Distribution of the p66Shc Adaptor Protein Among Mitochondrial and Mitochondria-Associated Membranes Fractions in Normal and Oxidative Stress Conditions

Abstract

p66Shc is an adaptor protein and one of the cellular fate regulators since it modulates mitogenic signaling pathways, mitochondrial function, and reactive oxygen species (ROS) production. p66Shc is localized mostly in the cytosol and endoplasmic reticulum (ER); however, under oxidative stress, p66Shc is post-translationally modified and relocates to mitochondria. p66Shc was found in the intermembrane space, where it interacts with cytochrome c, contributing to the hydrogen peroxide generation by the mitochondrial respiratory chain. Our previous studies suggested that p66Shc is localized also in mitochondria-associated membranes (MAM). MAM fraction consists of mitochondria and mostly ER membranes. Contact sites between ER and mitochondria host proteins involved in multiple processes including calcium homeostasis, apoptosis, and autophagy regulation. Thus, p66Shc in MAM could participate in processes related to cell fate determination. Due to reports on various and conditional p66Shc intracellular localization, in the present paper, we describe the allocation of p66Shc pools in different subcellular compartments in mouse liver tissue and HepG2 cell culture. We provide additional evidence for p66Shc localization in MAM. In the present study, we use precisely purified subcellular fraction isolated by differential centrifugation-based protocol from control mouse liver tissue and HepG2 cells and from cells treated with hydrogen peroxide to promote mitochondrial p66Shc translocation. We performed controlled digestion of crude mitochondrial fraction, in which the degradation patterns of p66Shc and MAM fraction marker proteins were comparable. Moreover, we assessed the distribution of the individual ShcA isoforms (p46Shc, p52Shc, and p66Shc) in the subcellular fractions and their contribution to the total ShcA in control mice livers and HepG2 cells. In conclusion, we showed that a substantial pool of p66Shc protein resides in MAM in control conditions and after oxidative stress induction.

Keywords: fractionation; mitochondria; mitochondria—ER contact sites; oxidative stress.

Figure 1

p66Shc distribution in cellular fractions isolated from mouse liver. (A) The levels of marker proteins in the fractions: for mitochondria (including crude mitochondria (MC) and pure mitochondria (MP))—mitochondrial superoxide dismutase (SOD2), voltage-dependent anion channel (VDAC), mitochondrial import inner membrane translocase (Tim23), and cytochrome c (Cyt c), for ER—calreticulin (Clrt), for mitochondria-associated membranes (MAM) - sigma non-opioid intracellular receptor 1 (Sigma R1), and for cytosol (including crude cytosol (CC) and pure cytosol (CP))—glyceraldehyde-3-phosphate dehydrogenase (GAPDH); (B) the level of ShcA proteins: p66Shc, p52Shc, and p46Shc in fractions isolated from mice livers shown in the representative Western blot picture; (C) quantification of p66Shc in each fraction compared to the p66Shc signal in total homogenate (H); box plots show the medians (lines), means are indicated with (+); n = 6; statistical significance evaluated with ordinary one-way ANOVA with Tukey’s method based multiple comparisons (**** p < 0.0001, * p < 0.05, ns—no significance); (D) heat map showing an enrichment of p66Shc in each fraction in reference to the fraction of origin: homogenate for crude cytosol and crude mitochondria, crude mitochondria for MAM, and purified mitochondria and crude cytosol for purified cytosol and ER; (E) graphs show the percentage contribution of p66Shc in each fraction in the total (100%) of p66Shc content calculated as a sum of p66Shc signals from MP, MAM, CP, and ER fractions (pie chart shows mean percentage representation of p66Shc in each fraction and box plot shows the medians (lines) and means indicated with (+) with SD); n = 6; Statistical significance evaluated with ordinary one-way ANOVA with Tukey’s method based multiple comparisons (**** p < 0.0001, ** p < 0.005, * p < 0.05, ns—no significance); (F) Western blot showing the levels of p66Shc in 25 µg of the total homogenate (H), pure mitochondria (MP), and MAM fractions samples as references and after immunoprecipitation (IP) with anti—ShcA antibodies in MP and MAM fractions from mice livers (IgG, heavy chain of the IP antibodies is visible together with p52Shc at approximately 52 kDa), n = 2; and (G) diagram illustrating the concept of the distribution of the investigated proteins across the intracellular fractions mentioned in the study, described in detail in the text.

Figure 2

p66Shc distribution in cellular fractions isolated from HepG2 cell cultures. (A) A comparison of the p66Shc protein levels in crude fractions: homogenate (H), post-mitochondrial supernatant—crude cytosolic fraction (CC), and crude mitochondrial fraction (MC) in HepG2 cells untreated and treated with the H₂O₂ followed by the quantification of p66Shc ratios between MC from H₂O₂ treated HepG2 to untreated cells after normalization to the level in the total homogenate (input) samples; t-test ** p < 0.005; (B,C) the levels of ShcA proteins: p66Shc, p52Shc, and p46Shc in fractions isolated from untreated HepG2 cells (B) and in fractions isolated from HepG2 cells treated with 1 mM H₂O₂ for 24 h (C) followed by p66Shc fraction shares quantification showed as the percentage contribution of p66Shc in each fraction in the total (100%) of p66Shc content calculated as a sum of p66Shc signals from MP, MAM, CP, and ER fractions; pie charts show mean percentage representation of p66Shc in each fraction and respective box plots show the mean with (SD); plots show the median with SD, means are indicated with (+); statistical significance evaluated with ordinary one-way ANOVA with Tukey’s method based multiple comparisons (*** p < 0.0005, ** p < 0.005, and * p < 0.05, ns—no significance); levels of marker proteins: for mitochondria—mitochondrial superoxide dismutase (SOD2) and cytochrome c (Cyt c), ER—calreticulin (Clrt), MAM: long-chain-fatty-acid-CoA ligase 4 (ACSL4), cytosol (CC and CP)—GAPDH; (D) heat map presenting quantification of a p66Shc ratio in each fraction to p66Shc in homogenate; and (E) heat map presenting quantification of a p66Shc ratio in each fraction to p66Shc in the respective fraction of origin: homogenate for MC and CC, CC for ER and cytosol, and MC for MAM and MP.

Figure 3

p66Shc digestion with trypsin in crude mitochondrial fraction isolated from the HepG2 cell line. p66Shc protein and fractions markers: SigmaR1 for MAM, VDAC for outer mitochondrial membrane (OMM), Cyt c for intermembrane space (IMS), and SOD2 for mitochondrial matrix (MM) detected by Western blot in the residual samples after trypsin digestion of 100 µg of MC isolated from (A) control untreated HepG2, and (B) HepG2 cells treated with 1 mM H₂O₂ for 24 h. Quantification presented below the representative Western blots shows the mean ratio of p66Shc (A,B) and fractions markers (C) in trypsinized samples to the level of p66Shc (A,B) or each fraction marker (C) in the input untreated MC sample (value is assigned as 1) with SD. Statistical significance was calculated with one sample t-test (where value = 1 refers to the input—undigested MC sample), p-value (**** p < 0.0001, *** p < 0.0005, and ** p < 0.005, * p < 0.05, ns—no significance).