Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene, SMN2 that produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by two-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-quinazoline probe, we identified the scavenger decapping enzyme, DcpS as a potential binder. We show that the C5-quinazolines potently inhibit DcpS decapping activity, and that the potency of inhibition correlates with potency for SMN2 promoter induction. Binding of C5-quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m7GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.
The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.
The papillomavirus E5 proteins are short, hydrophobic transforming proteins. The transmembrane E5 protein encoded by bovine papillomavirus transforms cells by activating the platelet-derived growth factor b receptor tyrosine kinase in a ligand-independent fashion. The bovine papillomavirus E5 protein forms a stable complex with the receptor, thereby inducing receptor dimerization and activation, trans-phosphorylation, and recruitment of cellular signaling proteins to the receptor. The E5 proteins of the human papillomaviruses also appear to a ect the activity of growth factor receptors and their signaling pathways. The interaction of papillomavirus E5 proteins with a subunit of the vacuolar ATPase may also contribute to transformation. Further analysis of these unique mechanisms of viral transformation will yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways. Oncogene (2001) 20, 7866 ± 7873.
Quantitative epidermal growth factor (EGF)-binding experiments have shown that the EGF-receptor (EGFR)is displayed on the surface of intact cells in two forms, a minority of high-affinity and a majority of low-affinity EGFRs. On the basis of the threedimensional structure of the extracellular ligand binding domain of the EGFR, it was proposed that the intramolecularly tethered and autoinhibited configuration corresponds to the low-affinity receptor, whereas the extended configuration accounts for the highaffinity EGFRs on intact cells. Here we test this model by analyzing the properties of EGFRs mutated in the specific regions responsible for receptor autoinhibition and dimerization, respectively. Our results show that mutagenic disruption of the autoinhibitory tether in EGFR results in a decrease in the dissociation rate of EGF without a detectable change in EGFR activation and signaling through EGFR even in response to stimulation with low concentrations of EGF. Mutagenic disruption of the dimerization arm, on the other hand, increased the rate of EGF dissociation and impaired EGFR activation and signaling via the EGFR. This study demonstrates that the extended configuration of EGFR does not account for the apparent high-affinity EGF-binding to EGFR on intact cells. Furthermore, the autoinhibition conferred by the tethered configuration of the extracellular ligand-binding domain provides only a limited control of EGFR function.
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