Contribution of Apaf-1 to the pathogenesis of cancer and neurodegenerative diseases

Highlights

• Alteration in Apaf-1 level is frequently observed in many human cancers.

• The contribution of Apaf-1 to pathology of neurodegenerative diseases is not as clear.

• Designing screening assays of apoptosome assembly/disassembly will be useful in finding novel therapeutic agents.

• More attention should be made to the contribution of Apaf-1 isoforms to the above mentioned pathologies.

Abstract

Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.

Introduction

Apoptosis is a well-organized active process of cell death playing a central role in the development and homeostasis of multicellular organisms. Apoptosis is characterized by morphological hallmarks including cellular shrinkage, chromatin condensation, membrane blebbing, nuclear fragmentation, and apoptotic body formation engulfed by phagocytic cells [1]. Apoptosis is induced by extrinsic and intrinsic stimuli and as such is classified into the receptor and mitochondrial-mediated pathways, also known as the extrinsic and the intrinsic pathway, respectively [2]. In both pathways, all morphological changes result from the action of cysteine aspartyl proteases, known as caspases. These enzymes are synthesized as inactive procaspases and stored in the cytosol, suggesting the requirement for a fast response should the need arise. The two major classes of apoptotic caspases are initiator caspases of caspases-2, -8, -9, -10 and effector caspases of caspases-3, -6, -7. All initiator caspases require a specific complex for activation. Caspase-2, the most highly conserved caspase across all species, is activated by a large protein complex termed the PIDDosome in response to a range of different stresses including heat, metabolic disruption, and DNA damage [[3], [4], [5], [6]]. Caspase-2 has been shown to mediate and amplify mitochondria-dependent apoptosis via cleaving BID, a Bcl-2 family protein with a BH3 domain only [7]. The truncated BID acts by both enhancing channel formation by Bax and Bak to release cytochrome c from the mitochondria and neutralizing antiapoptotic Bcl-2 proteins [8]. Caspase-9 is the initiator caspase of the intrinsic pathway or the mitochondrial pathway, while the caspases of −8 and −10 are the initiator caspases of the extrinsic or receptor-mediated pathways. These pathways merge at the effector caspases of caspase-3 and to some extent caspase-7. Proteolytic processing of the effector procaspases by the initiator caspases leads to their activation. These events are followed by the cleavage of vital cellular proteins at specific sites, culminating in cell death [9]. Procaspase-6 acts as one of many substrates of caspase-3 contributing to the apoptotic events in the nucleus by cleaving nuclear mitotic apparatus protein (NuMA) and Lamin A [10]. The activation of the initiator caspase-9 is achieved through a specific complex named apoptosome. Unlike caspase-9, the activation of caspase-8 and -10 is achieved through the formation of various complexes whose main component is a distinct subset of the tumor necrosis factor receptor (TNFR) superfamily members characterized by the presence of death domain (DD) [11]. Diversity of TNFR, their interacting ligands, and intracellular regulatory elements create many complexes with various signaling outcomes. The formation of death inducing signaling complexes, DISCs, is associated with the activation of mostly caspase-8 and in some instances caspase-10 leading to apoptosis. Other complexes can also form, the most notable of which is the 2-megadalton ripoptosome complex. Once again, various ripoptosomes can form with RIP kinase 1 acting as a major common component. Homo association of caspase-8 in the complex leads to apoptosis, while its hetero association with a caspase-8 homolog FLIP_L, which lacks active site cysteine, prevents cell death. Necroptosis can also occur if caspase-8 heterodimerizes with the shorter form of FLIP [12]. Moreover, caspase-8 participates in inflammation through a complex known as FADDosome. FADDosome is a complex of caspase-8, FADD and RIP1 which are assembled in response to Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) and leads to NF-κB-dependent expression of pro-inflammatory cytokines and chemokines. Caspase-8 appears to be a scaffold for the formation of this complex, because the activity of this complex is not dependent on the enzymatic activity of caspase-8 [13]. On other hand, it has been reported that FADDosome is an apoptosis-inducing complex in cancer cells via caspase-8 activation, which occurs in response to DNA single-strand breaks. It has been proposed that the presence of caspase-10 is essential for FADDosome assembly and caspase-8 activity in this complex [14].

Apoptosome is a mega-complex initially formed by cytochrome c, Apoptotic protease activating factor 1 (Apaf-1), (d)ATP and procaspase-9 assembly, although other proteins like X-linked inhibitor of apoptosis protein (XIAP) and caspase-3 have also been found in association with the complex. Apaf-1 is present in a locked and inactive monomeric form in the cytosol. In response to intracellular stresses such as hypoxia, growth factor deprivation, cell detachment and stress signals, such as drug-induced DNA damage, cytochrome c is released from the mitochondrial intermembrane space into the cytosol. Interaction of the cytochrome c with the WD40 domain of Apaf-1 activates the protein leading to the formation of the Apaf-1 apoptosome platform. This heptameric complex recruits procaspase-9 through a homotypic CARD-CARD association forming the apoptosome complex. Caspase-9 is a holoenzyme requiring the formation of the apoptosome complex for its activation. Activated procaspase-9 cleaves itself at D315, which in turn activates effector caspases (Fig. 1) [15]. The exact number of procaspase-9 molecules per apoptosome complex is a matter of debate but it is generally believed to be between 2 and 5 molecules per seven Apaf-1 molecules in the apoptosome depending upon the condition [[16], [17], [18], [19], [20]]. The apoptosome is also responsible for amplifying extrinsic cell death pathways through the truncation of BID, a process similar to the above-discussed mechanism for caspase-2 [21].

Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders and cancer. At one end of the spectrum lies inadequate apoptosis seen in cancer and in the other end lies excessive apoptosis occurring in neurodegenerative diseases [22]. Interestingly, a gradual decrease in Apaf-1 expression occurs during brain development, seemingly rendering neurons resistant to apoptosis [23]. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 protein [[24], [25], [26]]. Consequently, down-regulation of Apaf-1, as a key molecule in the intrinsic pathway of apoptosis, appears to be the strategy of choice to prevent apoptosis in non-dividing cells, which is accomplished through various mechanisms such as endogenous inhibitors, microRNAs and epigenetic alterations. In this context, it is important to examine whether or not Apaf-1 upregulation is a mechanism behind enhanced cell death in neurodegenerative diseases, and its downregulation acts as a mechanism for the reduced cell death in cancers. Pinpointing the extent of Apaf-1 participation in apoptosis dysregulation in cancer in general and neurodegenerative disorders in particular is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to these pathologies and shed light on the therapeutic aspects of Apaf-1 as an important target in these diseases.