Subcellular mislocalization and aggregation of the human FUS protein occurs in neurons of patients with subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS is one of several ...RNA-binding proteins that can functionally self-associate into distinct liquid-phase droplet structures. It is postulated that aberrant interactions within the dense phase-separated state can potentiate FUS's transition into solid prion-like aggregates that cause disease. FUS is post-translationally modified at numerous positions, which affect both its localization and aggregation propensity. These modifications may influence FUS-linked pathology and serve as therapeutic targets.
Abstract Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease caused by loss of upper and lower motor neurons. The majority of ALS cases are classified as sporadic (80–90%), with the ...remaining considered familial based on patient history. The last decade has seen a surge in the identification of ALS-causing genes – including TARDBP (TDP-43), FUS , MATR3 (Matrin-3), C9ORF72 and several others – providing important insights into the molecular pathways involved in pathogenesis. Most of the protein products of ALS-linked genes fall into two functional categories: RNA-binding/homeostasis and protein-quality control ( i.e. autophagy and proteasome). The RNA-binding proteins tend to be aggregation-prone with low-complexity domains similar to the prion-forming domains of yeast. Many also incorporate into stress granules (SGs), which are cytoplasmic ribonucleoprotein complexes that form in response to cellular stress. Mutant forms of TDP-43 and FUS perturb SG dynamics, lengthening their cytoplasmic persistence. Recent evidence suggests that SGs are regulated by the autophagy pathway, suggesting a unifying connection between many of the ALS-linked genes. Persistent SGs may give rise to intractable aggregates that disrupt neuronal homeostasis, thus failure to clear SGs by autophagic processes may promote ALS pathogenesis.
FUS (fused in sarcoma) is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form ...distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prionlike domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prionlike domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prionlike domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser-26 and Ser-30). Both sites were usually phosphorylated in a subpopulation of cellular FUS following a variety of DNA-damaging stresses but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multiphosphorylation of FUS's prionlike domain does not cause cytoplasmic localization.
Many proteins involved in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) are remarkably similar to proteins that form prions in the yeast
. These ALS-associated proteins are not ...orthologs of yeast prion proteins, but are similar in having long, intrinsically disordered domains that are rich in hydrophilic amino acids. These so-called prion-like domains are particularly aggregation-prone and are hypothesized to participate in the mislocalization and misfolding processes that occur in the motor neurons of ALS patients. Methods developed for characterizing yeast prions have been adapted to studying ALS-linked proteins containing prion-like domains. These yeast models have yielded major discoveries, including identification of new ALS genetic risk factors, new ALS-causing gene mutations and insights into how disease mutations enhance protein aggregation.
Objective: To review the literature and place into a quantified context the relationship of Parkinson’s disease diagnosis to a subsequent diagnosis of malignant melanoma, and to briefly explore ...potential molecular associations between the two diseases.
Methods: The Medline database was queried with terms related to Parkinson’s disease (PD) and malignant melanoma, with use of Boolean operator AND to identify studies involving both diseases. Studies were divided into primary and meta-analyses, with exclusive evaluation of those quantifying risk of malignant melanoma after an established diagnosis of Parkinson’s disease. Critical studies were identified using Medline searches to identify established quantified risk metrics between classic melanoma risk factors and subsequent development of malignant melanoma.
Results: Twelve primary studies and three meta-analyses were evaluated and their risk metrices tabulated. Three studies offered estimated risk of development of malignant melanoma in patients with classic melanoma risk factors. These metrices were also tabulated and compared with the metrices established by the twelve primary studies. This demonstrated a similarity in overall risk of developing malignant melanoma in a patient with a diagnosis of Parkinson’s disease as compared to a patient with classical melanoma risk factors.
Limitations: Relatively few studies identified specifically quantified the classic risk factors for melanoma, and relatively few studies specifically quantified the degree of risk for developing melanoma after an established Parkinson’s disease diagnosis.
Conclusion: It is wise to consider the presence of Parkinson’s disease in a patient as one factor when clinicians decide on the appropriateness of regular full body screening examinations.
Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast ...prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.
Synopsis
Self‐association of the ALS and FTD neurodegenerative disease‐linked RNA‐binding protein FUS is regulated by phosphorylation of its low complexity domain.
FUS low complexity (LC) domain can be phosphorylated at several sites: by DNA‐PK at 12 sites in vitro, and in human cells after DNA damage at additional sites.
FUS LC phosphorylation and phosphomimetic substitution discourages phase separation and aggregation.
FUS LC phosphomimetic substitution decreases FUS aggregation in yeast and human cell models.
FUS LC phosphomimetic substitution reduces FUS toxicity in the yeast model.
Post‐translational modification emerges as a cellular mechanism for controlling physiological assembly of the ALS and FTD neurodegenerative disease‐linked RNA‐binding protein FUS.
Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other ...RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.
Neuronal inclusions of aggregated RNA-binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation-prone, yeast ...prion-like, low sequence-complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in-cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation-prone/prion-like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self-interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity. Hence, post-translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.
The Cryptosporidium antigen CpMuc4 expressed in Toxoplasma tachyzoites (A) binds to get epithelial cells in a similar pattern to native CpMuc4 antigen (B).
•CpMuc4 is a C. parvum antigen involved in ...parasite host cell invasion.•Native CpMuc4 antigen was isolated and characterized.•CpMuc4 expressed in T. gondii (rTgCpMuc4) is similar to native antigen.•Binding of rTgCpMuc4 to host cells parallels binding of native antigen.•Post-translational modifications are required for CpMuc4 binding to host cells.
Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a Cryptosporidium parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in Escherichia coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in Toxoplasma gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.
Although the NASP 2020 Standards emphasize consultation as a core competency, few studies have examined consultation preservice training on consultant outcomes. This article describes the content and ...implementation of a training program focused on ecological-positive behavior consultation, called Positive Environment and Behavior for Building Learning and Engagement (PEBBLE). PEBBLE incorporates (a) didactic instruction related to consultation and ecological-positive support strategies, (b) evidence-based strategy and consultation implementation resources, and (c) supervised in-vivo practice. Outcome measures assessed both trainee outcomes, including self-reported efficacy, knowledge, and confidence, and classroom outcomes (direct observations). Results show PEBBLE led to statistically significant improvements in trainees' self-reported efficacy, knowledge, and confidence and had a positive impact on classroom environments. Additionally, students who participated in PEBBLE in their first year of school psychology training had self-efficacy and competency ratings comparable to more advanced students. Implications for consultative training based on ecological-positive support approaches and future research are addressed.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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