Development of a fluorescent monoclonal antibody‐based assay to measure the allosteric effects of synthetic peptides on self‐oligomerization of AGR2 protein

Many regulatory proteins are homo‐oligomeric and designing assays that measure self‐assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer‐associated oncoprotein AGR2. A two site‐sandwich microtiter assay (^2SMTA) was designed using a DyLight800‐labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self‐peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N‐terminal region of AGR2 increase in trans oligomer stability as defined using the ^2SMTA assay. A DSS‐crosslinking assay that traps the AGR2 dimer through K95‐K95 adducts confirmed that Δ45‐AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt‐AGR2, Δ45‐AGR2 (more stable dimer), and monomeric AGR2^E60A revealed that Δ45‐AGR2 was more active in binding to Reptin than either wt‐AGR2 or the AGR2^E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization

Death-associated protein kinase 1 (DAPK-1) is a Ca²⁺/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.     

Nuclear gene trees and the phylogenetic relationships of the mangabeys (Primates: Papionini)

Phylogenetic relationships of mangabeys within the Old World monkey tribe Papionini are inferred from analyses of nuclear DNA sequences from five unlinked loci. The following conclusions are strongly supported, based on congruence among trees derived for the five separate gene regions: (1) mangabeys are polyphyletic within the Papionini; (2) Cercocebus is the sister taxon to the genus Mandrillus; and (3) Lophocebus belongs to a clade with Papio and Theropithecus, with Papio as its most likely sister taxon. Morphologically based phylogenies positing mangabey monophyly were evaluated by mapping the sequences for each locus on these trees. The data seem to fit these trees poorly in both maximum-parsimony and likelihood analyses. Incongruence among nuclear gene trees occurred in the interrelationships among Lophocebus, Papio, and Theropithecus. Several factors that may account for this incongruence are discussed, including sampling error, random lineage sorting, and introgression.      

PPCMatrix: a PowerPC dotmatrix program to compare large genomic sequences against protein sequences

Summary : An interactive dotmatrix program for the MacOS was designed that allows comparison of DNA to protein sequences using nested 3-frame translations. Availability : Shareware, available at http://copan.bioz.unibas.ch/software/ Contact : burglin@ubaclu. unibas.ch