Predictable fold switching by the SARS‐CoV‐2 protein ORF9b

Extant fold‐switching proteins remodel their secondary structures and change their functions in response to environmental stimuli. These shapeshifting proteins regulate biological processes and are associated with a number of diseases, including tuberculosis, cancer, Alzheimer''s, and autoimmune disorders. Thus, predictive methods are needed to identify more fold‐switching proteins, especially since all naturally occurring instances have been discovered by chance. In response to this need, two high‐throughput predictive methods have recently been developed. Here we test them on ORF9b, a newly discovered fold switcher and potential therapeutic target from the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Promisingly, both methods correctly indicate that ORF9b switches folds. We then tested the same two methods on ORF9b1, the ORF9b homolog from SARS‐CoV‐1. Again, both methods predict that ORF9b1 switches folds, a finding consistent with experimental binding studies. Together, these results (a) demonstrate that protein fold switching can be predicted using high‐throughput computational approaches and (b) suggest that fold switching might be a general characteristic of ORF9b homologs.     

Dorsal Anterior Cingulate Thickness Is Related to Alexithymia in Childhood Trauma-Related PTSD

Alexithymia, or “no words for feelings”, is highly prevalent in samples with childhood maltreatment and posttraumatic stress disorder (PTSD). The dorsal anterior cingulate cortex (dACC) has been identified as a key region involved in alexithymia, early life trauma, and PTSD. Functional alterations in the dACC also have been associated with alexithymia in PTSD. This study examined whether dACC morphology is a neural correlate of alexithymia in child maltreatment-related PTSD. Sixteen adults with PTSD and a history of childhood sexual abuse, physical abuse, or exposure to domestic violence, and 24 healthy controls (HC) completed the Toronto Alexithymia Scale 20 (TAS–20) and underwent magnetic resonance imaging. Cortical thickness of the dACC was measured using FreeSurfer, and values were correlated with TAS–20 scores, controlling for sex and age, in both groups. Average TAS–20 score was significantly higher in the PTSD than the HC group. TAS–20 scores were significantly positively associated with dACC thickness only in the PTSD group. This association was strongest in the left hemisphere and for TAS–20 subscales that assess difficulty identifying and describing feelings. We found that increasing dACC gray matter thickness is a neural correlate of greater alexithymia in the context of PTSD with childhood maltreatment. While findings are correlational, they motivate further inquiry into the relationships between childhood adversity, emotional awareness and expression, and dACC morphologic development in trauma-related psychopathology.     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

Molecular Characterization of Melanoma Cases in Denmark Suspected of Genetic Predisposition

Both environmental and host factors influence risk of cutaneous melanoma (CM), and worldwide, the incidence varies depending on constitutional determinants of skin type and pigmentation, latitude, and patterns of sun exposure. We performed genetic analysis of CDKN2A, CDK4, BAP1, MC1R, and MITFp.E318K in Danish high-risk melanoma cases and found CDKN2A germline mutations in 11.3% of CM families with three or more affected individuals, including four previously undescribed mutations. Rare mutations were also seen in CDK4 and BAP1, while MC1R variants were common, occurring at more than twice the frequency compared to Danish controls. The MITF p.E318K variant similarly occurred at an approximately three-fold higher frequency in melanoma cases than controls. To conclude, we propose that mutation screening of CDKN2A and CDK4 in Denmark should predominantly be performed in families with at least 3 cases of CM. In addition, we recommend that testing of BAP1 should not be conducted routinely in CM families but should be reserved for families with CM and uveal melanoma, or mesothelioma.     

Consequences of Seed Origin and Biological Invasion for Early Establishment in Restoration of a North American Grass Species

Local, wild-collected seeds of native plants are recommended for use in ecological restoration to maintain patterns of adaptive variation. However, some environments are so drastically altered by exotic, invasive weeds that original environmental conditions may no longer exist. Under these circumstances, cultivated varieties selected for improved germination and vigor may have a competitive advantage at highly disturbed sites. This study investigated differences in early establishment and seedling performance between wild and cultivated seed sources of the native grass, Poa secunda, both with and without competition from the invasive exotic grass, Bromus tectorum. We measured seedling survival and above-ground biomass at two experimental sites in western Montana, and found that the source of seeds selected for restoration can influence establishment at the restoration site. Cultivars had an overall advantage when compared with local genotypes, supporting evidence of greater vigor among cultivated varieties of native species. This advantage, however, declined rapidly in the presence of B. tectorum and most accessions were not significantly different for growth and survival in competition plots. Only one cultivar had a consistent advantage despite a strong decline in its performance when competing with invasive plants. As a result, cultivated varieties did not meet expectations for greater establishment and persistence relative to local genotypes in the presence of invasive, exotic species. We recommend the use of representative local or regional wild seed sources in restoration to minimize commercial selection, and a mix of individual accessions (wild, or cultivated when necessary) in highly invaded settings to capture vigorous genotypes and increase the odds native plants will establish at restoration sites.     

A rapid and affordable screening platform for membrane protein trafficking

Background

Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed.

Results

This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane.

Conclusion

The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.

     

Growth of Chitinophaga pinensis on Plant Cell Wall Glycans and Characterisation of a Glycoside Hydrolase Family 27 β-l-Arabinopyranosidase Implicated in Arabinogalactan Utilisation

The genome of the soil bacterium Chitinophaga pinensis encodes a diverse array of carbohydrate active enzymes, including nearly 200 representatives from over 50 glycoside hydrolase (GH) families, the enzymology of which is essentially unexplored. In light of this genetic potential, we reveal that C. pinensis has a broader saprophytic capacity to thrive on plant cell wall polysaccharides than previously reported, and specifically that secretion of β-l-arabinopyranosidase activity is induced during growth on arabinogalactan. We subsequently correlated this activity with the product of the Cpin_5740 gene, which encodes the sole member of glycoside hydrolase family 27 (GH27) in C. pinensis, CpArap27. Historically, GH27 is most commonly associated with α-d-galactopyranosidase and α-d-N-acetylgalactosaminidase activity. A new phylogenetic analysis of GH27 highlighted the likely importance of several conserved secondary structural features in determining substrate specificity and provides a predictive framework for identifying enzymes with the less common β-l-arabinopyranosidase activity.