No abstract

No abstract

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (⌬F508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ⌬F508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ⌬F508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ⌬F508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ⌬F508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.Cystic fibrosis causes lung, liver, pancreas, and reproductive tract disorders, typically leading to death prior to middle age from deterioration in pulmonary function (1). CFTR 1 protein is composed of two membrane spanning domains (MSD1 and MSD2), two nucleotide-binding domains (NBD1 and NBD2), and a regulatory region (R). Although it functions as an ATPgated anion channel, CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (2) based on high sequence similarity between the NBDs and canonical ABC domains. Understanding the exact molecular pathology caused by the ⌬F508 mutation in CFTR is of great importance in the development of drugs to treat cystic fibrosis because of the prevalence of this mutation in the human population. ⌬F508 CFTR fails to mature appropriately in the endoplasmic reticulum and is poorly populated in the epithelial membrane (3-6). It has been proposed that the primary effect of the ⌬F508 mutation is to cause misfolding of NBD1, which leads to aberrant transport and ultimately targeted proteolytic degradation of CFTR (7,8). Channels harboring the deletion show enhanced sensitivity to proteolytic degradation (9) but have at least partial wild-type chloride conductance properties (4, 10). Canonical ABC domain structures are composed of three subdomains, a central F1-type ATP-binding core subdomain, an antiparallel ␤-sheet (ABC␤) s...

Summary Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we reported a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a “histone H4 mimic” GK-X-GK motif that is di-acetylated by Tip60. The di-acetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructs an activated Twist/BRD4/P-TEFb/RNA-PolII complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.