Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
Extensive literature is available on how focal adhesions sense and respond to environmental cues during fast migration in polarized cells. In contrast, little is known about how cells feel and translate environmental cues during slow mesenchymal migration through tissues (e.g. dendritic cells, macrophages). These cells form many more podosomes than focal adhesions and use them as mechanosensors to probe and remodel the extracellular matrix. Each podosome consists of a dense, protrusive actin core and an adhesive ring of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Podosomes are highly dynamic, and their actin content continuously fluctuates mediating their protruding activity. Podosomes are organized in large clusters. Electron microscopy showed that podosomes are interconnected by cytoskeletal fibers, suggesting the existence of a mesoscale organization. By combining image correlation spectroscopy (ICS) techniques such as raster ICS (RICS) and timeresolved spatiotemporal ICS (trSTICS), we investigated the collective dynamic behavior of podosome clusters. We compared the dynamic behavior of the mechanosensitive protein vinculin and the mechano-insensitive protein talin. RICS showed similar diffusion (D=2.4 and 2.0mm 2 /s) and binding (t=0.024 and 0.026s) properties for vinculin and talin. In contrast trSTICS revealed significant differences in their flow patterns. While waves of correlated flow, with speeds ranging from 0.01-0.16 mm/min, are visible throughout the podosome cluster for vinculin, talin dynamics show no clear directionality. Moreover, podosome formation and dissolution are accompanied by characteristic flow patterns of vinculin, suggesting that localized recruitment of mechanosensitive proteins could coordinate podosome protrusive forces. Finally, using cross correlation STICS we show that vinculin, but not talin, dynamics correlate to flow patterns in the actin network. Taken together, our data demonstrate coordinated behavior of mechanosensitive adhesion proteins in podosome clusters providing evidence for mesoscale coordinated protrusive dynamics.
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