Dystrophin missense mutations alter focal adhesion tension and mechanotransduction.

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin-glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-Tags.

HUH-tags have emerged as versatile fusion partners that mediate sequence specific protein-ssDNA bioconjugation through a simple and efficient reaction. Here we present HUHgle, a python-based interactive tool for the visualization, design, and optimization of substrates for HUH-tag mediated covalent labeling of proteins of interest with ssDNA substrates of interest. HUHgle streamlines design processes by integrating an intuitive plotting interface with a search function capable of predicting and displaying protein-ssDNA bioconjugate formation efficiency and specificity in proposed HUH-tag/ssDNA sequence combinations. Validation demonstrates that HUHgle accurately predicts product formation of HUH-tag mediated bioconjugation for single- and orthogonal-labeling reactions. In order to maximize the accessibility and utility of HUHgle, we have implemented it as a user-friendly Google Colab notebook which facilitates broad use of this tool, regardless of coding expertise.

Molecular basis of proteolytic cleavage regulation by the extracellular matrix receptor dystroglycan.

The dystrophin-glycoprotein-complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix and actin cytoskeleton. Breaking this connection is associated with diseases such as muscular dystrophy, yet cleavage of dystroglycan by matrix-metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We determined the crystal structure of the membrane-adjacent domain (amino acids 491-722) of E. coli expressed human dystroglycan to understand MMP cleavage regulation. The structural model includes tandem immunoglobulin-like (IGL) and sperm/enterokinase/agrin-like (SEAL) domains, which support proteolysis in diverse receptors to facilitate mechanotransduction, membrane protection, and viral entry. The structure reveals a C-terminal extension that buries the MMP site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate structure-guided and disease-associated mutations disrupt proteolytic regulation using a cell-surface proteolysis assay. Thus disrupted proteolysis is a potentially relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis.

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the ß-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the ß-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.