Mitochondria-Nucleus Communication by Shuttle Proteome in the RPE under Oxidative Stress

Abstract

Purpose: : Constant crosstalk between mitochondria and the nucleus is essential to control cellular biochemical pathways under altered oxygen environment. Mitochondria-generated signals, including reactive oxygen species play a key role in early downstream network of caspase dependant reactions, and they trigger chromatin condensation and DNA degradation as nuclear apoptosis. Despite extensive efforts to understand RPE cell death, the mechanisms underlying mitochondria-nucleus communication are still elusive. Comprehensive identifications of protein-protein/protein-lipid interactions, including post-translational, binding affinity changes, may disclose a new rate-determining step in RPE apoptosis. Our proteomic approach to understand protein translocalization between mitochondria and nuclei may suggest subcellular communication mechanism in aging and diabetic.

Methods: : Mitochondria and nuclear proteins were extracted and fractioned by differential centrifugation from ARPE-19 cells. Interacting proteins were resolved by Co-immunoprecipitation and separated by 2D electrophoresis. Western blotting and immunocytochemical analysis were performed to visualize the protein translocalization. Proteomic data from aging and diabetic rat model were compared to age-matching control. The functional role of shuttle proteome was further studied using protein-lipid interaction assay.

Results: : Previously, we demonstrated that prohibitin shuttles from mitochondria to the nucleus as cardiolipin chaperon, transcription coactivator, and anti-apoptotic protein. Our current study shows that prohibitin interacts with optic atrophy 3 (OPA3), small heat shock proteins (HSPC072), and palmitoyltransferase (ZDHHC18) in mitochondria as well as p53 and Rb in the nucleus. In addition, series of protein-protein interaction assays demonstrated that prohibitin may interact with RNA polymerase II transcription factor SIII A3 and BCL2-binding protein. Prohibitin-lipid binding switch mediated by cardiolipin and phosphotadylinositol seems to control dynamic translocalization of prohibitin between mitochondria and the nucleus.

Conclusions: : Our protein-protein/protein-lipid interaction map provides an overview of mitochondrial and nuclear binding partners with prohibitin in response to oxidative stress. Prohibitin may act as an anti-apoptotic chaperone as the default function in mitochondria, whereas it becomes a transcriptional co-activator with p53/Rb under prolonged or repeated stress conditions. Apoptotic/transcriptional dual function of prohibitin might be the crucial mechanism for controlling apoptotic signaling as a cell death determinant from uncontrolled proliferation.

Keywords: mitochondria • retinal pigment epithelium • proteomics