Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.
Fluoride ion channels of the Fluc family selectively export F− ions to rescue unicellular organisms from acute F− toxicity. Crystal structures of bacterial Fluc channels in complex with synthetic monobodies, fibronectin-derived soluble β-sandwich fold proteins, show 2-fold symmetric homodimers with an antiparallel transmembrane topology. Monobodies also block Fluc F− current via a pore blocking mechanism. However, little is known about the energetic contributions of individual monobody residues to the affinity of the monobody—channel complex or whether the structural paratope corresponds to functional reality. This study seeks to structurally identify and compare residues interacting with Fluc between two highly similar monobodies and subjects them to mutagenesis and functional measurements of equilibrium affinities via a fluorescence anisotropy binding assay to determine their energetic contributions. The results indicate that the functional and structural paratopes strongly agree and that many Tyr residues at the interface, while playing a key role in affinity, can be substituted with Phe and Trp without large disruptions.
The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.
Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Despite multimodal therapy, resistant GBM stem-like cells (GSCs) inevitably mediate disease recurrence. To identify novel vulnerabilities of GSCs, we performed an arrayed CRISPR/Cas9 screen against select adhesion G protein-coupled receptors (aGPCRs), many of which we found to be de novo expressed in GBM. Knockout of CD97 (ADGRE5), previously implicated in GBM cell migration, produced the most striking proliferative disadvantage in patient-derived GBM cultures (PDGC) among aGPCRs tested. We found high CD97 surface expression in all our PDGCs, while levels remained nearly undetectable in non-neoplastic brain cells, confirming that CD97 is de novo expressed in GBM. Upon shRNA-mediated knockdown of CD97 in PDGCs from all three TCGA transcriptional subtypes, we observed significantly reduced proliferation, as measured by Ki67 and Hoechst cell cycle analysis, and significantly diminished surface expression of CD133, a GSC marker. Notably, CD97 knockdown also significantly reduced tumorsphere initiation capacity in six PDGCs, as measured by extreme limiting dilution assays. These findings suggest that CD97 regulates GSC self-renewal in vitro. RNA-sequencing and GSEA pathway analysis from PDGCs following CD97 knockdown indicate an enrichment of aerobic respiratory gene sets, suggesting one of the major regulatory roles of CD97 is metabolic regulation. Indeed, metabolic assays show that CD97 knockdown alters oxygen consumption and glycolysis rates in PDGCs. Lastly, we have developed human synthetic antibodies to target CD97 in order to investigate its therapeutic potential. We have observed internalization of some of these antibodies, thus identifying candidates for the development of antibody-drug conjugates. In addition, other clones reduced GBM cell proliferation and elicited expression of various differentiation markers. Overall; our studies identify novel roles of CD97 in regulating the cellular hierarchy in GBM and tumor cell metabolism, and provide a strong scientific rationale for developing biologics to target CD97 in GBM.
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