cells Review FAIM: An Antagonist of Fas-Killing and Beyond Jianxin Huo 1 , Shengli Xu 1,2 and Kong-Peng Lam 1,3,4,* 1 Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 138668, Singapore; [email protected] (J.H.); [email protected] (S.X.) 2 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore 3 Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore 4 School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore * Correspondence: [email protected] Received: 15 May 2019; Accepted: 30 May 2019; Published: 4 June 2019 Abstract: Fas Apoptosis Inhibitory Molecule (FAIM) is an anti-apoptotic protein that is up-regulated in B cell receptor (BCR)-activated B cells and confers upon them resistance to Fas-mediated cell death. Faim has two alternatively spliced isoforms, with the short isoform ubiquitously expressed in various tissues and the long isoform mainly found in the nervous tissues. FAIM is evolutionarily conserved but does not share any significant primary sequence homology with any known protein. The function of FAIM has been extensively studied in the past 20 years, with its primary role being ascribed to be anti-apoptotic. In addition, several other functions of FAIM were also discovered in different physiological and pathological conditions, such as cell growth, metabolism, Alzheimer’s disease and tumorigenesis. However, the detailed molecular mechanisms underlying FAIM’s role in these conditions remain unknown. In this review, we summarize comprehensively the functions of FAIM in these different contexts and discuss its potential as a diagnostic, prognostic or therapeutic target. Keywords: FAIM; B cells; Fas-mediated apoptosis; TCR-mediated apoptosis; metabolism; Alzheimer’s disease; Multiple myeloma; Akt; c-FLIP 1. Introduction Apoptosis is a form of programmed cell death that plays important roles in various physiological processes to maintain homeostasis by controlling the number of cells in the tissues. It is an irreversible process that can be initiated through either an intrinsic or extrinsic pathway. In the intrinsic pathway, the cell “kills” itself upon sensing cell stress, whereas in the extrinsic pathway, the cell “kills” itself after receiving death signals from other cells. Both pathways trigger a cascade of enzymatic activation of proteins called caspases (cysteine-aspartic proteases) that destroy the cell by degrading proteins indiscriminately. Fas (CD95/APO-1/TNFRSF6), a member of the tumor necrosis factor (TNF) receptor superfamily, is a death receptor that mediates the extrinsic apoptosis of cells [1]. Engagement of Fas receptors by FasL (CD178) leads to Fas trimerization and the formation of the intracellular death-inducing signalling complex (DISC) that comprises adapter protein Fas-associated death domain protein (FADD) and procaspase-8 (FADD-like interleukin-1β-converting enzyme (FLICE)). Pro-caspase-8 is activated by auto-cleavage within DISC and subsequently leads to the activation of the downstream caspase-3 followed by the execution of apoptosis. In the immune system, Fas plays important roles in regulating lymphocyte maturation, receptor repertoire selection and the deletion of autoreactive lymphocytes to ensure a functional and innocuous immune system in humans. Fas-mediated apoptosis is tightly regulated by both positive and negative signals to ensure proper Cells 2019, 8, 541; doi:10.3390/cells8060541 www.mdpi.com/journal/cells Cells 2019, 8, 541 2 of 12 Fas killing of lymphocytes for the regulation of lymphocyte growth and differentiation [2,3]. Fas apoptosis inhibitory molecule (FAIM) has been identified as a negative regulator of Fas signalling [4] and was subsequently found to play multifaceted roles in many physiological processes. In this review, we will discuss the physiological role of FAIM and its involvement in pathological situations such as tumorigenesis and metabolic disorders. 2. The Discovery of FAIM FAIM was first identified as a negative regulator of Fas signalling in BCR-stimulated B lymphocyte. Using mRNA differential display, Schneider et al. found that faim gene was inducibly expressed in murine primary B lymphocytes stimulated with antibodies directed against their surface immunoglobulin M (IgM) and in the presence of soluble dimeric CD40 ligand (CD40L). Consequently, FAIM is upregulated and enables the B cells to be resistant to Fas-mediated apoptosis. It was also found that FAIM is a relatively small molecule of 179 amino acids in length and is ubiquitously expressed [4]. Subsequent study revealed that faim gene, which consists of six exons and is located at chromosome 9f1 in mice (syntenic region 3q22.3 in humans), gives rise to two alternatively spliced RNA isoforms that share part of the 50UTR in exon I but have two different translation initiation sites in exons II and exon III, respectively. The longer splicing variant of faim (FAIM-L) contains 22 additional amino acids on the N-terminus compared to the smaller variant (FAIM-S) [5]. FAIM-S is found to be ubiquitously expressed in all tissues, whereas FAIM-L is specifically expressed in neural tissues, such as the cortex, cerebellum, hippocampus, hindbrain and spinal cord [6]. More recently, Coccia et al. found two additional FAIM isoforms, FAIM-S_2a and FAIM-L_2a, which have the same sequence as FAIM-S and FAIM-L but include exon 2a. Similar to the expression patterns of FAIM-S and FAIM-L, FAIM-S_2a is ubiquitously expressed in all tissues, whereas FAIM-L_2a is exclusively expressed in the nervous system. Interestingly, in contrast to cytosolic proteins FAIM-S and FAIM-L, FAIM-S_2a and FAIM-L_2a are able to localize to the nucleus, indicating that these two FAIM isoforms may have additional neural-related functions (Figure1). Importantly, FAIM-S_2a and FAIM-L_2a increased neurite outgrowth in NGF-treated PC12 cells, indicating their functional role in neuronal development and differentiation [7]. Figure 1. Structure and comparison of four human Fas apoptosis inhibitory molecule (FAIM) isoforms. Schematic representation of FAIM gene structure shown with exon 1a, 1b, 2a, 2b, 3, 4, 5 and 6. In contrast to the 50 and 30 untranslated regions (UTRs), coding sequence (CDS) regions are indicated with a darker color. TSS, Transcriptional start site. 3. Structural Study of FAIM Although FAIM is highly conserved evolutionarily, it does not share significant primary sequence homology with any known protein. NMR spectroscopy study of murine FAIM-S revealed that its N- Cells 2019, 8, 541 3 of 12 and C-regions can fold independently of each other and clearly constitute distinct domains [8]. Detailed analysis revealed that the C-terminal domain of FAIM shares some topological features with the “up-and-down barrel” motif found in fatty acid binding protein (FABP) and the P2 family of proteins. However, unlike FABP, the C-terminal domain of FAIM appears to be too small to accommodate a fatty acid residue within its cavity and probably has no role in lipid binding. The architecture of the C-terminal domain (CTD) of FAIM is also similar to that of type III fibronectin domains except for the lack of β-strands found in the latter. The lack of structural homology to other proteins suggests that the FAIM-CTD structure cannot directly shed light on the molecular mechanisms of FAIM [8]. Another study of the structure of human FAIM-S showed that the crystal structure of the N-terminal domain (NTD) of FAIM and the NMR solution structure of the C-terminal domain adopt a similar protein fold containing eight antiparallel β-strands which form two sheets. Further structural and biochemical analyses implied that the N-terminal domain exists as a dimer and the C-terminal domain exist as a monomer and that they can interact with each other. A mutation study suggested that residues Glu38, Arg110 and Asn123 are important for the interaction of the two domains and the anti-apoptotic activity of FAIM, suggesting that the inter-domain interaction of FAIM is important for FAIM’s anti-apoptotic function [8]. 4. Physiological Functions of FAIM 4.1. FAIM’s Role in Fas-Mediated Apoptosis of B Cells, Thymocytes and Hepatocytes FAIM was initially found to be up-regulated in BCR-stimulated B cells and antagonized Fas-mediated apoptosis of B cells. Overexpression of FAIM in the B lymphoma cell line BAL-17 confers these cells with resistance to Fas-killing [4]. In contrast, FAIM-deficient B cells exhibited increased sensitivity to Fas-triggered apoptosis in vitro [9]. In addition, FAIM-deficient thymocytes and hepatocytes were also more susceptible to Fas-killing, and FAIM-deficient mice suffered greater mortality and exhibited exacerbated liver damage in response to Fas engagement in vivo. In Fas-mediated apoptosis, trimerized Fas receptor recruits the adapter molecule FADD and caspase-8 into the DISC, resulting in caspase-8 activation. A molecule with sequence homology to caspase-8, termed cellular FLICE-inhibitory protein (c-FLIP), possesses apoptosis-inhibiting functions by binding to DISC to block caspase-8 activation and thereby inhibits Fas-mediated apoptosis [3,10]. Detailed biochemical analyses further revealed decreased expression of c-FLIP (L) (long) and c-FLIP (R) (short) in FAIM-deficient thymocytes and hepatocytes and increased association of caspase-8 with Fas in the mutant cells. These results suggest that FAIM modulates Fas-mediated apoptosis through influencing the expression of c-FLIP and regulating the physical association of caspase-8 and DISC [9] (Figure2). Figure 2. Schematic representation of FAIM’s involvement in various signaling pathways and physiological or disease outcomes. Cells 2019, 8, 541 4 of 12 4.2. FAIM’s Role in TCR-Mediated Apoptosis of Thymocytes Apoptosis also plays an important role in lymphocyte development and homeostasis [11]. T cells develop in the thymus and undergo a process of positive and negative selection triggered by the engagement of their T cell receptors (TCR) to select for functional and non-auto reactive T cells [12,13].