Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis

Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

“…There are over one‐hundred published MDM2‐binding proteins that have been discovered using a large range of cell types, methodologies, and experimental conditions . This vast number of MDM2‐interactors has not been integrated into MDM2 “signaling pathways.” These target proteins could act as biomarkers to predict MDM2 drug responses but identifying such “core” MDM2‐binding proteins using tissue culture approaches is complicated by the fact that cell density can have a significant effect on p53 protein synthesis [28] and on the steady‐state levels of MDM2 protein through phosphorylation of its pseudo‐substrate motif . In addition, cell density changes created by changing the cell number at time of seeding also can attenuate p53‐dependent biological processes; this effect of cell density is not a consequence of density‐dependent cell‐cycle changes but rather are linked to cadherin‐mediated cell‐cell junctions .…”

“…The allosteric effect of Nutlin‐3 on MDM2 functions. (A) MDM2 has multiple functional domains including an N‐terminal peptide‐binding groove that is the binding site of Nutlin‐3 ; a central domain that responds allosterically to N‐terminal MDM2‐binding ligands (like Nutlin‐3) to increase p53 binding and ubiquitination (arrows) , and a RING domain that is required for E2 docking and allosteric control of ubiquitination by the E2, UBCH5 . N‐terminal domain Nutlin‐3 binding by MDM2 can stimulate p53 ubiquitination , stimulate ubiquitin‐dependent protein activation of Notch , and direct binding to alter target protein oligomerization of NPM .…”

“…Our hypothesis is focused on the concept that Nutlin‐3 can promote changes in the MDM2‐interactome by either dissociating existing PPIs or induction of new MDM2 interactions. Our experimental design included cells grown at two different densities and treated with Nutlin‐3, since cell density changes can alter MDM2 steady‐state levels and cell density can impact on p53 activity . We identified mitochondrial proteins as a group of proteins responding early to Nutlin‐3 treatment and have validated key changes in the protein–protein interaction partners of one mitochondrial protein within the pyruvate dehydrogenase complex.…”

Structural and dynamic characterization of the Golgi Reassembly and Stacking Protein (GRASP) in solution

“…This property is explored by the cell in the processes of assembly and disassembly of the Golgi cisternaes. One convenient approach to study the effect of single site-directed phosphorylation is based on the use of phosphomimetic mutations [199,200]. In this approach, the specific target amino acid residue is mutated to a negatively charged amino acid (aspartic or glutamic acid), thus inducing a permanent phosphorylated-like protein.…”

Aggregation and conformation of alpha-synuclein: effects of ligand binding and phosphomimetics

“…Phosphomimic mutants are now extensively used in research for a variety of applications (Dissmeyer and Schnittger, 2011;Fraser et al, 2014), including conformational studies (Hara et al, 2006), mass spectrometry (Jovcevski et al, 2015) and studies into the effects of phosphorylation on αsyn aggregation (Chen (Chen et al, 2016). Concentrations of several transition metals, including copper and iron, are known to be increased in aging brains (Morita et al, 2001), and the levels of these metals, along with zinc, are also known to be elevated in the brains of patients with PD and AD, and are found in increased concentrations within the LBs of PD patients (Dexter et al, 1989;Riederer et al, 1989;Hirsch et al, 1991;Jenner and Olanow, 1996;Lovell et al, 1998).…”