Deubiquitinase-targeting chimeras for targeted protein stabilization.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

Regeneration of airway epithelial cells to study rare cell states in cystic fibrosis.

Pathological remodeling of the airway epithelium is commonly observed in cystic fibrosis (CF). Thus, tissue repair is critical to restore integrity and maintenance of the epithelial barrier function. Epithelial repair is a multi-step process initiated by progenitor cell migration into the injured area, proliferation, and re-differentiation into all of the cell types that contribute to the function of a normal airway epithelium. Recent technological advances applied to relevant animal and cell injury models have helped in understanding the complexity of progenitor cell differentiation. This short review will introduce the current knowledge of the mechanisms regulating airway epithelial cell (AEC) regeneration and repair, with a focus on the specification of two rare cell types/states: ionocytes and deuterosomal cells.

Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche.

Adult stem cells are often found in specialized niches, where the constituent cells direct self-renewal of their stem cell pool. The niche is therefore crucial for both normal homeostasis and tissue regeneration. In many mammalian tissues, niche cells have classically been difficult to identify, which has hampered any understanding of how tissues first construct niches during development. Fortunately, the Drosophila germline stem cell (GSC) niche is well defined, allowing for unambiguous identification of both niche cells and resident stem cells. The testis niche first forms in the early embryo, during a late stage of gonadogenesis. Here, using live-imaging both in vivo and ex vivo, we follow pro-niche cells as they assemble and assume their final form. We show that after ex vivo culture the niche appears fully functional, as judged by enrichment of adhesion proteins, the ability to activate STAT in adjacent GSCs, and to direct GSCs to divide orthogonally to the niche, just as they would in situ. Collectively, our imaging has generated several novel insights on niche morphogenesis that could not be inferred from fixed images alone. We identify dynamic processes that constitute an assembly phase and a compaction phase during morphogenesis. The compaction phase correlates with cell neighbor exchange among the assembled pro-niche cells, as well as a burst of divisions among newly recruited stem cells. Before compaction, an assembly phase involves the movement of pro-niche cells along the outer periphery of the gonad, using the extracellular matrix (ECM) to assemble at the anterior of the gonad. Finally, live-imaging in integrin mutants allows us to define the role of pro-niche cell-ECM interaction with regard to the new assembly and compaction dynamics revealed here.

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.

The functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury-repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the 'pulmonary ionocyte', which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium.

TRRAP is a central regulator of human multiciliated cell formation.

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.