The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II

Autophagy. 2018;14(12):2117-2138. doi: 10.1080/15548627.2018.1505153. Epub 2018 Sep 6.

Abstract

Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Keywords: Autophagy; B lymphocytes; LC3 adaptor; mitochondria; mitophagy; p66SHC.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / metabolism
  • B-Lymphocytes / physiology*
  • Cell Differentiation / genetics
  • Cell Differentiation / immunology
  • Cell Survival / genetics
  • Cell Survival / immunology
  • Cells, Cultured
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mice
  • Mice, 129 Strain
  • Mice, Knockout
  • Microtubule-Associated Proteins / metabolism*
  • Mitochondria / metabolism*
  • Mitochondria / physiology
  • Mitochondrial Membranes / metabolism*
  • Mitochondrial Membranes / pathology
  • Mitophagy / genetics*
  • Oxidants / metabolism
  • Permeability
  • Prohibitins
  • Protein Binding
  • Reactive Oxygen Species / metabolism
  • Src Homology 2 Domain-Containing, Transforming Protein 1 / genetics
  • Src Homology 2 Domain-Containing, Transforming Protein 1 / physiology*

Substances

  • MAP1LC3B protein, human
  • Microtubule-Associated Proteins
  • Oxidants
  • PHB protein, human
  • Prohibitins
  • Reactive Oxygen Species
  • SHC1 protein, human
  • Shc1 protein, mouse
  • Src Homology 2 Domain-Containing, Transforming Protein 1

Grants and funding

This work was supported by the Associazione Italiana per la Ricerca sul Cancro [IG 2014-15220]; Associazione Italiana per la Ricerca sul Cancro [IG2016- 18906]; Associazione Italiana per la Ricerca sul Cancro [IG 2017-20148]; Kræftens Bekæmpelses Videnskabelige Udvalg (KBVU) [R146-A9364]; Kræftens Bekæmpelses Videnskabelige Udvalg (KBVU) [R146-A9471]; Fondazione Telethon [GGP1102]; Istituto Toscano Tumori;Lundbeckfonden [R209–2015–3505]; Novo Nordisk Research Foundation [22544]; Fondazione Roma; Bjarne Saxhof Foundation [7559].