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

Abstract

Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network

“…MDM2 is described to bind to over 100 proteins, yet little is known about how all these interactions relate to the cell biology of MDM2. Small molecules targeting MDM2 alter the binding of its associated proteins by either directly disrupting the MDM2 interface required for association ---and/or ---promote numerous allosteric changes in multiple MDM2 domains directly, thus altering motifs to that either gain or lose capacity to interact with proteins (Janssen et al, 2006;Way et al, 2016). Here, we noted that Nutlin-3A could enhance the interaction between MDM2 and NDUFS1 (Fig.…”

“…Therefore, we examined the impact of SOD1 co-expression on MDM2 WT -and MDM2 1-101 -induced mitochondrial ROS production, DNA damage, and subsequent apoptosis in H1299. As demonstrated before, MDM2 WT or MDM2 1-101 lead to increased mitochondrial ROS, H2AX accumulation, and apoptosis; and co-expression of SOD1 was effective in preventing the majority of these stress phenotypes Several studies have highlighted the importance of gaining a broader perspective of the MDM2 interactome, and its regulation by small molecules like Nutlin-3A (Janssen et al, 2006;Way et al, 2016). MDM2 is described to bind to over 100 proteins, yet little is known about how all these interactions relate to the cell biology of MDM2.…”

“…1G). Numerous mitochondrial proteins (e.g., mitochondrial pyruvate dehydrogenase complex) and functions (e.g., nucleotide exchange and electron transport) are regulated in a Nutlin-3A-dependent manner with little mechanistic or biological insights (Janssen et al, 2006;Way et al, 2016;Schneider et al, 2016), and there are notions in the literature that Nutlin-3A can regulate both cellular metabolism and signaling independent of Page 16 11/30/18 the p53 pathway (Ye et al, 2017;Fåhraeus et al, 2014). A key area of future investigation is to determine under which circumstances cells promote the MDM2·NDUFS1 interaction to regulate mitochondrial respiration, and what are the upstream signals that promote MDM2 stabilization and function in these settings.…”

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

“…The Nutlin-3 responsive proteins can be stratified with respect to p53-like "BOX-I" homology motifs that identify a relatively large set of p53-like MDM2 binding proteins whose equilibrium binding to MDM2 is disturbed by Nutlin-3 [26]. Mass spectrometry demonstrated that mitochondrial protein interactions are perturbed by Nutlin-3, which induces an increase of MDM2 binding to the dihydrolipoamide dehydrogenase (DLD) subunit of mitochondrial pyruvate dehydrogenase in the nucleus [27]. Thus, the balance between tumor suppressor induction and oncoprotein activation by MDM2-targeted drugs could tip the balance towards tumor regression or tumor survival, respectively.…”

Dynamic post‐translational modifications in obesity

“…We hypothesized that these proteins were acetylated and crotonylated according to their function in lipid metabolism. 9,10 We then summarized the dynamic changes of acetylation and crotonylation sites in Dld and Acat1 proteins.…”