Mathematical modeling of the regulation of caspase-3 activation and degradation

Caspases are thought to be important players in the execution process of apoptosis. Inhibitors of apoptosis (IAPs) are able to block caspases and therefore apoptosis. The fact that a subgroup of the IAP family inhibits active caspases implies that not each caspase activation necessarily leads to apoptosis. In such a scenario, however, processed and enzymically active caspases should somehow be removed. Indeed, IAP-caspase complexes covalently bind ubiquitin, resulting in degradation by the 26S proteasome. Following release from mitochondria, IAP antagonists (e.g. second mitochondrial activator of caspases (Smac)) inactivate IAPs. Moreover, although pro-apoptotic factors such as irradiation or anti-cancer drugs may release Smac from mitochondria in tumor cells, high cytoplasmic survivin and ML-IAP levels might be able to neutralize it and, consequently, IAPs would further be able to bind activated caspases. Here, we propose a simple mathematical model, describing the molecular interactions between Smac deactivators, Smac, IAPs, and caspase-3, including the requirements for both induction and prevention of apoptosis, respectively. In addition, we predict a novel mechanism of caspase-3 degradation that might be particularly relevant in long-living cells.     

Mammalian mitochondrial IAP binding proteins

Four mitochondrial proteins have been identified that immunoprecipitate with the mammalian inhibitor of apoptosis (IAP) protein XIAP. Each of them interacts via a processed amino terminus that resembles those of the insect pro-apoptotic IAP binding proteins Grim, HID, Reaper, and Sickle. Two, Diablo/Smac and HrtA2/Omi, have been extensively characterized. Both Diablo and HtrA2 can bind to IAPs and promote apoptosis when over-expressed in transfected cells, but unlike the insect IAP antagonists, to date there is scant evidence that they are important regulators of apoptosis in more physiological circumstances.     

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization

The X-linked inhibitor of apoptosis protein (XIAP) is a potent cellular inhibitor of apoptosis. Designing small-molecule inhibitors that target the BIR3 domain of XIAP, where Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI) and caspase-9 bind, is a promising strategy for inhibiting the antiapoptotic activity of XIAP and for overcoming apoptosis resistance of cancer cells mediated by XIAP. Herein, we report the development of a homogeneous high-throughput assay based on fluorescence polarization for measuring the binding affinities of small-molecule inhibitors to the BIR3 domain of XIAP. Among four fluorescent probes tested, a mutated N-terminal Smac peptide (AbuRPFK-(5-Fam)-NH(2)) showed the highest affinity (Kd =17.92 nM) and a large dynamic range (deltamP = 231 +/- 0.9), and was selected as the most suitable probe for the binding assay. The binding conditions (DMSO tolerance and stability) have been investigated. Under optimized conditions, a Z' factor of 0.88 was achieved in a 96-well format for high-throughput screening. It was found that the popular Cheng-Prusoff equation is invalid for the calculation of the competitive inhibition constants (Ki values) for inhibitors in the FP-based competitive binding assay conditions, and accordingly, a new mathematical equation was developed, validated, and used to compute the Ki values. An associated Web-based computer program was also developed for this task. Several known Smac peptides with high and low affinities have been evaluated under the assay conditions and the results obtained indicated that the FP-based competitive binding assay performs correctly as designed: it can quantitatively and accurately determine the binding affinities of Smac-based peptide inhibitors with a wide range of affinities, and is suitable for high-throughput screening of inhibitors binding to the XIAP BIR3 domain.     

Differentiation-induced HL-60 cell apoptosis: A mechanism independent of mitochondrial disruption?

HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.     

SPION-Smac mimetic nano-conjugates: Putative pro-apoptotic agents in oncology

Non-covalent (NP-1/3) and covalent (NP-A-1/3) pro-apoptotic SPION-Smac mimetic nano-conjugates antitumor agents are reported. The solution synthesis of key Smac mimetics, their support onto SPIONs through non-covalent adsorption (NP-1/3) or APTES-mediated covalent binding (NP-A-1/3), the analytical characterization of SPION-Smac mimetic conjugates, their target affinity in cell-free assays, and their cytotoxicity against tumor cells are thoroughly described.     

Smac/DIABLO在过氧化氢所致C2C12肌原细胞凋亡中的作用

为探讨Smac/DIABLO在过氧化氢(H₂O₂）所致C₂C₁₂肌原细胞凋亡中的作用,采用Hoechst 33258染色,观察H₂O₂ (0.5 mmol/L)处理C₂C₁₂肌原细胞不同时间后,细胞核形态学改变并计算凋亡核百分率,DNA抽提及琼脂糖电泳观察凋亡特征性梯状带,利用细胞成分分离后蛋白质印迹分析H₂O₂是否导致Smac/DIABLO从线粒体释放,采用Caspase检测试剂盒及蛋白质印迹分析Caspase-3和Caspase-9的活化,转染Smac/DIABLO基因,观察Smac/DIABLO过表达对H₂O₂所致的C₂C₁₂肌原细胞凋亡的影响.结果表明:H₂O₂处理1 h后,Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放入胞浆,2 h更明显;H₂O₂处理4 h后,Caspase-3和Caspase-9活化,12 h达高峰;H₂O₂处理24 h后,C₂C₁₂肌原细胞显示特征性的凋亡形态改变,凋亡核百分率明显升高,DNA电泳出现明显“梯状”条带.与单纯过氧化氢损伤组相比,Smac/DIABLO高表达的C₂C₁₂肌原细胞经过氧化氢损伤组的Caspase-3和Caspase-9的活化、凋亡核百分率的升高、“梯状”条带的出现均更明显.结果表明,H₂O₂可导致Smac/DIABLO从C₂C₁₂肌原细胞线粒体释放,促进Caspase-9和Caspase-3的活化而促进细胞凋亡的发生.