hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.
Liquid-liquid phase separation of proteins and nucleic acids into membraneless organelles (MLOs) spatially organizes cellular components and reactions. The RNA-binding protein heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) carries mRNA targets in MLOs called transport granules in neurons and oligodendrocytes. At sites of local translation, hnRNPA2 is phosphorylated by the tyrosine protein kinase Fyn, releasing the mRNA for translation. Fyn recognizes targets through its SH3 domain (Fyn-SH3). However, hnRNPA2 lacks canonical SH3binding sequences, raising the question of how Fyn-SH3 binds hnRNPA2 in phase-separated transport granules. Here, we characterize the structural details of the interaction of the hnRNPA2 low-complexity domain (LC) with Fyn-SH3 and the effect of Fyn-SH3 on hnRNPA2 phase separation. We combined in vitro microscopy and solution NMR spectroscopy to evaluate assembly of hnRNPA2 and Fyn-SH3 into in vitro phase-separated granules and probe the structural details of their interaction. We observed that Fyn-SH3 induces hnRNPA2 LC phase separation and that Fyn-SH3 is incorporated into in vitro hnRNPA2 LC granules. Moreover, we identified hnRNPA2 LC interaction sites on the surface of Fyn-SH3. Our data offer a structural view of how hnRNPA2 LC may interact with Fyn. To our knowledge, our study provides the first example of a single globular domain inducing phase separation of a disordered MLO scaffold protein.
Objective To determine the clinical and molecular features in patients with amyotrophic lateral sclerosis 4 (ALS4) due to mutations in the senataxin (SETX) gene and to develop tools for evaluating SETX variants. Methods Our study involved 32 patients, including 31 with mutation in SETX at c.1166 T>C (p.Leu389Ser) and 1 with mutation at c.1153 G>A (p.Glu385Lys). Clinical characterization of the patients included neurological examination, blood tests, magnetic resonance imaging (MRI), and dual‐energy x‐ray absorptiometry (DEXA). Fibroblasts and motor neurons were obtained to model the disease and characterize the molecular alteration in senataxin function. Results We report key clinical features of ALS4. Laboratory analysis showed alteration of serum creatine kinase and creatinine in the Leu389Ser ALS4 cohort. MRI showed increased muscle fat fraction in the lower extremities, which correlates with disease duration (thigh fat fraction R2 = 0.35, p = 0.01; lower leg fat fraction R2 = 0.49, p < 0.01). DEXA measurements showed lower extremities are more affected than upper extremities (average fat z scores of 2.1 and 0.6, respectively). A cellular assay for SETX function confirmed that like the Leu389Ser mutation, the Glu385Lys variant leads to a decrease in R loops, likely from a gain of function. Interpretation We identified clinical laboratory and radiological features of ALS4, and hence they should be monitored for disease progression. The molecular characterization of R‐loop levels in patient‐derived cells provides insight into the disease pathology and assays to evaluate the pathogenicity of candidate mutations in the SETX gene. ANN NEUROL 2020;87:547–555
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that has significant overlap with frontotemporal dementia (FTD). Mutations in specific genes have been identified that can cause and/or predispose patients to ALS. However, the clinical variability seen in ALS patients suggests that additional genes impact pathology, susceptibility, severity, and/or progression of the disease. To identify molecular pathways involved in ALS, we undertook a meta-analysis of published genetic modifiers both in patients and in model organisms, and undertook bioinformatic pathway analysis. From 72 published studies, we generated a list of 946 genes whose perturbation (1) impacted ALS in patient populations, (2) altered defects in laboratory models, or (3) modified defects caused by ALS gene ortholog loss of function. Herein, these are all called modifier genes. We found 727 modifier genes that encode proteins with human orthologs. Of these, 43 modifier genes were identified as modifiers of more than one ALS gene/model, consistent with the hypothesis that shared genes and pathways may underlie ALS. Further, we used a gene ontology-based bioinformatic analysis to identify pathways and associated genes that may be important in ALS. To our knowledge this is the first comprehensive survey of ALS modifier genes. This work suggests that shared molecular mechanisms may underlie pathology caused by different ALS disease genes. Surprisingly, few ALS modifier genes have been tested in more than one disease model. Understanding genes that modify ALS-associated defects will help to elucidate the molecular pathways that underlie ALS and provide additional targets for therapeutic intervention.
hnRNPA2 is a major component of mRNA transport granules in oligodendrocytes and neurons. However, the structural details of how hnRNPA2 binds the A2 recognition element (A2RE) and if this sequence stimulates granule formation by enhancing phase separation of hnRNPA2 has not yet been studied. Using solution NMR and biophysical studies, we find that each of the two individual RRMs retain the domain structure observed in complex with RNA but are not rigidly confined (i.e. they move independently) in solution in the absence of RNA. hnRNPA2 RRMs bind the minimal rA2RE11 weakly but at least, and most likely, two hnRNPA2 molecules are able to simultaneously bind the longer 21mer myelin basic protein A2RE. Upon binding of the RNA, NMR chemical shift deviations are observed in both RRMs, suggesting both play a role in binding the A2RE11. Interestingly, addition of short A2RE RNAs or longer RNAs containing this sequence completely prevents in vitro phase separation of full-length hnRNPA2 and aggregation of the disease-associated mutants. These findings suggest that RRM interactions with specific recognition sequences alone do not account for nucleating granule formation, consistent with models where multivalent protein:RNA and protein:protein contacts form across many sites in granule proteins and long RNA transcripts.
Background. Iatrogenic Cushing’s syndrome is commonly seen as a complication of chronic steroid use. While most often associated with the use of prescription oral steroids, rare cases result from unintentional steroid exposure. In particular, numerous complementary and alternative medicines have been found to contain steroids not previously known to users. Case Presentation. Here, we present a case of iatrogenic Cushing’s syndrome caused by prolonged ingestion of dexamethasone found within an over-the-counter arthritis supplement called Artri King. Conclusion. A thorough history of medication use to include over-the-counter medications and supplements may be required to identify the source of exogenous glucocorticoids in iatrogenic Cushing’s syndrome.
Background Evolutionary psychologists have demonstrated that humans are attracted to individuals who possess average anatomy for the population. Objectives It is the aim of this study to prove that a composite of average facial features would be more attractive to raters than the cohort utilized to create the composite. Methods Our male and female cohorts each consisted of 41 standardized frontal-view monochrome photos, with one composite image derived from the other forty real images. We utilized Amazon Mechanical Turk, a widely used crowdsourcing platform, to receive ratings of images ranging from 1-7, with 1 and 7 being least and most attractive, respectively. The strength of the preference for the composite over the real images was assessed by the difference between the mean rating of the composite and real images. Results We received 870 respondents and 876 respondents for the male and female cohorts respectively. For the male and female cohorts, the composite image was rated significantly higher than the rest of the cohort overall and across all ages, genders, and countries of residence (all p<0.0001). For both cohorts, the strength of the preference was significantly higher for European voters and lower for South American and non-binary raters (all p<0.05). Conclusions Our study reveals that average facial anatomy is perceived as most attractive across all demographics, which we hope will serve as a steppingstone for further studies that will lead to objective cosmetic quantifications and integrating evidence-based medicine into aesthetic surgery.
Background Triple-negative (TN) and luminal A breast cancer molecular subtypes have divergent clinical and prognostic characteristics for breast cancer patients. Our study aims to compare the reconstructive choice of these two groups from the time they receive a tissue expander (TE) to the time they complete autologous or implant-based breast reconstruction. Methods A total of 255 patients who underwent delayed-immediate breast reconstruction with TE placement from 2013 to 2017 diagnosed with either TN (n = 73) or luminal A (n = 182) invasive breast cancer subtype seen by two surgeons at a single institution were identified. Preference of autologous and implant-based reconstruction was analyzed, along with TE complications, race, age, body mass index (BMI), smoking, adjuvant therapy, and comorbidities. Results There was a significant difference in the choice of implant- or autologous-based reconstruction among these two groups (p < 0.05). A greater proportion of luminal A patients underwent implant-based reconstruction (63.47%) and a greater proportion of TN patients underwent autologous-based reconstruction (53.13%). With regard to TE outcomes, there was no significant difference between the two groups with regard to duration of TE placement by reconstructive type or TE surgical complications. Significantly, more TN patients underwent radiation therapy (p < 0.01) and neoadjuvant chemotherapy (p < 0.0001) than luminal A patients. BMI, comorbidities, radiation therapy, and overall TE complications were identified as predictive factors of patients electing for autologous reconstruction over implants. Conclusion TN breast cancer patients mostly chose autologous-based reconstruction, while luminal A patients chose implant-based reconstruction. Both patient groups carried their TEs for similar duration with similar complication profile. Radiation therapy is likely a major factor in the decision for the type of delayed-immediate reconstruction among this population.
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