Lebiedzinska-Arciszewska Magdalena, Pakula Barbara, Bonora Massimo, Missiroli Sonia, Potes Yaiza, Jakubek-Olszewska Patrycja, Simoes Ines C M, Pinton Paolo, Wieckowski Mariusz R
Laboratory of Mitochondrial Biology and Metabolism, Nencki Institute of Experimental Biology of Polish Academy of Sciences, 02-093 Warsaw, Poland.
Department of Medical Sciences, Section of Experimental Medicine, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, 44121 Ferrara, Italy.
Int J Mol Sci. 2024 Nov 29;25(23):12835. doi: 10.3390/ijms252312835.
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.
p66Shc是一种衔接蛋白，也是细胞命运调节因子之一，因为它能调节有丝分裂信号通路、线粒体功能和活性氧（ROS）生成。p66Shc主要定位于细胞质和内质网（ER）；然而，在氧化应激下，p66Shc会发生翻译后修饰并重新定位于线粒体。p66Shc存在于线粒体外膜间隙，在那里它与细胞色素c相互作用，促进线粒体呼吸链产生过氧化氢。我们之前的研究表明，p66Shc也定位于线粒体相关膜（MAM）。MAM组分由线粒体和主要是内质网的膜组成。内质网和线粒体之间的接触位点存在参与多种过程的宿主蛋白，包括钙稳态、细胞凋亡和自噬调节。因此，MAM中的p66Shc可能参与与细胞命运决定相关的过程。由于有关于p66Shc在细胞内不同定位和条件性定位的报道，在本文中，我们描述了p66Shc在小鼠肝脏组织和HepG2细胞培养物不同亚细胞区室中的分布情况。我们为p66Shc定位于MAM提供了额外证据。在本研究中，我们使用通过基于差速离心的方案从对照小鼠肝脏组织、HepG2细胞以及用过氧化氢处理以促进线粒体p66Shc易位的细胞中精确纯化的亚细胞组分。我们对粗制线粒体组分进行了对照消化，其中p66Shc和MAM组分标记蛋白的降解模式具有可比性。此外，我们评估了单个ShcA异构体（p46Shc、p52Shc和p66Shc）在亚细胞组分中的分布及其对对照小鼠肝脏和HepG2细胞中总ShcA的贡献。总之，我们表明在对照条件下以及氧化应激诱导后，大量的p66Shc蛋白存在于MAM中。
