Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase

NM23 proteins: innocent bystanders or local energy boosters for CFTR?

NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies

Immunisation

SIGLEAvailable from British Library Document Supply Centre-DSC:GPE/2402 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

High-throughput functional analysis of CFTR and other apically localized proteins in iPSC-derived human intestinal organoids

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport