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

Abstract

p66SHC is a pro-oxidant member of the SHC family of protein adaptors that acts as a negative regulator of cell survival. In lymphocytes p66SHC exploits both its adaptor and its reactive oxygen species (ROS)-elevating function to antagonize mitogenic and survival signaling and promote apoptosis. As a result, p66SHC deficiency leads to the abnormal expansion of peripheral T and B cells and lupus-like autoimmunity. Additionally, a defect in p66SHC expression is a hallmark of B cell chronic lymphocytic leukemia, where it contributes to the accumulation of long-lived neoplastic cells. We have recently provided evidence that p66SHC exerts a further layer of control on B cell homeostasis by acting as a new mitochondrial LC3-II receptor to promote the autophagic demise of dysfunctional mitochondria. Here we discuss this finding in the context of the autophagic control of B cell homeostasis, development, and differentiation in health and disease.

Keywords: B lymphocytes; ROS; autophagy; mitophagy; p66SHC.

FIGURE 1

p66SHC structure and functions in B lymphocytes. (A) Schematic representation of the modular organization of p66SHC. The scheme shows the N-terminal collagen-homology domain (CH2), the CYCS-binding domain (CB), the phosphotyrosine-binding domain (PTB), the collagen homology domain (CH1), and the C-terminal SRC-homology domain 2 (SH2) together with the phosphorylatable tyrosine and serine residues, and the LIR motifs involved in the interaction with LC3B. (B) Role of p66SHC in BCR and chemokine signaling and apoptosis in B lymphocytes. Left panel, After BCR engagement p66SHC inhibits cell activation and survival by impairing Ras/MAPK and Akt activation. Middle panel, p66SHC promotes the assembly of a complex with the phosphatases SHP-1 and SHIP-1 on CXCR4 or CXCR5 which inhibits cytoskeleton remodeling and hence B cell adhesion and migration. In addition, p66SHC decreases the surface levels of these chemokine receptors slowing down their recycling to the plasma membrane (dotted lines), and, as a result, negatively regulates their ability to signal in B lymphocytes. Right panel, p66SHC increases B cell susceptibility to apoptosis by inducing production of ROS and modulating the expression of several members of the BCL-2 family of apoptosis-regulating proteins. The wide-ranging functions of p66Shc depicted in this scheme, which are also present in T cells, are associated to pathogenic outcomes. Indeed, p66Shc deficiency is a causal factor for the development of lupus-like autoimmunity in mice and for the onset and severity of human CLL.

FIGURE 2

Regulation of B cell development, activation and differentiation by autophagy. B cells develop and differentiate into an array of peripheral B cell subsets through a stepwise process starting from a hematopoietic stem cell (HSC). The figure shows a simplified outline of B cell lineage differentiation with a particular focus on the stages in which autophagy and mitochondria have been implicated in the regulation of B cell survival, fate and/or functions. In the bone marrow autophagy maintains the metabolism of HSCs by preventing the accumulation of mitochondrial ROS, which regulate their self-renewal and differentiation (Ho et al., 2017). The HSC gives rise to progenitors of two distinct populations of B lymphocytes, called B1 and B2 cells. B1 B cell generation is restricted to fetal and early neonatal life and thereafter a population of B1 B cells is preserved in the periphery by self-renewal. The ability of B1 B cells to self-renew is dependent on autophagy and its role in the regulation of cellular metabolism as well as lipid and mitochondrial homeostasis. Conversely, the production of B2 B cells is constant throughout life, thus autophagy is dispensable for their survival and self-renewal, but required at the pro-B to pre-B transition, when immunoglobulin gene rearrangements lead to the expression of a pre-B cell receptor (BCR) on the cell surface. Within secondary lymphoid tissues (i.e., lymph nodes and spleen) activation of a mature B cell and its subsequent differentiation into an antibody-secreting plasma cell or a memory B cell requires the specific recognition of a foreign antigen (Ag) by the BCR and critical signals derived from an antigen-specific CD4⁺ T cell. After Ag-BCR binding, the Ag undergoes receptor-dependent internalization and delivered to the endosomal compartment, where it is degraded into peptides, then associated to MHC-II molecules and transferred back to the surface, where it is presented to a matched helper T cell. Autophagy participates in antigen presentation by favoring the degradation and modification of foreign antigens. Some activated B cells develop into short-lived plasmablasts that rely on autophagy for pruning mitochondrial content, while others proliferate within a germinal center (GC), where an increased non-canonical autophagy has been implicated in the control of mitochondrial mass and ROS production. Since ROS act as signaling molecules for B cell fate, autophagy and related changes in mitochondrial content and ROS levels are crucial for driving GC cell differentiation. At later stages autophagy limits endoplasmic reticulum (ER) stress caused by the robust antibody secretory activity of plasma cells, while it favors the maintenance of immunological memory against viral infections by protecting memory B cells from oxidative stress and lipid peroxidation toxicity. In this scenario, p66SHC participates in the regulation of B cell fate by promoting the differentiation of activated B cells to isotype-switched memory cells through its mitochondria-depolarizing and ROS-generating activity.

FIGURE 3

Role of p66SHC-dependent autophagy/mitophagy in B cells. In the presence of p66SHC, B cells undergo oxidative stress with insufficient ATP production and metabolic imbalance. The increased ADP/ATP ratio activates AMPK and enhances the autophagic flux. p66SHC impairs ATP production not only by modulating the AMPK pathway but also by dissipation of the mitochondrial membrane potential and ROS generation. The loss of mitochondrial integrity activates the PINK1-PRKN/PARKIN-mediated mitophagy pathway and increases the ubiquitination of OMM proteins, such as the Voltage-Dependent Anion Channel (VDAC) protein. The ubiquitinated proteins are recognized by the autophagic adaptor SQSTM1/p62 that mediates the recruitment of phagophore membranes through LC3 interaction. p66SHC is also able to interact with membrane-associated LC3 through an LC3-interacting region (LIR) motif and form a complex with active AMPK. Although the mitochondrial pool of p66SHC is localized in the intermembrane space, ubiquitination of OMM proteins causes OMM rupture, making mitochondrial p66SHC accessible for interaction with LC3-II to promote phagophore membrane recruitment to damaged mitochondria. Hence, p66SHC impacts cellular metabolism and mitochondrial function in B cells acting as an LC3 receptor to recruit autophagosomal membranes to dysfunctional mitochondria primed to be removed via mitophagy.