Computational Study on Apoptosis-Inducing Factor (AIF)-Mediated Modulation of Menadione using Molecular Docking and Parametrized Quantum Methods

Scythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe(-/-) cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe(-/-) cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF.

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.

Purpose of the study. To investigate the level of protein AIF in the mitochondria of tumor cells and visually unchanged tissues of the colon in male and female patients with colorectal cancer.Materials and methods. The study included results, obtained from 132 patients with stage T2–3N0M0 colon cancer, comprising 52 women and 80 men. Mitochondria were isolated from human colon and tumor tissue cells using differential centrifugation in a high-speed refrigerated centrifuge. The concentration of protein AIF (pg/mg protein) in mitochondria was determined using ELISA «Human AIF Elisa Kit» (Cloud-CloneCorp., China).Results. It was established that in males, the AIF level in the mitochondria of rectal, sigmoid colon and ascending colon tumor cells was 2.4 times, 1.9 times (p < 0.05) and 3.1 times higher, respectively, than in the mitochondria of the corresponding tissues not affected by the tumor. In the mitochondria of the intestinal tissue not affected by the tumor, significant differences in the AIF content were observed, with levels varying depending on the anatomical location. In the sigmoid colon, the level of this factor was found to be 1.9 (p < 0.05) and 2.6 times higher than in the rectum and ascending colon, respectively. Concurrently, no notable discrepancies in the AIF concentration within the mitochondria of conditionally unimpaired tissues were observed in the female subjects. The AIF content was found to be higher in the mitochondria of tumor cells in women than in conditionally intact tissues. Specifically, it was observed to be 2.1 times higher in rectal tumors, 4.4 times higher in sigmoid colon tumors and 1.7 times (p < 0.05) higher in ascending colon tumors. Significant discrepancies in the AIF content between men and women, as well as between the mitochondria of tumor sample cells, were identified. In the rectal and ascending colon tumor, the AIF level in women was found to be markedly elevated in comparison to men, exhibiting a ratio of 1.3 (p < 0.05) and 2.4, respectively.Conclusion. In patients with colorectal cancer, the content of AIF in tumor mitochondria is observed to increase. This can be considered to represent stimulation mechanism of tumor proliferative activity due to its NADH/NADPH oxidase function, which promotes the survival of malignant cells.

Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

Overexpression of Apoptosis-Inducing Factor (AIF) and Pro-Survival Function in AML.

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Apoptosis-inducing factor (AIF) is a caspase-independent death effector. Normally residing in the mitochondrial intermembrane space, AIF is released and translocated to the nucleus in response to proapoptotic stimuli. Nuclear AIF binds to DNA and induces chromatin condensation and DNA fragmentation, characteristics of apoptosis. Until now, it remained to be clarified how the mitochondrial-nuclear translocation of AIF is regulated. Here we report that steroid receptor coactivator-interacting protein (SIP) interacts directly with AIF in mitochondria and specifically inhibits caspase-independent and AIF-dependent apoptosis. Challenging cells with apoptotic stimuli leads to rapid degradation of SIP, and subsequently AIF is liberated from mitochondria and translocated to the nucleus to induce apoptosis. Together, our data demonstrate that SIP is a novel regulator in caspase-independent and AIF-mediated apoptosis.

Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing

Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide-containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space whose specific enzymatic activity remains unknown. Upon an apoptotic insult, AIF undergoes proteolysis and translocates to the nucleus, where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner. Besides playing a key role in execution of caspase-independent cell death, AIF has emerged as a protein critical for cell survival. Analysis of in vivo phenotypes associated with AIF deficiency and defects, and identification of its mitochondrial, cytoplasmic, and nuclear partners revealed the complexity and multilevel regulation of AIF-mediated signal transduction and suggested an important role of AIF in the maintenance of mitochondrial morphology and energy metabolism. The redox activity of AIF is essential for optimal oxidative phosphorylation. Additionally, the protein is proposed to regulate the respiratory chain indirectly, through assembly and/or stabilization of complexes I and III. This review discusses accumulated data with respect to the AIF structure and outlines evidence that supports the prevalent mechanistic view on the apoptogenic actions of the flavoprotein, as well as the emerging concept of AIF as a redox sensor capable of linking NAD(H)-dependent metabolic pathways to apoptosis.

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.

Causal Role of Apoptosis-Inducing Factor for Neuronal Cell Death Following Traumatic Brain Injury

Inhibition of Nuclear Translocation of Apoptosis-Inducing Factor Is an Essential Mechanism of the Neuroprotective Activity of Pigment Epithelium-Derived Factor in a Rat Model of Retinal Degeneration

Exploration of binding mechanism of triclosan towards cancer markers using molecular docking and molecular dynamics

Dysregulation of apoptosis in diffuse B-large cell lymphoma (DLBCL) contributes to increased tumor proliferation, as well as the formation of resistance to treatment. One of the most relevant areas of molecular research is the study of the mechanisms of programmed cell death - apoptosis. The AIF (Apoptosis Inducing Factor) protein participates in the regulation of the caspase-independent pathway of cell death and is a potential predictor of the course of DLBCL. To evaluate the prognostic value of AIF expression in DLBCL. A retrospective study included 100 patients with newly diagnosed DLBCL. All patients received standard 1st line therapy according to the R-CHOP scheme. Using histological, immunohistochemical studies of lymph nodes and/or other organs involved in the pathological process were performed. A morphometric calculation of the data was carried out with the determination of the percentage of AIF-expressing tumor cells. The threshold level of expression of these proteins was calculated using by the ROC analysis (57%). Based on the threshold, patients were divided into groups with high (above threshold) and low (subthreshold) protein levels. The calculation of overall (OS) and progression-free (PFS) survival was performed using the Kaplan-Meier method with graphical construction of the corresponding curves, the levels of factors were compared using the log-rank test criterion. The independent influence of factors on the course of DLBCL was determined using multivariate Cox regression analysis. The subthreshold level of AIF was associated with low rates of OS and PFS of patients (34.9 and 48.8%, respectively). As a result of multivariate Cox analysis, it was found that the AIF protein is an independent prognostic marker for the course of DLBCL. The subthreshold value of AIF expression is associated with low OS and PFS in patients.

AIF, reactive oxygen species, and neurodegeneration: A “complex” problem