Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi/post-Golgi compartments.

MUC16, precursor of the most widely used ovarian cancer biomarker CA125, is up regulated in multiple malignancies and is associated with poor prognosis. While the pro-tumorigenic and metastatic roles of MUC16 are ascribed to the cell-associated carboxyl-terminal MUC16 (MUC16-Cter), the exact biochemical nature of MUC16 cleavage generating MUC16-Cter has remained unknown. Using different lengths of dual-epitope (N-terminal FLAG- and C-terminal HA-Tag) tagged C-terminal MUC16 fragments, we demonstrate that MUC16 cleavage takes place in the juxta-membrane ectodomain stretch of twelve amino acids that generates a ~17 kDa cleaved product and is distinct from the predicted sites. This was further corroborated by domain swapping experiment. Further, the cleavage of MUC16 was found to take place in the Golgi/post-Golgi compartments and is dependent on the acidic pH in the secretory pathway. A similar pattern of ~17 kDa cleaved MUC16 was observed in multiple cell types eliminating the possibility of cell type specific phenomenon. MUC16-Cter translocates to the nucleus in a cleavage dependent manner and binds to the chromatin suggesting its involvement in regulation of gene expression. Taken together, we demonstrate for the first time the oft-predicted cleavage of MUC16 that is critical in designing successful therapeutic interventions based on MUC16.

Characterization of the connexin45 carboxyl-terminal domain structure and interactions with molecular partners.

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.