HDAC Inhibitors as Epigenetic Regulators

The rescued cells were cultured in Gibco high glucose Dulbeccos Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. == Cell aggregation assay == A431 N-Bis(2-hydroxypropyl)nitrosamine Pcad-mCherry cells were detached using 0.05% trypsin diluted with calcium and magnesium-free solution (CMFS). anti-cadherin antibodies for intracellular drug delivery. Subject terms:Molecular conformation, Cadherins P-cadherins are cell adhesion proteins that are overexpressed in cancers. This study offers insights into the regulation of cell adhesion and the design of drug delivery antibodies by showing that an antibody which traps P-cadherin in a distinct extracellular structure triggers cadherin endocytosis. == Introduction == P-cadherin (placental cadherin; Pcad), a member of the classical cadherin family N-Bis(2-hydroxypropyl)nitrosamine of cell-cell adhesion proteins, is usually overexpressed in malignant cancers1, including cancers that originate from the breast2, pancreas3and lung4. Consequently, Pcad is recognized as a promising monoclonal antibody (Mab) target for anti-cancer drug delivery5. In therapeutic applications, drug conjugated Mabs specifically target the extracellular regions of adherent Pcads at cell-cell junctions6. Upon N-Bis(2-hydroxypropyl)nitrosamine binding to Pcad ectodomains, the Mabs are internalized for payload release7. However, the molecular linkage between extracellular cadherin conformations and cadherin endocytosis is usually unknown. Consequently, molecular guidelines for engineering Mabs that bind to Pcad ectodomains and promote cadherin internalization have not been established. Like all classical cadherins, Pcad ectodomains bind in two distinct adhesive conformations: X-dimers and strand-swap dimers (S-dimers)8,9. Due to their faster on-rate and weaker binding strength, X-dimers are thought to be an intermediate in the formation and dissociation of S-dimers911. Notably, cells expressing mutant cadherins trapped in an X-dimer conformation, exhibit more dynamic cell junctions due to increased cadherin turnover10. However, the mechanism by which X-dimer formation promotes cadherin internalization is usually unknown. A grasp regulator of cadherin internalization is usually p120-catenin (p120), a protein that binds to the cadherin cytoplasmic region12. By acting as an endocytosis inhibitor, p120 stabilizes cadherins around the cell surface. Conversely, the dissociation of p120 triggers cadherin endocytosis and promotes their internalization13. However, it is unclear if X-dimer formation triggers cytoplasmic p120 dissociation and promotes cadherin endocytosis. Studying this outside-in mechanism is challenging due to a lack of methods to trap cadherins in an X-dimer conformation without introducing mutations into the cadherin extracellular region. One strategy to trap cadherins in distinct binding conformations is the use of Mabs. For instance, two E-cadherin (Ecad) specific Mabs, 19A11 and 66E8, have been shown to bind and stabilize S-dimers1416. The stabilization of Ecad S-dimers around the cell surface has been shown to strengthen cell adhesion and promote the dephosphorylation of p12017,18, suggesting a possible outside-in relationship between Ecad extracellular binding conformations and intracellular signaling. Conversely, the Mab CQY684, part of the antibody-drug conjugate PCA062, was recently shown to recognize the ectodomains of Pcad. Binding of CQY684 resulted in the endocytosis of the Mab-Pcad complex, N-Bis(2-hydroxypropyl)nitrosamine thereby facilitating drug delivery19. Despite the potential of CQY684 Rabbit Polyclonal to MAP2K7 (phospho-Thr275) as a drug delivery platform and its usage in Phase-I clinical trials20, the molecular mechanism by which CQY684 triggers Pcad endocytosis is usually unknown. In this study, we combine biophysical (atomic pressure microscopy, and bead aggregation), computational (molecular dynamics and steered molecular dynamics simulations), biochemical (surface biotinylation, phos-tag gels, and co-immunoprecipitation), and cell biological (cell adhesion assays, confocal imaging, and fluorescence recovery after photobleaching) measurements to demonstrate that CQY684 traps Pcad in an X-dimer conformation and stabilizes this binding structure. We show that this stabilization of X-dimers triggers the phosphorylation of p120 which results in its dissociation from the Pcad cytoplasmic region. The dissociation of p120 targets the antibody-Pcad complex to the lysosome. Our results establish an outside-in N-Bis(2-hydroxypropyl)nitrosamine signaling mechanism that provides fundamental insights into how cells regulate adhesion. Cadherin outside-in signaling can also be exploited by antibodies for intracellular drug delivery. == Results == == CQY684 selectively strengthens Pcad X-dimers == Since a previous structural study has shown that CQY684 binding does not interfere with formation of either Pcad S-dimers or X-dimers19, we used atomic pressure microscopy (AFM) to measure the effect of CQY684 around the stability of these distinct Pcad conformations. We used three constructs in our experiments: human Pcad W2A mutant (trapped in an X-dimer conformation)9, human Pcad K14E mutant (trapped in an S-dimer conformation)9, and wild-type human Pcad (WT). We immobilized the complete extracellular region of each Pcad construct (EC15) on an AFM cantilever and glass substrate functionalized with polyethylene glycol (PEG) tethers, and measured PcadPcad interactions with or without 40 nM CQY684 in the buffer (+CQY or CQY, Fig.1a, upper panel). == Fig. 1. CQY684 Fab strengthens Pcad X-dimers. == aUpper panel: scheme for AFM experiments carried out in the absence of CQY684 Fab (CQY), and in the presence of CQY684.