Cytochrome b5 reductases: Redox regulators of cell homeostasis.

The cytochrome-b5 reductase (CYB5R) family of flavoproteins is known to regulate reduction-oxidation (redox) balance in cells. The five enzyme members are highly compartmentalized at the subcellular level and function as "redox switches" enabling the reduction of several substrates, such as heme and coenzyme Q. Critical insight into the physiological and pathophysiological significance of CYB5R enzymes has been gleaned from several human genetic variants that cause congenital disease and a broad spectrum of chronic human diseases. Among the CYB5R genetic variants, CYB5R3 is well-characterized and deficiency in expression and activity is associated with type II methemoglobinemia, cancer, neurodegenerative disorders, diabetes, and cardiovascular disease. Importantly, pharmacological and genetic-based strategies are underway to target CYB5R3 to circumvent disease onset and mitigate severity. Despite our knowledge of CYB5R3 in human health and disease, the other reductases in the CYB5R family have been understudied, providing an opportunity to unravel critical function(s) for these enzymes in physiology and disease. In this review, we aim to provide the broad scientific community an up-to-date overview of the molecular, cellular, physiological, and pathophysiological roles of CYB5R proteins.

Augmenter of liver regeneration promotes mitochondrial biogenesis in renal ischemia-reperfusion injury.

Mitochondria are the center of energy metabolism in the cell and the preferential target of various toxicants and ischemic injury. Renal ischemia-reperfusion (I/R) injury triggers proximal tubule injury and the mitochondria are believed to be the primary subcellular target of I/R injury. The promotion of mitochondrial biogenesis (MB) is critical for the prevention I/R injury. The results of our previous study showed that augmenter of liver regeneration (ALR) has anti-apoptotic and anti-oxidant functions. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. To gain further insight into the role of ALR in MB, human kidney (HK)-2 cells were treated with lentiviruses carrying ALR short interfering RNA (siRNA) and a model of hypoxia reoxygenation (H/R) injury in vitro was created. We observed that knockdown of ALR promoted apoptosis of renal tubular cells and aggravated mitochondrial injury, as evidenced by the decrease in the mitochondrial respiratory proteins adenosine triphosphate (ATP) synthase subunit ß, cytochrome c oxidase subunit 1, and nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) beta subcomplex 8. Meanwhile, the production of reactive oxygen species was increased and ATP levels were decreased significantly in HK-2 cells, as compared with the siRNA/control group (p < 0.05). In addition, the mitochondrial DNA copy number and membrane potential were markedly decreased. Furthermore, critical transcriptional regulators of MB (i.e., peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, mitochondrial transcription factor A, sirtuin-1, and nuclear respiratory factor-1) were depleted in the siRNA/ALR group. Taken together, these findings unveil essential roles of ALR in the inhibition of renal tubular cell apoptosis and attenuation of mitochondrial dysfunction by promoting MB in AKI.

ERV1/ChemR23 Signaling Protects Against Atherosclerosis by Modifying Oxidized Low-Density Lipoprotein Uptake and Phagocytosis in Macrophages.

BACKGROUND: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein-coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. METHODS: Lipidomic plasma analysis were performed after EPA supplementation in Apoe-/- mice. Erv1/Chemr23-/- xApoe-/- mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. RESULTS: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe-/- mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1-mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. CONCLUSIONS: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Augmenter of liver regeneration potentiates doxorubicin anticancer efficacy by reducing the expression of ABCB1 and ABCG2 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is highly chemoresistant and therefore challenges both physicians and patients. Augmenter of liver regeneration (ALR), previously also known as 'hepatic stimulator substance', is reported to inhibit the epithelial-mesenchymal transition (EMT) in HCC, one of the frequent events that occur in cancer metastasis, suggesting that ALR is involved in HCC. In this study, we report for the first time that the transfection of ALR enhances the antitumor effect of chemotherapy with doxorubicin, a typical anticancer drug, on HCC in vitro and in vivo. The efflux of doxorubicin from ALR-transfected HCC cells is efficiently suppressed. This implies the intracellular retention of doxorubicin in tumor cells, which is at least partly attributable to the effective inhibition of ABCB1 and ABCG2 transporter expression in ALR-expressing cells. The downregulation of ALR expression by short hairpin RNA diminishes the antitumor effect of ALR. We further demonstrate that ALR inhibits the AKT/Snail signaling pathway, resulting in the downregulation of ABCB1 and ABCG2 expression. In conclusion, our results suggest that ALR is a potential chemotherapeutic agent against HCC.

Clinical Implications of Augmenter of Liver Regeneration in Cancer: A Systematic Review.

BACKGROUND/AIM: Hepatocellular carcinoma is a substantial healthcare burden with high prevalence and poor prognosis. As such, efforts are continually made to uncover molecules relevant in cancer biology, that are exploitable as targets for therapy. The mitochondrion is the powerhouse of the cell and exhibits altered functionality in the malignant state, including aberrant regulation of apoptosis and cellular respiration. Augmenter of liver regeneration (ALR) is a multifunctional mitochondrial protein that demonstrates anti-oxidative and anti-apoptotic properties and plays a key role in liver regeneration. MATERIALS AND METHODS: The present study systematically reviews the available literature on the role of ALR in cancer. RESULTS: Systematic search of PubMed resulted in 12 studies discussing ALR in multiple types of cancer. More specifically, ALR appears to be up-regulated in malignant cells and tissues. Furthermore, treatment of cells with exogenous ALR shows an anti-apoptotic effect while silencing or inhibiting ALR decreases cell and tumor survival. CONCLUSION: ALR clearly plays a role in cancer biology and demonstrates potential as a therapeutic target.

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection.

The recent discovery of small molecules targeting the cytochrome bc1 :aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1 :aa3 consists of a menaquinone:cytochrome c reductase (bc1 ) and a cytochrome aa3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1 :aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

Augmenter of liver regeneration (ALR) exhibits a dual signaling impact on hepatic acute-phase response.

The acute-phase response (APR) is an inflammatory process triggered mainly by IL-6 in response to neoplasm, tissue injury, infection or inflammation. Signaling of IL-6 is transduced by activating STAT3 which rapidly results in production of acute-phase proteins (APPs) such as fibrinogen ß (FGB) and haptoglobin (HP). Augmenter of liver regeneration (ALR), a hepatotrophic factor supporting liver regeneration, was reported to be upregulated after liver damage. In this study we analyzed the role of ALR for IL-6 signaling and APR. Thus, we investigated the expression and release of APPs in human liver cells under conditions of increased exogenous or endogenous ALR. HepG2 cells and ALR-reexpressing HepG2 cells were treated with IL-6 in the presence or absence of exogenous ALR for different time points. The mRNA expression and release of both FGB and HP were measured by RT-PCR and ELISA. We found that exogenously applied ALR attenuated the IL-6-induced mRNA expression and protein secretion of both FGB and HP. In contrast, IL-6 stimulation in HepG2 cells which re-express ALR, revealed elevated APR shown by increased mRNA expression and secretion of FGB and HP. Furthermore, we found that ALR-mediated regulation of IL-6-induced APP production is accompanied by altered STAT3 activity. While exogenous ALR reduced the IL-6-induced phosphorylation of STAT3, endogenous ALR enhanced STAT3 activity in liver cells. In conclusion, ALR, dependent on its localization, changes APR at least in part, by modifying STAT3 activation. This study shows a dual signaling of ALR and suggests that ALR is pivotal for the regulation of APR, a crucial event in liver injury and regeneration.

Hepatic expression of miR-122 and antioxidant genes in patients with chronic hepatitis B.

INTRODUCTION: The pathogenesis of chronic hepatitis B depends on both, the immune response and oxidative stress. AIM OF THE STUDY: To assess the hepatic expression of miR-122 and the antioxidant genes: HMOX-1, NQO1 and GFER1, in liver biopsy specimens obtained from patients with chronic hepatitis B, with regard to selected clinical and histological parameters, using RT-PCR. RESULTS: The study group comprised 34 HBV-infected patients. Statistically significant associations were found between lower hepatic expression of HMOX-1 and greater severity of liver inflammation (p=0.04). However, significantly higher expression of NQO1 was observed in patients with advanced liver fibrosis (p=0.035). Hepatic expression of miR-122 in HBV patients was not associated with viral load or liver injury. CONCLUSION: The hepatic expression of HMOX-1and NQO1 may be associated with liver injuries in chronic hepatitis B. However, hepatic expression of miR-122 does not seem to correspond to progression of the liver disease.

[Over-expression of augmenter of liver regeneration promotes proliferation and suppresses hydrogen peroxide-induced apoptosis in LO2 cells].

OBJECTIVE: To investigate the effects of over-expression of 23 kDa augmenter of liver regeneration (ALR) on cell proliferation and apoptosis in the normal human hepatic cell line LO2. METHODS: The recombinant plasmid expressing 23 kDa ALR (pcDNA6/23 kDa ALR) was constructed and transfected into LO2 cells with MegaTran 1.0 transfection reagent. The expressions of ALR mRNA and protein in LO2 cells were detected by real-time quantitative PCR and Western blotting, respectively; MTS assay was used to detect the cell proliferation of LO2 cells; cell cycle and apoptosis of LO2 cells were measured by flow cytometry. RESULTS: The recombinant expression plasmid pcDNA6/23 kDa ALR was constructed successfully, and the expression of the target protein 23 kDa ALR increased significantly in the transfected cells. Compared with pcDNA6-myc/HisA group, the transient transfection of pcDNA6/23 kDa ALR into LO2 cells promoted cell proliferation and inhibited cell apoptosis induced by H2O2, however, no significant differences were detected in G0 phase and S phase. CONCLUSION: The over-expression of 23 kDa ALR in LO2 cells promoted the cell proliferation and enhanced cell resistance to H2O2.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

AIM: To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). METHODS: Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. RESULTS: Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. CONCLUSION: ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

Augmenter of liver regeneration, a protective factor against ROS-induced oxidative damage in muscle tissue of mitochondrial myopathy affected patients.

Mitochondria-related myopathies (MM) are a group of different diseases defined by a varying degree of dysfunctions of the mitochondrial respiratory chain which leads to reactive oxygen species (ROS) generation followed by oxidative stress and cellular damage. In mitochondrial myopathy muscle tissue an overexpression of antioxidant enzymes has been documented probably as an attempt to counteract the free radical generation. We previously documented, in human non-pathological muscle fibres, the expression of the augmenter of liver regeneration (ALR), a sulfhydryl oxidase enzyme, whose presence is related to the mitochondria; indeed it has been demonstrated that ALR mainly localizes in the mitochondrial inter-membrane space. Furthermore we reported, in different experimental models, in vivo and in vitro, the anti-apoptotic and anti-oxidative capacities of ALR, achieved by up-regulating Bcl-2 anti-apoptotic family factors and the anti-apoptotic/anti-oxidative secretory isoform of clusterin (sClu). With the present study we aimed to determine ALR, Bcl-2 protein, clusterin and ROS expression in muscle tissue biopsies from MM-affected patients. Non-pathological muscle tissue was used as control. Enzymatic, histochemical, immunohistochemical and immune electron microscopy techniques were performed. The data obtained revealed in MM-derived muscle tissue, compared to non-pathological tissue, the over-expression of ROS, ALR and Bcl-2 and the induction of the nuclear, pro-apoptotic, isoform of clusterin (nCLU).

Biogenesis of yeast Mia40 - uncoupling folding from import and atypical recognition features.

The discovery of the mitochondrial intermembrane space assembly (MIA) pathway was followed by studies that focused mainly on the typical small substrates of this disulfide relay system and the interactions between its two central partners: the oxidoreductase Mia40 and the FAD-protein Erv1. Recent studies have revealed that more complex proteins utilize this pathway, including Mia40 itself. In the present study, we dissect the Mia40 biogenesis in distinct stages, supporting a kinetically coordinated sequence of events, starting with (a) import and insertion through the Tim23 translocon, followed by (b) folding of the core of imported Mia40 assisted by the endogenous Mia40 and (c) final interaction with Erv1. The interaction with endogenous Mia40 and the subsequent interaction with Erv1 represent kinetically distinguishable steps that rely on completely different determinants. Interaction with Mia40 proceeds very early (within 30 s) and is characterized by no Cys-specificity, an increased tolerance to mutations of the hydrophobic substrate-binding cleft and no apparent dependence on glutathione as a proofreading mechanism. All of these features illustrate a very atypical behaviour for the Mia40 precursor compared to other substrates of the MIA pathway. By contrast, interaction with Erv1 occurs after 5 min of import and relies on a more stringent specificity.

Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells.

Oxidation of cysteine residues to disulfides drives import of many proteins into the intermembrane space of mitochondria. Recent studies in yeast unraveled the basic principles of mitochondrial protein oxidation, but the kinetics under physiological conditions is unknown. We developed assays to follow protein oxidation in living mammalian cells, which reveal that import and oxidative folding of proteins are kinetically and functionally coupled and depend on the oxidoreductase Mia40, the sulfhydryl oxidase augmenter of liver regeneration (ALR), and the intracellular glutathione pool. Kinetics of substrate oxidation depends on the amount of Mia40 and requires tightly balanced amounts of ALR. Mia40-dependent import of Cox19 in human cells depends on the inner membrane potential. Our observations reveal considerable differences in the velocities of mitochondrial import pathways: whereas preproteins with bipartite targeting sequences are imported within seconds, substrates of Mia40 remain in the cytosol for several minutes and apparently escape premature degradation and oxidation.

A small molecule inhibitor of redox-regulated protein translocation into mitochondria.

The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.

(77)Se enrichment of proteins expands the biological NMR toolbox.

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable (77)Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate (77)Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, (77)Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.

Targeting and import mechanism of coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) into the mitochondrial intermembrane space.

Coiled-coil helix coiled-coil helix domain-containing protein 3 (ChChd3) is a mitochondrial inner membrane (IM) protein facing toward the intermembrane space (IMS). In the IMS, ChChd3 complexes with multiple proteins at the crista junctions and contact sites and plays a key role in maintaining crista integrity. ChChd3 is myristoylated at the N terminus and has a CHCH domain with twin CX(9)C motifs at its C terminus. The CHCH domain proteins are traditionally imported and trapped in the IMS by using a disulfide relay system mediated by Mia40 and Erv1. In this study, we systematically analyzed the role of the myristoylation and the CHCH domain in the import and mitochondrial localization of ChChd3. Based on our results, we predict that myristoylation promotes binding of ChChd3 to the outer membrane and that the CHCH domain translocates the protein across the outer membrane. By analysis of the CHCH domain cysteine mutants, we further show that they have distinct roles in binding to Mia40 in the IMS and proper folding of the protein. The transient disulfide-bonded intermediate with Mia40 is formed preferentially between the second cysteine in helix 1, Cys(193), and the active site cysteine in Mia40, Cys(55). Although each of the four cysteines is essential for folding of the protein and binding to mitofilin and Sam50, they are not involved in import. Together our results indicate that both the myristoylation and the CHCH domain are essential for the import and mitochondrial localization of ChChd3. Once imported, ChChd3 binds to Mia40 for further folding and assembly into macromolecular complexes.

Protein oxidative folding in the intermembrane mitochondrial space: more than protein trafficking.

The process of oxidative folding in the intermembrane mitochondrial space (IMS) is an exciting field of research because folding is simultaneously coupled to protein translocation and functional regulation. Contrary to the endoplasmatic reticulum ER where several chaperones of the disulfide isomerase family exist, oxidative folding in the IMS is exclusively catalyzed by the oxoreductase Mia40 that recognizes a group of proteins with characteristic cysteine motifs organized in twin CX(3)C, twin CX(9)C or CX(2)C motifs. In this review, we discuss the structural and biochemical studies leading to our current understanding of the Mia40 pathway as well as the open questions on the field. In fact, despite significant advances, several key points on the Mia40 pathway remain to clarify namely on the molecular mechanism trough which substrate oxidative folding is catalyzed. This issue is receiving increasing attention since failures in the import, sorting and folding of mitochondrial proteins is related to an increasing number of debilitating human disorders.

Decreased expression of the augmenter of liver regeneration results in increased apoptosis and oxidative damage in human-derived glioma cells.

The mammalian growth factor erv1-like (GFER) gene encodes a sulfhydryl oxidase enzyme, named Augmenter of Liver Regeneration (ALR). Recently it has been demonstrated that ALR supports cell proliferation acting as an anti-apoptotic factor. This effect is determined by ALR ability to support the anti-apoptotic gene expression and to preserve cellular normoxic conditions. We recently demonstrated that the addition of recombinant ALR (rALR) in the culture medium of H(2)O(2)-treated neuroblastoma cells reduces the lethal effects induced by the hydrogen peroxide. Similar data have been reported in the regenerating liver tissue from partially hepatectomized rats treated with rALR. The purpose of the present study was to evaluate the effect of the GFER inhibition, via the degradation of the complementary mRNA by the specific siRNA, on the behaviour of the apoptosis (apoptotic gene and caspase expression and apoptotic cell number) and of the oxidative stress-induced parameters (reactive oxygen species (ROS), clusterin expression and mitochondrial integrity) in T98G glioma cells. The results revealed a reduction of (i) ALR, (ii) clusterin and (iii) bcl-2 and an increase of (iv) caspase-9, activated caspase-3, ROS, apoptotic cell number and mitochondrial degeneration. These data confirm the anti-apoptotic role of ALR and its anti-oxidative properties, and shed some light on the molecular pathways through which ALR modulates its biological effects.

Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space.

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.