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

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.

Kaempferol Protects Cell Damage in In Vitro Ischemia Reperfusion Model in Rat Neuronal PC12 Cells

“…In this study, we also found that OGD-induced oxidative stress was significantly inhibited by KFL, suggesting a substantial role of KFL in the redox regulation under OGD condition. P66 isoform of SHC1 (p66shc) proapoptotic ROS-elevating SHC family adaptor is an important regulator of redox homeostasis [31]. Genetic ablation of the p66Shc has been shown to reverse cognitive deficits and improve mitochondrial function in an APP transgenic mouse model of Alzheimer's disease [32].…”

<p>p66shc siRNA Nanoparticles Ameliorate Chondrocytic Mitochondrial Dysfunction in Osteoarthritis</p>

“…43 However, p66shc disrupts mitochondrial function via its CYCS (cytochrome c, somatic) binding domain, impairing ATP production, activating AMP-activated protein kinase (AMPK) and enhancing autophagic flux. 44 ROS are generated by the Nox family of enzyme complexes, which catalyze the transfer of electrons from NADPH to molecular oxygen, generating O 2 . 45,46 Thus, p66Shc deficiency reduced the activation of the PHOX complex, decreasing superoxide production.…”

Regulation of Selective B Cell Autophagy by the Pro-oxidant Adaptor p66SHC

“…As we will discuss in the following sections, we have identified a new survival-related function of p66Shc as regulator of B lymphocyte autophagy (Onnis et al, 2018), a process that regulates B cell fate, survival and differentiation, highlighting p66SHC as a pleiotropic master regulator of lymphocyte survival.…”

“…Not surprisingly, p66SHC deficiency is associated with pathogenic outcomes, including autoimmunity and leukemia (Finetti et al, 2008;Capitani et al, 2010;Patrussi et al, 2019). We have recently reported a new role for p66SHC as regulator of B-cell autophagy/mitophagy and differentiation (Onnis et al, 2018). Here we will summarize our current understanding of the multifaceted role of p66SHC in lymphocyte survival and discuss the pro-autophagic/mitophagic function of p66SHC in the context of the autophagic control of B-cell development and differentiation.…”

“…We have recently reported that p66SHC affects B cell survival not only by antagonizing survival signaling by the BCR and promoting apoptosis, but unexpectedly also through selective autophagy/mitophagy (Onnis et al, 2018). Based on the ability of p66SHC to inhibit glycolysis in fibroblasts, which leads to a decrease in ATP production (Soliman et al, 2014), we asked whether p66SHC modulates metabolism in B cells.…”