditions results in the generation of fibrils with different structures, morphologies and stabilities, which severely limits the possibility of cross-study comparisons as well as result interpretations. In this work, we examine the condition-structure relationship of insulin amyloid aggregation under a range of commonly used pH and ionic strength conditions as well as solution components. We demonstrate the correlation between the reaction solution properties and the resulting aggregation kinetic parameters, aggregate secondary structures, morphologies, stabilities and dye-binding modes.

Lyso-phosphatidylcholine as an interfacial stabilizer for parenteral monoclonal antibody formulations.

Therapeutic proteins suffer from physical and chemical instability in aqueous solution. Polysorbates and poloxamers are often added for protection against interfacial stress to prevent protein aggregation and particle formation. Previous studies have revealed that the hydrolysis and oxidation of polysorbates in parenteral formulations can lead to the formation of free fatty acid particles, insufficient long-term stabilization, and protein oxidation. Poloxamers, on the other hand, are considered to be less effective against protein aggregation. Here we investigated two lyso-phosphatidylcholines (LPCs) as potential alternative surfactants for protein formulations, focusing on their physicochemical behavior and their ability to protect against the formation of monoclonal antibody particles during mechanical stress. The hemolytic activity of LPC was tested in varying ratios of plasma and buffer mixtures. LPC effectively stabilized mAb formulations when shaken at concentrations several orders of magnitude below the onset of hemolysis, indicating that the potential for erythrocyte damage by LPC is non-critical. LPC formulations subjected to mechanical stress through peristaltic pumping exhibited comparable protein particle formation to those containing polysorbate 80 or poloxamer 188. Profile analysis tensiometry and dilatational rheology indicated that the stabilizing effect likely arises from the formation of a viscoelastic film at approximately the CMC. Data gathered from concentration-gradient multi-angle light scattering and isothermal titration calorimetry support this finding. Surfactant desorption was evaluated through sub-phase exchange experiments. While LPCs readily desorbed from the interface, resorption occurred rapidly enough in the bulk solution to prevent protein adsorption. Overall, LPCs behave similarly to polysorbate with respect to interfacial stabilization and show promise as a potential substitute for polysorbate in parenteral protein formulations.

Exploring the Neuroprotective Potential of Icariin through Modulation of Neural Pathways in the Treatment of Neurological Diseases.

Neuropathological diseases involve the death of neurons and the aggregation of proteins with altered properties in the brain. Proteins are used at the molecular level to categorize neurodegenerative disorders, emphasizing the importance of protein-processing mechanisms in their development. Natural herbal phytoconstituents, such as icariin, have addressed these neurological complications. Icariin, the principal compound in Epimedium, has been studied for its antineuroinflammatory, anti-oxidative, and antiapoptotic properties. Recent scientific investigations have shown that icariin exhibits promising therapeutic and preventive properties for mental and neurodegenerative disorders. In preclinical, icariin has been shown to inhibit amyloid development and reduce the expression of APP and BACE-1. Previous preclinical studies have demonstrated that icariin can regulate proinflammatory responses in neurological conditions like Parkinson's disease, depression, cerebral ischemia, ALS, and multiple sclerosis. Studies have shown that icariin possesses neuroprotective properties by modulating signaling pathways and crossing the blood-brain barrier, suggesting its potential to address various neurocomplications. This review aims to establish a foundation for future clinical investigations by examining the existing literature on icariin and exploring its potential therapeutic implications in treating neurodegenerative disorders and neuropsychiatric conditions. Future research may address numerous concerns and yield captivating findings with far-reaching implications for various aspects of icariin.

PLK2-mediated phosphorylation of SQSTM1 S349 promotes aggregation of polyubiquitinated proteins upon proteasomal dysfunction.

Dysregulation in protein homeostasis results in accumulation of protein aggregates, which are sequestered into dedicated insoluble compartments so-called inclusion bodies or aggresomes, where they are scavenged through different mechanisms to reduce proteotoxicity. The protein aggregates can be selectively scavenged by macroautophagy/autophagy called aggrephagy, which is mediated by the autophagic receptor SQSTM1. In this study, we have identified PLK2 as an important regulator of SQSTM1-mediated aggregation of polyubiquitinated proteins. PLK2 is upregulated following proteasome inhibition, and then associates with and phosphorylates SQSTM1 at S349. The phosphorylation of SQSTM1 S349 strengthens its binding to KEAP1, which is required for formation of large SQSTM1 aggregates/bodies upon proteasome inhibition. Our findings suggest that PLK2-mediated phosphorylation of SQSTM1 S349 represents a critical regulatory mechanism in SQSTM1-mediated aggregation of polyubiquitinated proteins.

Insight into the effects of ultrasound-assisted intermittent tumbling on the gelation properties of myofibrillar proteins: Conformational modifications, intermolecular interactions, rheological properties and microstructure.

The aim of the present study was to evaluate the effects of ultrasound-assisted intermittent tumbling (UT) at 300 W, 20 kHz and 40 min on the conformation, intermolecular interactions and aggregation of myofibrillar proteins (MPs) and its induced gelation properties at various tumbling times (4 and 6 h). Raman results showed that all tumbling treatments led the helical structure of MPs to unfold. In comparison to the single intermittent tumbling treatment (ST), UT treatment exerted more pronounced effects on strengthening the intermolecular hydrogen bonds and facilitating the formation of an ordered ß-sheet structure. When the tumbling time was the same, UT treatment caused higher surface hydrophobicity, fluorescence intensity and disulfide bond content in the MPs, inducing the occurrence of hydrophobic interaction and disulfide cross-linking between MPs molecules, thus forming the MPs aggregates. Additionally, results from the solubility, particle size, atomic force microscopy and SDS-PAGE further indicated that, relative to the ST treatment, UT treatment was more potent in promoting the polymerization of myosin heavy chain. The MPs aggregates in the UT group were more uniform than those in the ST group. During the gelation process, the pre-formed MPs aggregates in the UT treatment increased the thermal stability of myosin, rendering it more resistant to heat-induced unfolding of the myosin rod region. Furthermore, they improved the protein tail-tail interaction, resulting in the formation of a well-structured gel network with higher gel strength and cooking yield compared to the ST treatment.

Comprehensive evaluation of pathogenic protein accumulation in fibroblasts from all subtypes of Sanfilippo disease patients.

Sanfilippo disease is a lysosomal storage disorder from the group of mucopolysaccharidoses (MPS), characterized by storage of glycosaminoglycans (GAGs); thus, it is also called MPS type III. The syndrome is divided into 4 subtypes (MPS III A, B, C and D). Despite the storage of the same GAG, heparan sulfate (HS), the course of these subtypes can vary considerably. Here, we comprehensively evaluated the levels of protein aggregates (APP, ß-amyloid, p-tau, α-synuclein, TDP43) in fibroblasts derived from patients with all MPS III subtypes, and tested whether lowering GAG levels results in a decrease in the levels of the investigated proteins and the number of aggregates they form. Elevated levels of APP, ß-amyloid, tau, and TDP43 proteins were evident in all MPS III subtypes, and elevated levels of p-tau and α-synuclein were demonstrated in all subtypes except MPS IIIC. These findings were confirmed in the neural tissue of MPS IIIB mice. Fluorescence microscopy studies also indicated a high number of protein aggregates formed by ß-amyloid and tau in all cell lines tested, and a high number of aggregates of p-tau, TDP43, and α-synuclein in all lines except MPS IIIC. Reduction of GAG levels by genistein led to the decrease of levels of all tested proteins and their aggregates except α-synuclein, indicating a relationship between GAG levels and those of some protein aggregates. This work describes for the first time the problem of deposited protein aggregates in all subtypes of Sanfilippo disease and suggests that GAGs are partly responsible for the formation of protein aggregates.

Mutation/metal deficiency in the "electrostatic loop" enhanced aggregation process in apo/holo SOD1 variants: implications for ALS diseases.

Despite the many mechanisms it has created to prevent unfolding and aggregation of proteins, many diseases are caused by abnormal folding of proteins, which are called misfolding diseases. During this process, proteins undergo structural changes and become stable, insoluble beta-sheet aggregates called amyloid fibrils. Mutations/disruptions in metal ion homeostasis in the ALS-associated metalloenzyme superoxide dismutase (SOD1) reduce conformational stability, consistent with the protein aggregation hypothesis for neurodegenerative diseases. However, the exact mechanism of involvement is not well understood. Hence, to understand the role of mutation/ metal deficiency in SOD1 misfolding and aggregation, we investigated the effects of apo/holo SOD1 variants on structural properties using biophysical/experimental techniques. The MD results support the idea that the mutation/metal deficiency can lead to a change in conformation. The increased content of ß-sheet structures in apo/holo SOD1 variants can be attributed to the aggregation tendency, which was confirmed by FTIR spectroscopy and dictionary of secondary structure in proteins (DSSP) results. Thermodynamic studies of GdnHCl showed that metal deficiency/mutation/intramolecular S-S reduction together are required to initiate misfolding/aggregation of SOD1. The results showed that apo/holo SOD1 variants under destabilizing conditions induced amyloid aggregates at physiological pH, which were detected by ThT/ANS fluorescence, as well as further confirmation of amyloid/amorphous species by TEM. This study confirms that mutations in the electrostatic loop of SOD1 lead to structural abnormalities, including changes in hydrophobicity, reduced disulfide bonds, and an increased propensity for protein denaturation. This process facilitates the formation of amyloid/amorphous aggregates ALS-associated.

Ezetimibe Lowers Risk of Alzheimer's and Related Dementias over Sevenfold, Reducing Aggregation in Model Systems by Inhibiting 14-3-3G::Hexokinase Interaction.

Numerous factors predispose to progression of cognitive impairment to Alzheimer's disease and related dementias (ADRD), most notably age, APOE(ε4) alleles, traumatic brain injury, heart disease, hypertension, obesity/diabetes, and Down's syndrome. Protein aggregation is diagnostic for neurodegenerative diseases, and may be causal through promotion of chronic neuroinflammation. We isolated aggregates from postmortem hippocampi of ADRD patients, heart-disease patients, and age-matched controls. Aggregates, characterized by high-resolution proteomics (with or without crosslinking), were significantly elevated in heart-disease and ADRD hippocampi. Hexokinase-1 (HK1) and 14-3-3G/γ proteins, previously implicated in neuronal signaling and neurodegeneration, are especially enriched in ADRD and heart-disease aggregates vs. controls (each P<0.008), and their interaction was implied by extensive crosslinking in both disease groups. Screening the hexokinase-1::14-3-3G interface with FDA-approved drug structures predicted strong affinity for ezetimibe, a benign cholesterol-lowering medication. Diverse cultured human-cell and whole-nematode models of ADRD aggregation showed that this drug potently disrupts HK1::14-3-3G adhesion, reduces disease-associated aggregation, and activates autophagy. Mining clinical databases supports drug reduction of ADRD risk, decreasing it to 0.14 overall (P<0.0001; 95% C.I. 0.06-0.34), and <0.12 in high-risk heart-disease subjects (P<0.006). These results suggest that drug disruption of the 14-3-3G::HK1 interface blocks an early "lynchpin" adhesion, prospectively reducing aggregate accrual and progression of ADRD.

Two novel DnaJ chaperone proteins CG5001 and P58IPK regulate the pathogenicity of Huntington's disease related aggregates.

Huntington's disease (HD) is a rare neurodegenerative disease caused due to aggregation of Huntingtin (HTT) protein. This study involves the cloning of 40 DnaJ chaperones from Drosophila, and overexpressing them in yeasts and fly models of HD. Accordingly, DnaJ chaperones were catalogued as enhancers or suppressors based on their growth phenotypes and aggregation properties. 2 of the chaperones that came up as targets were CG5001 and P58IPK. Protein aggregation and slow growth phenotype was rescued in yeasts, S2 cells, and Drosophila transgenic lines of HTT103Q with these overexpressed chaperones. Since DnaJ chaperones have protein sequence similarity across species, they can be used as possible tools to combat the effects of neurodegenerative diseases.

Microglial Drivers of Alzheimer's Disease Pathology: An Evolution of Diverse Participating States.

Microglia, the resident immune-competent cells of the brain, become dysfunctional in Alzheimer's disease (AD), and their aberrant immune responses contribute to the accumulation of pathological proteins and neuronal injury. Genetic studies implicate microglia in the development of AD, prompting interest in developing immunomodulatory therapies to prevent or ameliorate disease. However, microglia take on diverse functional states in disease, playing both protective and detrimental roles in AD, which largely overlap and may shift over the disease course, complicating the identification of effective therapeutic targets. Extensive evidence gathered using transgenic mouse models supports an active role of microglia in pathology progression, though results vary and can be contradictory between different types of models and the degree of pathology at the time of study. Here, we review microglial immune signaling and responses that contribute to the accumulation and spread of pathological proteins or directly affect neuronal health. We additionally explore the use of induced pluripotent stem cell (iPSC)-derived models to study living human microglia and how they have contributed to our knowledge of AD and may begin to fill in the gaps left by mouse models. Ultimately, mouse and iPSC-derived models have their own limitations, and a comprehensive understanding of microglial dysfunction in AD will only be established by an integrated view across models and an appreciation for their complementary viewpoints and limitations.

Sensitive colorimetric immunosensor using AuNP-functionalized polymer film for picogram-level detection of Tau protein intermediate aggregates.

Here we demonstrate for the first time that an antibody-gold nanoparticles (AuNPs)-polymer conjugate thin-film biosensor can easily be fabricated to selectively capture Tau protein. Gold nanoparticles (AuNPs) are employed as sensing elements, thus capitalizing on their propensity to undergo assembly or disassembly in response to the adsorption or conjugation of various biomolecules on their surface, thereby forming robust interactions with the target analyte. We show that the Tau protein in its different aggregation phases can be detected, by restricting the reaction area on the solid thin polymer film and thus reducing the diffusion effects usually encountered in immunosensors. A limit of detection (LOD) of 460 pg/mL was reached, demonstrating a great potential for detecting Tau in aggregation states. This sensor based on thin polymer film could open new routes for sensing and monitoring Tau protein in biological assays and biomedical diagnosis.

Utilizing A hydrophobic primary container surface to reduce the formation of subvisible particles in monoclonal antibody solution caused by fluid shear.

The exposure of protein molecules to interfaces may cause protein aggregation and particle formation in protein formulations, especially hydrophobic interfaces, which may promote protein aggregation in solution. In this study, we found that modification of the surface properties by application of a hydrophobic Octadecyltrichlorosilane (OTS) could reduce the generation of protein aggregates and particles in protein solution induced by fluid shear. A stable protein adsorption layer was formed at the hydrophobic interface through the strong hydrophobic interaction between the protein and hydrophobic surface, which could prevent the aggregated protein from falling off into the bulk solution to form subvisible particles and insoluble protein aggregates. In addition, human complement enzyme linked immunosorbent assay results showed that the particles that were generated in the OTS-coated container did not activate human complement which indicated the OTS-coated container could be used as primary containers for certain types of monoclonal antibody formulation.

Catchers of folding gone awry: a tale of small heat shock proteins.

Small heat shock proteins (sHsps) are an important part of the cellular system maintaining protein homeostasis under physiological and stress conditions. As molecular chaperones, they form complexes with different non-native proteins in an ATP-independent manner. Many sHsps populate ensembles of energetically similar but different-sized oligomers. Regulation of chaperone activity occurs by changing the equilibrium of these ensembles. This makes sHsps a versatile and adaptive system for trapping non-native proteins in complexes, allowing recycling with the help of ATP-dependent chaperones. In this review, we discuss progress in our understanding of the structural principles of sHsp oligomers and their functional principles, as well as their roles in aging and eye lens transparency.

Unravelling aggregation propensity of rotavirus A VP6 expressed as E. coli inclusion bodies through in silico prediction.

The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.

Acute stress and multicellular development alter the solubility of the Dictyostelium Sup35 ortholog ERF3.

Among sequenced organisms, the genome of Dictyostelium discoideum is unique in that it encodes for a massive amount of repeat-rich sequences in the coding region of genes. This results in the Dictyostelium proteome encoding for thousands of repeat-rich proteins, with nearly 24% of the Dictyostelium proteome encoding Q/N-rich regions that are predicted to be prion like in nature. To begin investigating the role of prion-like proteins in Dictyostelium, we decided to investigate ERF3, the Dictyostelium ortholog of the well-characterized yeast prion protein Sup35. ERF3 lacks the Q/N-rich region required for prion formation in yeast, raising the question of whether this protein aggregates and has prion-like properties in Dictyostelium. Here, we found that ERF3 formed aggregates in response to acute cellular stress. However, unlike bona fide prions, we were unable to detect transmission of aggregates to progeny. We further found that aggregation of this protein is driven by the ordered C-terminal domain independently of the disordered N-terminal domain. Finally, we also observed aggregation of ERF3 under conditions that induce multicellular development, suggesting that this phenomenon may play a role in Dictyostelium development. Together, these findings suggest a role for regulated protein aggregation in Dictyostelium cells under stress and during development.IMPORTANCEPrion-like proteins have both beneficial and deleterious effects on cellular health, and many organisms have evolved distinct mechanisms to regulate the behaviors of these proteins. The social amoeba Dictyostelium discoideum contains the highest proportion of proteins predicted to be prion like and has mechanisms to suppress their aggregation. However, the potential roles and regulation of these proteins remain largely unknown. Here, we demonstrate that aggregation of the Dictyostelium translation termination factor ERF3 is induced by both acute cellular stress and by multicellular development. These findings imply that protein aggregation may have a regulated and functional role in the Dictyostelium stress response and during multicellular development.

ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes.

The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.