Release Of Pro-apoptotic Proteins Research Articles

Identification of Novel Potent Inhibitors Against Bcl-xL Anti-apoptotic Protein Using Docking Studies

Bcl-xL protein belongs to BCL-2 family which has either pro- or anti-apoptotic activities owing to their importance in the regulation of apoptosis, tumor genesis and cellular responses to anti-cancer therapy. Bcl-xL permeabilize the outer mitochondrial membrane of cells and inhibit these processes. Protein-inhibitor interactions play an important role in regulating the expression of Bcl-xL protein. Here, we report the docking studies that resulted in the identification of new inhibitors distinct from the previously reported inhibitor against this protein. The results have been validated using Sybyl surflux docking. New potent inhibitors from docking analysis are pentacyclic triterpenoid derivative (2S,4aR,6aR, 6bS,8aS,10R,12R,12aS,12bR,14bR,E)-10,12-dihydroxy-2,4a,14b-trimethyl-9-((((R)-3,4,5-trihydroxy-6-methyl-2H-pyran- 2-yl)oxy)methylene)-1,2,3,4,4a,5,6,6a,6b,8a,9,10,11,12,12a,12b,13,14b-octadecahydropicene-2-car-boxylic acid and 4- alkyl-4-methoxypiperidine derivative 8h (where R= 4-Cl-Ph) that promotes the release of pro-apoptotic proteins from the mitochondria which is a key event in cell death signaling. The compounds form stable complex with protein exhibiting highest binding affinity and Gibbs free energy. Pentacyclic triterpenoid derivatives compound-201 and piperidine derivative compound-39 are potent inhibitors with Ki value of 172.62nM and 175.24 nM high affinity and inhibitory potency. Salt bridge, pi-pi and hydrogen bonding interactions predominantly contribute towards the stability of the complexes. These compounds can further be exploited for their potential to enhance apoptosis. We have established the correlation between the experimental Ki value with our computational inhibition constant. The quantitative predictions in this study provide a scope for further experimental testing giving structural insights into the design and development of novel anticancer drugs.

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentrations, caused a dose-dependent loss of viability of hepatocytes. The death was apoptotic at moderate and necrotic at high concentrations of acrolein. Acrolein exposure rapidly and dramatically decreased intracellular glutathione and overall antioxidant capacity, and activated the stress-signaling MAP-kinases JNK, p42/44 and p38. Our data demonstrate for the first time in human hepatocytes, that acrolein triggered endoplasmic reticulum (ER) stress and activated eIF2α, ATF-3 and -4, and Gadd153/CHOP, resulting in cell death. Notably, the protective/adaptive component of ER stress was not activated, and acrolein failed to up-regulate the protective ER-chaperones, GRP78 and GRP94. Additionally, exposure to acrolein disrupted mitochondrial integrity/function, and led to the release of pro-apoptotic proteins and ATP depletion. Acrolein-induced cell death was attenuated by N-acetyl cysteine, phenyl-butyric acid, and caspase and JNK inhibitors. Our data demonstrate that exposure to acrolein induces a variety of stress responses in hepatocytes, including GSH depletion, oxidative stress, mitochondrial dysfunction and ER stress (without ER-protective responses) which together contribute to acrolein toxicity. Our study defines basic mechanisms underlying liver injury caused by reactive aldehyde pollutants such as acrolein.

Neuroprotective effect of Cyclosporin A on the development of early brain injury in a subarachnoid hemorrhage model: A pilot study

Cyclosporin A (CsA) has been demonstrated to be neuroprotective in ischemic and traumatic brain injuries by inhibiting mitochondrial permeability transition pore (mPTP) opening, thereby maintaining mitochondrial homeostasis and inhibiting pro-apoptotic protein release. The effects of CsA on early brain injury (EBI) after subarachnoid hemorrhage (SAH), however, have not been investigated. This study was designed to explore the effects of CsA on apoptotic signaling pathways and EBI after experimental SAH using four equal groups (n=36) of adult male SD rats, including the sham group, SAH+vehicle group, SAH+CsA2 group, and SAH+CsA10 group. The rat SAH model was induced by injection of 0.3ml non-heparinized arterial blood into the prechiasmatic cistern. In the SAH+CsA2 and SAH+CsA10 groups, a dose of 2mg/kg and 10mg/kg CsA was directly administered by intercarotid injection at 15min and again 24h after SAH induction. Cerebral tissue samples were extracted 48h after SAH. Increased expressions of Cytochrome C, apoptosis-inducing factor (AIF), and cleaved caspase-3 were observed in the cerebral cortex after SAH. Treatment with high dose (10mg/kg) CsA markedly decreased expressions of Cytochrome C, AIF, and cleaved caspase-3, and inhibited apoptosis pathways. Administration of CsA following SAH significantly ameliorated EBI, including cortical apoptosis, brain edema, blood-brain barrier (BBB) impairment, and neurobehavioral deficits. These findings suggest that early administration of CsA may ameliorate EBI and provide neuroprotection in the SAH model through potential mechanisms that include blockage of mPTP opening and inhibition of apoptotic cell death pathways.

Exercise Protects Cardiac Mitochondria against Ischemia–Reperfusion Injury

Three to five consecutive days of endurance exercise can protect the heart against an ischemia-reperfusion (IR) insult. However, the mechanisms responsible for this exercise-mediated cardioprotection remain unknown. Given the important role that mitochondria play in IR-induced cardiac myocyte injury, we hypothesized that exercise training promotes cardioprotection, at least in part, by increasing mitochondrial antioxidants, preventing mitochondrial release of reactive oxygen species, and protecting cardiac mitochondria against IR-induced oxidative damage and functional impairment. To test our hypothesis, Sprague-Dawley rats were assigned to either sedentary (n = 16) or exercise-trained (n = 16) groups. Exercise-trained animals performed 5 d of treadmill running for 60 min·d(-1) at 30 m·s(-1). Hearts were excised from sedentary and exercised-trained animals and were either perfused for 80 min or exposed to 40 min of global ischemia followed by 45 min of reperfusion by using an ex vivo isolated working heart model. After the protocol, cardiac subsarcolemmal and intermyofibrillar mitochondria were isolated and used to determine respiratory control ratio, reactive oxygen species emission, and indices of oxidative stress and apoptosis. Our results support our hypothesis because exercise training protected both cardiac subsarcolemmal and intermyofibrillar mitochondria from IR-induced uncoupling and oxidative damage. Specifically, the levels of cardiac mitochondrial 4-hydroxynonenal-conjugated proteins were elevated in hearts from sedentary animals exposed to IR compared with cardiac mitochondria isolated from exercise-trained animals. Exercise also resulted in an increase in mitochondrial antioxidant enzymes (copper-zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase) and prevented the IR-induced release of proapoptotic proteins from the mitochondria. Collectively, these novel findings reveal that exercise-induced cardioprotection is mediated, at least in part, through mitochondrial adaptations resulting in a mitochondrial phenotype that resists IR-induced damage.

Sensitivity of skeletal muscle to pro-apoptotic factors

In mononuclear cells, apoptosis leads to DNA fragmentation and cell destruction, regardless of the activated pathway. As regards multinuclear cells, e.g. skeletal muscle fibers, apoptosis rarely induces the death of the entire cell, and it generally affects single nuclei. This process, referred to as nuclear apoptosis, has a negative effect on the expression of genes in the myonuclear domain. Apoptosis may be initiated in muscle cells by external stimuli which activate cell membrane death receptors as well as by internal stimuli which stimulate the mitochondrial release of pro-apoptotic proteins. Reactive oxygen species also play an important role in the initiation of apoptosis. In muscle cells, ROS are produced in response to extracellular reactions or by cell mitochondria. It is, therefore, believed that mitochondria play a central role in apoptosis within skeletal muscle. Skeletal muscles have a well-developed system that protects them against oxidative damage. Myogenic stem cells are an integral part of multinucleated myofibers, and they are critically important for the maintenance of normal muscle mass, muscle growth, regeneration and hypertrophy. The latest research results indicate that myogenic cells are more sensitive to oxidative stress and pro-apoptotic factors than well-differentiated cells, such as myotubes. The complex structure and activity of skeletal muscle prompted research into the role of apoptosis and its intensity under various physiological and pathological conditions. This review summarizes the results of research investigating control mechanisms and the apoptosis process in skeletal muscle fibers, and indicates unresearched areas where further work is required.

Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca2+, and ROS

Mitochondrial permeability transition (MPT) and dysfunctions play a pivotal role in many patho-physiological and toxicological conditions. The interplay of mitochondrial thiol (mtThiol), MPT, Ca(2+) homeostasis, and resulting dysfunctions still remains controversial despite studies by several research groups. Present study was undertaken to ascertain the correlation between Ca(2+) homeostasis, mtThiol alteration and reactive oxygen species (ROS) in causing MPT leading to mitochondrial dysfunction. mtThiol depletion significantly enhanced Ca(2+) dependent MPT (swelling) and depolarization of mitochondria resulting in release of pro-apoptotic proteins like Cyt c, AIF, and EndoG. mtThiol alteration and Ca(2+) overload caused reduced mitochondrial electron flow, oxidation of pyridine nucleotides (NAD(P)H) and significantly enhanced ROS generation (DHE and DCFH-DA fluorescence). Studies with MPT inhibitor (Cyclosporin A), Ca(2+) uniport blocker (ruthenium red) and Ca(2+) chelator (BAPTA) indicated that mitochondrial dysfunction was more pronounced under dual stress of altered mtThiol and Ca(2+) overload in comparison with single stress of excessive Ca(2+). Transmission electron microscopy confirmed the changes in mitochondrial integrity under stress. Our findings suggest that the Ca(2+) overload itself is not solely responsible for structural and functional impairment of mitochondria. A multi-factorial cross-talk between mtThiol, Ca(2+) and ROS is responsible for mitochondrial dysfunction. Furthermore, minor depletion of mtThiol was found to be an important factor along with Ca(2+) overload in triggering MPT in isolated mitochondria, tilting the balance towards disturbed functionality.

Proapoptotic Agents in Lung Cancer

The session was introduced by Dr. Roy Herbst who defined apoptosis as a physiological process of programmed cell suicide, which eliminates cells that are no longer needed or have become irreparably damaged. Apoptosis is important in embryonic and perinatal development. In the adult-tissue homeostasis, apoptosis helps to replace ineffective or potentially damaging cells. It is distinct from other types of death such as necrosis and autophagy. Apoptosis is defined by specific morphologic and biochemical criteria and mediated by specialized proteases—caspases. Two key apoptosis signaling pathways are involved: the extrinsic pathway engages binding of a proapoptotic ligand to its “death” receptor (DR) and the activation of the caspase cascade. The ligand-independent intrinsic pathway is triggered by the tumor suppressor gene product P53 that acts through the release of proapoptotic proteins that activate caspase enzymes from the mitochondria. Apoptosis is dysregulated in cancer. Some cancer cells acquire genetic changes (e.g., p53, BclII, and inhibitors of apoptosis [IAP]) that circumvent apoptosis, permitting continued, unchecked proliferation. Strategies to reactivate apoptosis selectively in tumor cells have exciting potential for cancer therapy. Current efforts are focused on proapoptotic receptor agonists and on antagonists of apoptosis suppressors (e.g., inhibitors of apoptosis/IAP, survivin, and Bcl-2). SUMMARY

Antimycin A-induced mitochondrial apoptotic cascade is mitigated by phenolic constituents of Phyllanthus amarus aqueous extract in Hep3B cells

Antimycin A (AMA) treatment of cells blocks mitochondrial electron transport chain, and leads to elevated ROS generation, thereby causing damage to mtDNA, proteins and lipids, along with mitochondrial membrane depolarization, release of pro-apoptotic proteins into the cytoplasm, and induction of apoptosis. Prevention of such oxidative cellular damage by the aqueous extract of Phyllanthus amarus has been investigated in this study. The extract demonstrated significant potential in mitigating H 2O 2-induced membrane damage along with considerable recession in AMA-governed mitochondrial protein and lipid degradation in Hep3B cells. 8-OHdG analysis of mtDNA damage revealed substantial protective potential of the extract against mtDNA damage. SQ-PCR of selected mtDNA sequences confirmed the potential of the extract to alleviate levels of mtDNA damage. FACS analysis with JC-1 fluorescent dye established significant escalation of mitochondrial membrane potential by the extract in AMA-treated cells. Extract treatment resulted in a distinct decline in the degrees of AMA-induced release of cytochrome c and AIF into the cytoplasm along with consequent pacification of apoptosis. All protective efficiencies of the extract reported in this study were found to hold strong and significant ( P < 0.05) positive correlation to its total phenolic contents, thereby proving that polyphenolic constituents of P. amarus aqueous extract mitigate oxidative stress-induced cellular degeneration and aging.

Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy

Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments—air, vacuum or aqueous conditions. In addition, AFM force–distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force–distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria.

SOD1 and MitoTEMPO partially prevent mitochondrial permeability transition pore opening, necrosis, and mitochondrial apoptosis after ATP depletion recovery

Generation of excessive reactive oxygen species (ROS) leads to mitochondrial dysfunction, apoptosis, and necrosis in renal ischemia–reperfusion (IR) injury. Previously we showed that lentiviral vector-mediated overexpression of superoxide dismutase-1 (SOD1) in proximal tubular epithelial cells (LLC-PK 1) reduced cytotoxicity in an in vitro model of IR injury. Here, we examined the effects of SOD1 overexpression on mitochondrial signaling after ATP depletion–recovery (ATP-DR). To examine the role of mitochondrial ROS, a subset of cells was treated with the mitochondrial antioxidant MitoTEMPO. ATP-DR-mediated increase in mitochondrial calcium, loss of mitochondrial membrane potential, and increase in mitochondrial permeability transition pore (MPTP) were attenuated by SOD1 and MitoTEMPO ( P < 0.01). SOD1 prevented ATP-DR-induced mitochondrial Bax translocation, although the release of proapoptotic proteins from mitochondria was not prevented by SOD1 alone and required the presence of both SOD1 and MitoTEMPO. SOD1 suppressed the increase in c-jun phosphorylation, suggesting that JNK signaling regulates Bax translocation to mitochondria via ROS. ATP-DR-mediated changes in MPTP and mitochondrial signaling increased necrosis and apoptosis, both of which were partially attenuated by SOD1 and MitoTEMPO. These studies show that SOD1 and MitoTEMPO preserve mitochondrial integrity and attenuate ATP-DR-mediated necrosis and apoptosis.

Neuroprotective Effect of Bax-Inhibiting Peptide on Neonatal Brain Injury

Mitochondria play a critical role in mediating cell death in both the adult and immature brain. The cyclophilin D mitochondrial membrane permeability transition pore is critical in adult ischemia, whereas in neonatal hypoxic-ischemic (HI) brain injury, mitochondrial permeabilization appears to be primarily Bax-dependent. The aim of this study was to evaluate the neuroprotective effect of a cell-penetrating Bax-inhibiting peptide (BIP) on neonatal mouse HI brain injury. BIP (5 microL, 5 mg/mL) or a BIP-negative control (5 microL, 5 mg/mL) was injected intracerebroventricularly immediately before HI in postnatal day 9 mice. Mice were euthanized at different time points after HI for evaluation of brain injury, Bax activation, release of proapoptotic proteins, and caspase activation. The trace fear conditioning and cylinder tests were performed for evaluation of the functional recovery after BIP treatment. At 5 days after HI, there was a 41.2% reduction of brain injury in BIP-treated mice compared with BIP-negative control treated animals. Myelin basic protein and neurofilament quantification revealed that BIP reduced white matter injury. BIP treatment conferred improvement in both sensorimotor and memory functions at 7 weeks after HI. BIP protection was associated with a reduction of Bax activation, mitochondrial permeabilization, and downstream caspase activation. Bax inhibition provides neuroprotection and functional improvement in a neonatal mouse model of HI.

Targeting mitochondria for cancer therapy

Mitochondria are the cells' powerhouse, but also their suicidal weapon store. Dozens of lethal signal transduction pathways converge on mitochondria to cause the permeabilization of the mitochondrial outer membrane, leading to the cytosolic release of pro-apoptotic proteins and to the impairment of the bioenergetic functions of mitochondria. The mitochondrial metabolism of cancer cells is deregulated owing to the use of glycolytic intermediates, which are normally destined for oxidative phosphorylation, in anabolic reactions. Activation of the cell death machinery in cancer cells by inhibiting tumour-specific alterations of the mitochondrial metabolism or by stimulating mitochondrial membrane permeabilization could therefore be promising therapeutic approaches.

Mitochondria as decision‐makers in cell death

Mitochondria play an essential role in both cell health and death. Increasing experimental evidence suggests that mitochondria are involved in active control of cell death processes at several levels including (1) mitochondrial membrane permeabilization and release of proapoptotic proteins, (2) post-cytochrome c regulation of caspase activation, and (3) supply of energy for execution of death program. The purpose of this review is to discuss the main mechanisms by which alterations in mitochondrial outer membrane permit the translocation of proapoptotic proteins into cytosol, how mitochondria "make decisions" on the mode of cell death, and how they regulate caspase activation by changing the redox state of cytosolic cytochrome c. The interventions into these processes may constitute an important strategy for the pharmacological prevention of unwanted cell death in various pathologies or, conversely, for facilitation of anticancer therapy.

Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo

Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.

Nα-Tosyl-l-phenylalanine Chloromethyl Ketone Induces Caspase-dependent Apoptosis in Transformed Human B Cell Lines with Transcriptional Down-regulation of Anti-apoptotic HS1-associated Protein X-1

N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.

Mitochondrial targeting of tBid/Bax: a role for the TOM complex?

The release of pro-apoptotic proteins from the mitochondria is a key event in cell death signaling that is regulated by Bcl-2 family proteins. For example, cleavage of the BH3-only protein, Bid, by multiple proteases leads to the formation of truncated Bid that, in turn, promotes the insertion/oligomerization of Bax into the mitochondrial outer membrane, resulting in pore formation and the release of proteins residing in the intermembrane space. Bax, a monomeric protein in the cytosol is targeted to the mitochondria by a yet unknown mechanism. Several proteins of the outer mitochondrial membrane have been proposed to act as receptors for Bax, among them the voltage-dependent anion channel, VDAC, and the mitochondrial protein translocase of the outer membrane, the TOM complex. Alternatively, the unique mitochondrial phospholipid, cardiolipin, has been ascribed a similar function. Here, we review recent work on the mechanisms of activation and the targeting of Bax to the mitochondria and discuss the advantages and limitations of the methods used to study this process.

Intracellular Aβ and C99 aggregates induce mitochondria-dependent cell death in human neuroglioma H4 cells through recruitment of the 20S proteasome subunits

Recent studies have reported that neuronal apoptosis is induced not only by extracellular Aβ but also by intracellular Aβ; however, the mechanism by which intracellular Aβ contributes to the regulation of cell death associated with the pathogenesis of AD remains to be elucidated. Using immunological assays and a short-lived enhanced green fluorescent protein (d2EGFP) system, we showed that intracellular Aβ and C99 form perinuclear aggregates in the cytosol, and the resulting aggregates attenuate the activity of the 26S proteasome. In addition, the immunofluorescence assays (IFA) revealed that the 20S proteasome α-subunits are recruited into perinuclear aggregates in both human embryonic kidney (HEK293) and human neuroglioma H4 (H4) cells. Interestingly, we observed an increase in the levels of Bax, cleavage of PARP-1, and mitochondrial release of proapoptotic proteins, such as cytochrome c and HtrA2, in H4 cells with intracellular Aβ or C99 aggregates, but not in HEK293 cells with those aggregates. The results of the present study indicate that intracellular Aβ and C99 aggregates induce mitochondria-dependent apoptotic cell death via elevation of Bax levels as a result of proteasome inhibition in a cell type-specific manner.

The Pirh2–keratin 8/18 interaction modulates the cellular distribution of mitochondria and UV-induced apoptosis

Intermediate filaments (IFs) provide crucial structural support in higher eukaryotic cells. Accumulating evidences show that IFs also participate in various cellular activities including stress responses, cell growth, cell death and cell migration through dynamic interactions with various non-structure proteins. Here we report the identification of Pirh2, a RING-H2-type ubiquitin E3 ligase, as a novel binding partner of the cytoplasmic IF proteins keratin 8/18 (K8/18). Phosphorylation of either Pirh2 or K8/18 affects their association. Although Pirh2 was not found to influence the stability of K8/18, it displayed an unexpected role in regulating the organization of the network of K8/18 keratin filaments. Disruption of Pirh2-K8/K18 interaction by either UV irradiation or knockdown with Pirh2 or K18 led to the aggregation of K8/18 keratin filaments. It further induced mitochondrial redistribution, and this process is likely through a microtubule-mediated pathway. The abnormal localization of mitochondria in Pirh2-knockdown cells may partially account for its increased cell sensitivity to UV-induced apoptosis, probably through enhancing the release of pro-apoptotic proteins, such as cytochrome c and Smac/DIABLO to the cytosol. Overall, our data reveal the novel role of the Pirh2-K8/18 complex in governing the distribution of mitochondria.

Caspase-8 Mediates Mitochondrial Release of Pro-apoptotic Proteins in a Manner Independent of Its Proteolytic Activity in Apoptosis Induced by the Protein Synthesis Inhibitor Acetoxycycloheximide in Human Leukemia Jurkat Cells

The cysteine protease caspase-8 plays an essential role in apoptosis induced by death receptors. The protein synthesis inhibitor acetoxycycloheximide (Ac-CHX) has been previously shown to induce rapid apoptosis mediated by the release of cytochrome c in human leukemia Jurkat cells. In this study, the novel molecular mechanism that links caspase-8 to the mitochondrial release of pro-apoptotic proteins has been identified. Jurkat cells deficient in caspase-8 were more resistant to Ac-CHX than wild-type Jurkat cells and manifested decreased apoptosis induction and caspase activation as well as inefficient release of cytochrome c, Smac/DIABLO, and AIF into the cytosol. In contrast to Fas ligand stimulation, the general caspase inhibitor barely prevented the mitochondrial release of these pro-apoptotic proteins in Ac-CHX-treated cells, suggesting that caspase-8 activity is dispensable for triggering the mitochondrial pathway in Ac-CHX-induced apoptosis. Consistent with this notion, caspase-8-deficient Jurkat cells reconstituted with catalytically inactive caspase-8 became sensitive to Ac-CHX and exhibited apoptosis, caspase activation, the liberation of pro-apoptotic proteins into the cytosol, and Bak conformational change as efficiently as wild-type Jurkat cells. Unlike caspase-3, -6, -7, and -9, a small but significant portion of caspase-8 was found to localize in mitochondria before and after exposure to Ac-CHX. These results clearly demonstrate that caspase-8 is able to mediate the mitochondrial release of pro-apoptotic proteins in a manner independent of its proteolytic activity in Ac-CHX-induced apoptosis.

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

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.