Inhibitor of apoptosis proteins (IAPs) are an evolutionarily conserved class of multifunctional proteins (Srinivasula and Ashwell, 2008), which are defined by the presence of one to three tandem baculoviral IAP repeats (BIRs). The BIR domain is a highly conserved zinc binding fold of approximately 70 amino acids, harboring a CX2CX6WX3DX5HX6C consensus sequence (Hinds et al., 1999), which folds to a three stranded β-sheet surrounded by six short α-helices to coordinate a zinc ion chelated by three cysteine and one histidine residues (Birnbaum et al., 1994; Wu et al., 2000). The BIR domain is a prerequisite for protein–protein interaction (Hinds et al., 1999; Sun et al., 1999) and the inhibitory activity of IAPs in apoptosis (Takahashi et al., 1998).
The first members of IAPs were discovered in studies with baculoviruses, where binding and inhibiting cysteine-containing aspartate-specific proteases (caspases) contributed to the efficient infection and replication cycle in the host (Birnbaum et al., 1994; Crook et al., 1993).
The name IAP derived from the ability, to efficiently prevent apoptosis by binding and inhibiting active caspases and apoptotic protease activating factor 1 (APAF-1) through the BIR domains. Owing to this structural motif, IAPs are assigned to the BIR containing proteins (BIRPs) which can be classified into two groups: the IAP-like BIRPs, which are well established as apoptosis regulators and the survival-like BIRPs with important functions in cell cycle and cytokinesis (Verhagen et al., 2001).
Apoptosis, or programmed cell death, is a critical cellular process in normal development and homeostasis of multicellular organisms (Thompson, 1995) and caspases are the executioners in both, intrinsic and extrinsic pathways of apoptosis by cleaving a plethora of cellular components (Riedl and Shi, 2004). Several members of the IAP family can act both, upstream and downstream of caspase activation to promote cell survival (Deveraux and Reed, 1999). The action of IAPs can be opposed by certain pro-apoptotic factors, especially second mitochondria-derived activator of caspases (Smac/DIABLO) which promotes cytochrome-c dependent caspase activation (Vucic et al., 2002) or the evolutionarily conserved trimeric serin protease HtrA2/Omi (Martins, 2002; Suzuki et al., 2004; Vande Walle et al., 2008), which unleashes caspase activity in a biphasic process.
Introduction
35 It frees the active forms of caspases-3, -7 and -9 by proteolytically removing their natural inhibitors (Yang et al., 2003). Smac and HtrA2 are synthesized as larger cytosolic precursors that are proteolytically cleaved in mitochondria to expose their N-terminal tetrapeptide IAP binding motifs. In response to apoptotic stimuli, the processed Smac and HtrA2 are released into the cytosol where they can bind to a hydrophobic core at the BIR domain of IAPs (Liu et al., 2000; Vaux and Silke, 2003; Wu et al., 2000).
IAPs can effectively suppress apoptosis induced by a variety of stimuli, including death receptor activation, growth factor withdrawal, ionizing radiation, viral infection, and genotoxic damage. Indeed, IAPs are frequently overexpressed in many types of human cancer and are associated with chemoresistance, disease progression and poor patient survival (Hunter et al., 2007; LaCasse et al., 1998).
On the contrary, loss of IAPs is in some cases associated with the development of certain types of cancer. The physical association of the two best studied IAPs, XIAP and survivin, was found to convey the ubiquitin-dependent activation of NF-κB, which than drives the expression of genes important for cell migration, cell invasion and metastasis (Mehrotra et al., 2010).
Therefore, depending on the cellular context, IAPs seem to have both, pro-tumorigenic and anti-tumorigenic roles (Keats et al., 2007).
Moreover, several IAPs have been shown to promote proteasomal degradation of apoptotic proteins by catalyzing their ubiquitination and thereby regulating key components in cell death signaling cascades (Bartke et al., 2004; Vucic et al., 2011).
Recently, IAPs have emerged as broader regulators of cellular homeostasis, with functions extending beyond tumor development and apoptosis inhibition (Srinivasula and Ashwell, 2008), such as inflammatory signaling and immunity, mitogenic kinase signaling and mitosis (Dogan et al., 2008; Gyrd-Hansen et al., 2008; O'Riordan et al., 2008).
The activity of IAP is strictly regulated by different mechanisms. For RING domain containing IAPs a decreased half-life was observed due to the fact that they catalyze self-ubiquitination and proteasome-mediated degradation in a RING-dependent manner. This provides the interesting possibility that abundance is actively self-regulated (Yang et al., 2000). In addition, some IAP antagonists, such as Reaper, Grim and Hid, also stimulate IAP auto-ubiquitination and proteasomal degradation (Yoo et al., 2002).
36 3.3.1 BRUCE represents a special BIR containing protein (BIRP)
The inhibitor of apoptosis protein, BIR repeat containing ubiquitin-conjugating enzyme (BRUCE), represents a special member of the BIR containing protein (BIRP) family. It is a giant, highly conserved 528 kDa membrane associated protein which localizes mainly to the trans-golgi network (TGN) and perinuclear vesicles (Hauser et al., 1998).
It bears on one hand a single N-terminal BIR domain and on the other hand a C-terminal UBC-E2-domain, respectively. In contrast, most other BIRPs contain several BIR-domains and a C-terminal RING finger domain. Sequence analysis of BRUCE’s BIR domain revealed that BRUCE belongs to the survival-like BIRPs suggesting a role in cell cycle regulation and cytokinesis.
Remarkably, BRUCE is to date the only IAP described, which contains a functional C-terminal UBC-domain, characteristic for E2 enzymes of the ubiquitin proteasome system (Hauser et al., 1998). Due to the presence of this domain, BRUCE can form a thioester with Ub in vitro and mediate Ub-transfer to substrate proteins (Bartke et al., 2004; Hao et al., 2004; Hauser et al., 1998). These structural features indicate that BRUCE may combine properties of Ub-conjugating enzymes and IAP-like proteins and let suppose that the family of IAP-like proteins is functionally and structurally more diverse than previously expected.
The BRUCE cDNA was originally discovered in a homology screen for Ub conjugating enzymes in mice (Hauser et al., 1998). It is expressed in almost every mouse tissues with the highest expression in brain, liver and kidney (Hauser et al., 1998). The human homologue, Apollon, is like BRUCE widely expressed in almost every adult tissue and overexpressed in some drug-resistant cancer cells, suggesting that it may have an anti-apoptotic function (Chen et al., 1999). The importance and relevance of this antianti-apoptotic characteristic was further investigated and ascertained in numerous studies.
For instance, the expression level of BRUCE in rat brain and in cultured hippocampal neurons after treatment with the neurodegenerative component kainic acid was explored.
Thereby it was shown that BRUCE could promote survival of distinct neurons, whereas kainic acid treatment leads to BRUCE downregulation, leading to increased caspase-3 activation and cell death (Sokka et al., 2005). Downregulation of BRUCE expression in brain cancer cells with antisense oligonucleotides significantly sensitized the cells to apoptosis induced by DNA damaging agents, which is consistent with the fact that cancer cells over-expressing BRUCE show resistance to apoptosis inducing agents (Chen et al., 1999).
The most severe functional effect could be observed in gene-targeted BRUCE knockout mice, which led to impaired placenta development and growth retardation, resulting in embryonic lethality discernible after embryonic day 14.
Introduction
37 Despite the anti-apoptotic activities of BRUCE in cells, apoptosis was neither detected in mutant placenta nor in mouse embryonic fibroblasts (MEFs) (Lotz et al., 2004).
Further, also deletion of the C-terminal half of BRUCE, including the UBC domain, causes activation of caspases and apoptosis in the placenta and yolk sac, leading to embryonic lethality. This apoptosis is associated with up-regulation, stabilization and nuclear localization of the tumor suppressor p53, leading to the activation of Pidd, Bax and Bak, which might be specifically accompanied by mitochondrial-triggered apoptosis (Ren et al., 2005).
A unique function of BRUCE during mouse development lies in the regulation of spongiotrophoblast cell proliferation. Progressive loss of the spongiotrophoblast layer in Birc6/BRUCE mutants results in lethality at day 11.5 and 14.5 of embryonic development.
This further suggests a role for BRUCE, in addition to be an apoptosis inhibitor, as cell division regulator (Hitz et al., 2005). Genetic analysis in Drosophila has demonstrated that the Drosophila homologue dBRUCE, inhibits cell death induced by the essential Drosophila cell-death activators Reaper and Grim, resulting in viable but infertile male Drosophila BRUCE mutants (Vernooy et al., 2002). This argued for a role of dBRUCE in specialized pathways leading to cell death.
Moreover, the final stage of spermatid terminal differentiation in Drosophila requires the elimination of most of the cytoplasm, a process known as spermatid individualization, which is locally and temporary accompanied by an apoptosis like capase activation (Arama et al., 2003). Male sterile dBRUCE -/- flies failed to exclude the bulk cytoplasm which leads to sperm nucleus hyper-condensation and finally degeneration, indicating an uncontrolled or excessive apoptotic process. Interestingly, transient caspase activation is also involved during mammalian spermatogenesis, where spermatid cytoplasm needs to be eliminated and in consequence, there might be a need for the anti-apoptotic potential of BRUCE (Arama et al., 2003).
Despite the significant role for BRUCE in blocking apoptosis, very little is known about the regulation of BRUCE expression. Jansen and colleagues could show, that the treatment of myoblasts with prostaglandin F2alpha (PGF2α) reduces cell death and promotes myotube growth during myogenesis via upregulation of BRUCE through a pathway dependent on the nuclear factor of activated T cell 2 transcription factor (NFAT) (Jansen and Pavlath, 2008).
This finding describes a hitherto unrecognized relationship between NFAT signaling and regulation of IAP expression.
As aforementioned BRUCE belongs, with respect to its BIR-domain, to the survival-like BIRPs suggesting a role in cell cycle regulation and cytokinesis. Pohl and Jentsch reported in 2008, that BRUCE plays a crucial role at final stages of cytokinesis and particularly controls proper midbody ring formation which is required for cell cycle continuation.
38 During cell cycle, BRUCE concentrates in a pericentriolar compartment in interphase, moves partially to spindle poles in metaphase, and finally localizes to the spindle midzone and the midbody in telophase and during cytokinesis, where it serves as a platform for the membrane delivery machinery and binds mitotic regulators and components of the vesicle targeting machinery. BRUCE depletion causes cytokinesis defects and cytokinesis associated apoptosis. Notably, Ub relocalizes from midbody microtubules to the midbody ring during cytokinesis and depletion of BRUCE disrupts this process. Upon mitotic exit, BRUCE is targeted to the midbody ring via its C-terminus binding to mitotic kinesin-like protein 1 (MKLP1), which is a core component of the midbody ring. Both, BRUCE and MKLP1 are ubiquitinated and ubiquitin-specific protease Y (UBPY) was shown to serve as their deubiquitinating enzyme (Wu et al., 2004). This let suggest that BRUCE coordinates multiple steps required for abscission and ubiquitination of different protein targets may be a crucial trigger (Pohl and Jentsch, 2008). Further, Pohl and Jentsch could show that the midbody ring disposal by autophagy is a post-abscission event of cytokinesis, suggesting that autophagy is coupled to cytokinesis (Pohl and Jentsch, 2009).
Moreover, a novel regulatory role for BRUCE was uncovered in the interplay with the effector death caspase1 (Dcp-1) in starvation-induced autophagy during early Drosophila melanogaster oogenesis. These findings provide new insights into the molecular mechanisms that regulate autophagic and apoptotic events in vivo (Hou et al., 2008).
Interestingly, BRUCE itself can be degraded by autophagy and this controls DNA fragmentation and cell death in nurse cells during late oogenesis in Drosophila melanogaster. These results reveal autophagic degradation of an IAP as a mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death (Nezis et al., 2010).
BRUCE is a unique and an especial member of the BIRP family, due to its size, localization and its functions. BRUCE can act as a chimeric E2-E3 Ub ligase and can mono-ubiquitinate the IAP-antagonists Smac/Diablo, HtrA2 and active caspase-9 in vitro, which promotes their degradation (Bartke et al., 2004; Hao et al., 2004; Qiu and Goldberg, 2005). Therefore, BRUCE is thought to preserve cell survival by antagonizing apoptosis induced by spontaneously released pro-apoptotic factor Smac (Hao et al., 2004). Hereby, BRUCE can associate with precursors as well as mature forms of Smac by binding to regions in addition to the IAP binding motif, which promotes degradation of Smac and inhibits the activity of caspase-9 but not the effector caspase-3 (Qiu and Goldberg, 2005). Moreover, Smac as well as HtrA2 are able to compete for BRUCE-bound caspases. In response to apoptotic stimuli, BRUCE itself can be a substrate and cleaved by caspases and HtrA2 depending on the specific stimulus and the cell type (Sekine et al., 2005).
Introduction
39 Besides the regulation of caspases/proapoptotic factors, BRUCE itself seems to be regulated via ubiquitin-dependent degradation. Previous studies have implicated that the TRIM protein Nrdp1/RNF41 (neuregulin degrading protein 1/ring finger protein 41), a RING finger containing ubiquitin E3 ligase initially found to be involved in the degradation of ErbB/EGFR family of receptor tyrosine kinases (Diamonti et al., 2002; Qiu and Goldberg, 2002; Qiu et al., 2004), can act as a ubiquitin E3 ligase for BRUCE and mediate its proteasomal degradation.
Thereby cellular levels of BRUCE are maintained, impairing inhibition of apoptosis.
Overexpression of Nrdp1 was shown to decrease cellular levels of BRUCE; therefore Nrdp1 can be important in the initiation of apoptosis by catalyzing ubiquitination and degradation of BRUCE (Qiu et al., 2004).
Moreover, Nrdp1 regulates the turnover of the proteasome subunit hRpn13 and Parkin, which is a member of the E3 ubiquitin ligase family (Liu et al., 2007; Yu and Zhou, 2008).
Nrdp1 itself undergoes self-ubiquitination which leads to its proteasomal degradation.
Notably, Nrdp1 also specifically interacts and becomes stabilized by the deubiquitinating enzyme USP8/UBPY (ubiquitin-specific protease 8 or Y) (Wu et al., 2004), a cysteine protease implicated in cell cycle regulation, efficient downregulation of the EGF receptor and stability regulation of ESCRT-0 components Hrs and STAM2 (Cao et al., 2007; Clague and Urbe, 2006; Wright et al., 2011).
3.4 Autophagy
Autophagy, literally meaning ‘self-eating’, is an evolutionarily conserved intracellular catabolic process, in which portions of the cytoplasm are sequestered within cytosolic double-membrane vesicles called autophagosomes and subsequently delivered to the lysosome to allow degradation and recycling of the cargo (Kundu and Thompson, 2008). So far, the endoplasmic reticulum (ER), the Golgi, mitochondria, and plasma membrane have all been implicated in autophagosome formation (Hailey et al., 2010; Tooze and Yoshimori, 2010).
The autophagy-lysosome system is, beside the UPS, the other major protein degradation mechanism in eukaryotes (Kirkin et al., 2009; Nedelsky et al., 2008).
Autophagy embraces three major intracellular pathways in eukaryotic cells namely, macroautophagy, microautophagy, and chaperone mediated autophagy (CMA), which share a common destiny of lysosomal degradation, but are mechanistically different from one another (Cuervo, 2011; Klionsky, 2005; Li et al., 2011; Mijaljica et al., 2011) (Figure 7). The process of mammalian autophagy is divided into six principal steps: initiation, nucleation, elongation, closure, maturation and degradation or extrusion (Orsi et al., 2010).
40
Figure 7: Distinct types of autophagy.
Cytosolic proteins can enter the lysosome for degradation by at least three autophagic pathways.
(a) Macroautophagy is usually a catabolic process in which proteins, organelles or other cytosolic components are sequestered within cytosolic double-membrane vesicles called autophagosomes and subsequently delivered to the lysosome to allow degradation and recycling of the cargo. Selective variations of this process, in which distinct substrates (aggregate proteins or organelles) are targeted for degradation, and their names, are also depicted.
(b) During microautophagy the lysosomal membrane itself is envisaged as undergoing local rearrangement to directly engulf portions of cytoplasm or any constituents and are thereupon internalized after membrane scission and degraded in the lumen of the organelle. Cytosolic material can be sequestered ‘in bulk’ or selectively with the help of a cytosolic chaperone that recognizes the substrates.
(c) Chaperone-mediated autophagy (CMA) is a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. Soluble cytosolic proteins containing a targeting motif are recognized by the cytosolic heat shock cognate 70 (HSC70) chaperone and its co-chaperones, which deliver the substrate to the membrane of the lysosome. After docking onto the cytosolic tail of the lysosomal receptor, the substrate protein unfolds and crosses the lysosomal membrane through a multimeric complex. Substrate translocation requires a lumenal HSC70 chaperone and is followed by rapid degradation in the lysosomal lumen. CMA participates in quality control to maintain normal cell functions by clearing "old" proteins and provides energy to cells under nutritional stress. Figure taken from (Cuervo, 2011).
Introduction
41 During macroautophagy (autophagy), as depicted in Figure 7 (a), proteins, intact organelles (such as mitochondria) and portions of the cytosol are sequestered into a double-membrane vesicle, termed autophagosome. Subsequently, the completed autophagosome matures by fusing with an endosome and/or lysosome, thereby forming an autolysosome. This latter step exposes the cargo to lysosomal hydrolases to allow its breakdown and the resulting macromolecules are released back into the cytosol through membrane permeases for reuse (Beynon and Bond, 1986; Mortimore and Poso, 1987).
During microautophagy cytoplasmic materials are translocated into the lysosome or vacuole for degradation by direct invagination, protrusion, or septation of the lysosomal or vacuolar membrane, as depicted in Figure 7 (b) (Mijaljica et al., 2011).
By contrast, CMA translocates unfolded, soluble proteins directly across the limiting membrane of the lysosome (see Figure 7 (c)). CMA is activated as part of the cellular response to oxidative stress to target oxidized proteins to lysosomes without perturbing neighbouring unaffected proteins. Also, during prolonged starvation, the selectivity of CMA provides cells amino acids through selective degradation of expendable proteins (Kiffin et al., 2004). Through these diverging mechanisms cells ensure quality control, development and survival under nutrient deprivation and maintain cellular homeostasis by eliminating unnecessary proteins or damaged organelles (Cuervo, 2004; Klionsky, 2005; Li et al., 2011;
Mizushima et al., 2008).
Autophagy may be triggered under physiological conditions, such as nutrient starvation, or in response to a variety of stress stimuli, such as exposure to radiations or cytotoxic compounds. Moreover, autophagic processes have been described to play an important role in energetic balance, in cellular and tissue remodeling, aging and in the cellular defense against extracellular insults and pathogens.
Basal autophagy also plays a key role in eliminating defective organelles or aggregated proteins that may be resistant to the ubiquitin-proteasome degradation pathway and thereby sustain the cellular homeostasis (Komatsu et al., 2006; Ravikumar et al., 2002).
Although autophagy is basically a protective mechanism that sustains cell survival under adverse conditions, it has been demonstrated that the induction of autophagic process can contribute to a wide range of diseases, including cancer, neurodegeneration and microbial infection (Kundu and Thompson, 2008; Lee, 2009; Martinet et al., 2009). Therefore, as an intracellular self-destructive system, autophagy must be tightly regulated in order to adapt to different intracellular and extracellular stresses (see Figure 8).
42
Figure 8: Macroautophagy is extensively involved in cellular homeostasis
The morphological features of macroautophagy are depicted schematically. The initial sequestering compartment, the phagophore, expands into the double-membrane autophagosome. Fusion with an endosome generates the single-membrane amphisome, which subsequently fuses with a lysosome. The degraded cargo is released back into the cytosol through permeases. Some of the physiological connections between macroautophagy and human health and disease are indicated by the surrounding terms. Figure taken from (Klionsky, 2010).
Currently, there are over 34 proteins known which are required for autophagy. They were first identified in yeast and called autophagy related (Atg) proteins (Yang and Klionsky, 2010) and almost all of them are involved in the induction of autophagy, autophagosome nucleation, vesicle expansion and completion, and final retrieval of Atg proteins from mature autophagosomes.
One key Atg-containing complex is the autophagy-specific class III phosphatidylinositol 3-kinase (PI3P-3-kinase), also known as Vps34, which produces PI3P at the site of autophagosome formation (Simonsen and Tooze, 2009).
In mammals, activation of the membrane anchored kinase Vps34 is dependent on the formation of a multi-protein complex that consists of Beclin1 (Becn1), UV irradiation resistance-associated tumour suppressor gene (UVRAG), Endophilin B1 (Bif-1), activating molecule in Beclin1-regulated autophagy1 (Ambra1), and a myristylated serine kinase Vps15 (Fimia et al., 2007; Takahashi et al., 2007). The evolutionarily conserved Becn1 (the mammalian ortholog of Atg6), originally discovered as a Bcl-2-interacting protein (Liang et al., 1998) represents the first human protein shown to be indispensable for autophagy and exist in several complexes involved in autophagosome formation and maturation (Cao and Klionsky, 2007; Liang et al., 1999).
Introduction
43 It interacts with proteins that positively regulate autophagy, such as Atg14-like (Atg14L), UVRAG, Bif-1, run domain and cysteine-rich domain containing, Becn1-interacting protein (Rubicon), Ambra1, neuronal PDZ domain protein interacting specifically with TC10 (nPIST), (Orr, 2002), vacuole membrane protein 1 (VMP1) (Vaccaro et al., 2008), signaling lymphocyte-activation molecule (SAM) (Berger et al., 2010), inositol-1,4,5 trisphosphate receptor (IP(3)R) (Criollo et al., 2007), PTEN-induced kinase I (PINK-1) and survivin (Roca et al., 2008), to regulate the lipid kinase Vps-34 protein and promote formation of
43 It interacts with proteins that positively regulate autophagy, such as Atg14-like (Atg14L), UVRAG, Bif-1, run domain and cysteine-rich domain containing, Becn1-interacting protein (Rubicon), Ambra1, neuronal PDZ domain protein interacting specifically with TC10 (nPIST), (Orr, 2002), vacuole membrane protein 1 (VMP1) (Vaccaro et al., 2008), signaling lymphocyte-activation molecule (SAM) (Berger et al., 2010), inositol-1,4,5 trisphosphate receptor (IP(3)R) (Criollo et al., 2007), PTEN-induced kinase I (PINK-1) and survivin (Roca et al., 2008), to regulate the lipid kinase Vps-34 protein and promote formation of