Biology subjects

Hayward, A. N.

Publications and source records attributed to Hayward, A. N..

3 recordsLinked to original sources

bioRxiv2022

Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan

The dystrophin glycoprotein complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix to the actin cytoskeleton to drive critical aspects of development and adult homeostasis. Breaking this connection via mutation of the actin adaptor protein dystrophin or impaired glycosylation of dystroglycan are strongly associated with diseases such as Muscular Dystrophy, yet cleavage of the dystroglycan protein by matrix metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We solved X-ray structures of the membrane-adjacent domain of dystroglycan to understand the molecular underpinnings of dystroglycan MMP cleavage regulation. Dystroglycan proteolysis occurs within the versatile SEAL domain, which supports proteolysis in diverse receptors to facilitate mechanotransduction, protection of cell membranes, and even viral entry. The structure reveals a c-terminal extension of the SEAL domain that buries the MMP cleavage site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate that structure-guided and disease-associated mutations disrupt proteolytic regulation using a new cell-surface proteolysis assay. Finally, we find that disruption of proteolysis leads to altered cellular mechanics and migration using high-throughput DNA tension probe and wound healing assays. These findings highlight that disrupted proteolysis is a relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis and may offer new therapeutic avenues for dystroglycanopathies.

biochemistry↗

bioRxiv2018

Harnessing Notch signaling to decode mechanisms of proteolytic regulation in diverse cell-surface receptors

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in many diseases, yet decoding proteolytic regulation mechanisms of the estimated 400 receptors shed from the cell surface has been hindered by difficulties in controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where decades of study have revealed that intercellular forces drive exposure of a cryptic protease site within a juxtamembrane \"proteolytic switch\" domain to activate transcriptional programs inside the cell. Thus, we created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. Here, we identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate that SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish that the assay can be used to measure modulation of proteolysis by potential therapeutics.

systems biology↗

bioRxiv2018

Conformational regulation of proteolysis in the extracellular matrix receptor dystroglycan

The adhesion receptor dystroglycan provides a critical mechanical link between the extracellular matrix (ECM) and the actin cytoskeleton to help muscle cells withstand contraction and neural cells maintain the blood brain barrier. Disrupting the link is associated with diseases such as cancer and muscular dystrophy. Proteolysis of dystroglycan by Matrix Metalloproteases (MMPs) also breaks the mechanical anchor and is amplified in several pathogenic states. We use a combination of biochemical and cell-based assays to show that dystroglycan proteolysis is conformationally regulated by an extracellular, juxtamembrane \"proteolysis domain\", comprised of tandem Ig-like and SEA-like domains. The intact proteolysis domain is resistant to MMP cleavage, but structurally-disruptive muscular dystrophy-related mutations sensitize dystroglycan to proteolysis. Moreover, increased dystroglycan proteolysis correlates with faster cell migration, linking proteolysis to a disease-relevant cellular phenotype. Intriguingly, previously uncharacterized cancer-associated mutations that map to the proteolysis domain similarly lead to increases in proteolysis and rates of cell migration, potentially revealing a new pathogenic mechanism in cancer.

cell biology↗