Apoptosis-inducing Protein Research Articles

Expanding the toolbox of exosome-based modulators of cell functions

Exosomes are cell-derived extracellular vesicles and play important roles in mediating intercellular communications. Due to their unique advantages in transporting a variety of biomolecules, exosomes have been emerging as a new class of nanocarriers with great potential for therapeutic applications. Despite advancements in loading chemotherapeutics and interfering RNAs into exosomes, active incorporation of protein molecules into exosomes remains challenging owing to their distinctive physicochemical properties and/or a lack of knowledge of cargo sorting during exosome biogenesis. Here we report the generation of a novel type of engineered exosomes with actively incorporated membrane proteins or soluble protein cargos, named genetically infused functionally tailored exosomes (GIFTed-Exos). Through genetic fusion with exosome-associated tetraspanin CD9, transmembrane protein CD70 and glucocorticoid‐induced tumor necrosis factor receptor family‐related ligand (GITRL) could be displayed on exosome surface, resulting in GIFTed-Exos with excellent T-cell co-stimulatory activities. By genetically linking to a CD9-photocleavable protein fusion, fluorescent protein mCherry, apoptosis-inducing protein apoptin, and antioxidant enzyme catalase could be effectively packed into exosomes for light-controlled release. The generated GIFTed-Exos display notable in vitro and in vivo activities for delivering distinct types of protein cargos to target cells. As a possibly general approach, GIFTed-Exos provide new opportunities to create exosomes with new functions and properties for biomedical research.

Expression levels of MiRNA-16, SURVIVIN and TP53 in Preeclamptic and Normotensive women.

To measure and compare micro ribonucleic acid-16, survivin and tumour protein p53-regulated apoptosis-inducing protein 1 expression levels in preeclamptic and normotensive pregnancies, and to check the correlation of micro ribonucleic acid-16 with messenger ribonucleic acid expression of survivin and tumour protein p53. The observational cross-sectional comparative study was conducted at the Department of Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan, from 2016 to 2018, and comprised preeclamptic women in group A and normotensive women in group B. The preeclamptic patients were further categorised into early-onset preeclampsia subgroup A1and late-onset preeclampsia group A2. Expression of micro ribonucleic acid-16, messenger ribonucleic acid expression of survivin and tumour protein p53 in preeclamptic and normotensive pregnancies were analysed using real time polymerase chain reaction. Data was analysed using SPSS 22. Of the 54 patients, 27(50%) were in each of the two groups. Within group A, 14(52%) patients were in group A1 and 13(48%) in group A2. The expression of micro ribonucleic acid 16 showed significant increase in group A compared to group B (p<0.05). The difference was not significant between the subgroups A1 and A2. The levels of messenger ribonucleic acid expression of survivin and tumour protein p53 were deregulated in group A, with a decrease in survivin and an increase in tumour protein p53. The messenger ribonucleic acid expression of survivin and tumour protein p53 showed statistically significant differences across subgroups A1 and A2 (p<0.05). The micro ribonucleic acid-16 expression correlated negatively with messenger ribonucleic acid expression of survivin, but exhibited a positive correlation with tumour protein p53. Deregulated micro ribonucleic acid-16 along with differentially expressed apoptotic genes, survivin and tumour protein p53 might result in altered apoptosis implicated in the pathogenesis of preeclampsia.

Antitumor Effect of IL-2 and TRAIL Proteins Expressed by Recombinant Salmonella in Murine Bladder Cancer Cells.

Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49. The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action. The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed. The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer.

Oxidative stress-responsive apoptosis-inducing protein in patients with heterozygous familial hypercholesterolemia.

Oxidative stress, an inducer of apoptosis, plays a critical role in ischemia/reperfusion injury and atherosclerosis. We previously identified an apoptosis-inducing ligand, the post-translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A), 'oxidative stress-responsive apoptosis-inducing protein' (ORAIP). In this study, we investigated the role of ORAIP in patients with heterozygous familial hypercholesterolemia (HeFH), a leading cause of premature cardiovascular disease. We analyzed plasma ORAIP and oxidized low-density lipoprotein (oxLDL) levels in 60 patients with HeFH (60% male, 57.0 ± 13.6years of age) and 20 patients with LDL-C hypercholesterolemia (DL, 85% male, 64.1±13.3years of age). The coronary artery atherosclerosis from the patients with HeFH who had a coronary artery bypass graft was investigated by double immunostaining. The plasma ORAIP levels in the patients with HeFH were significantly elevated compared to those in the patients with DL (73.5 ± 46.0 vs. 48.3 ± 21.4ng/mL, p = 0.0277). The plasma oxLDL levels in HeFH patients were also elevated (156.8 ± 65.2 vs. 123.7 ± 46.6mg/dL, p = 0.0461) compared to those in DL patients and correlated with maxLDL-C levels (R = 0.4454, p = 0.00648). Double-immunostaining of ORAIP and oxLDL in the coronary artery from patients with HeFH who had a coronary artery bypass graft showed that ORAIP and oxLDL were colocalized with apoptotic vascular smooth muscle cells in the atherosclerotic plaque. ORAIP plays a role in the development of oxidative stress-induced atherosclerosis and may be an important therapeutic target for plaque rupture in patients with HeFH.

Apoptosis-Inducing Factor, Protein Expression, and Apoptosis Changes with Glutamine in Podocytes Cells Exposed with Cisplatin.

Cisplatin is a well-known chemotherapeutic drug. It is one of the most effective anticancer agents and is widely used for the treatment of several types of tumors. However, side effects in normal tissues and organs, such as nephrotoxicity that induces apoptosis in epithelial cells in the kidney, limit the use of cisplatin. Glutamine is a substrate for the synthesis of glutathione as an antioxidant and promotes HSP70 release, protecting cells from apoptosis induced by different stimuli. In the present study, we investigated the protective effect of glutamine on cisplatin nephrotoxicity in the kidney. Mice were divided into three groups such as a group of control (P0), a group of intraperitoneal injection of a single dose cisplatin 20 mg/kg BW at 7th day (P1), and a group of intravenous glutamine injection 100 mg/kg BW at days 1–7 and given an intraperitoneal injection of single dose cisplatin 20 mg/kg BW at 7th day (P2). Measurement of AIF expression and apoptotic cells was carried out by immunohistochemical methods. The number of AIF expressions and apoptotic cells is expressed in the Allred score. AIF expression result is as follows: P0: 3.29 ± 0.79, P1: 5.32 ± 0.68, and P2: 4.49 ± 0.47. Apoptosis result is as follows: P0: 3.04 ± 0.70, P1: 5.26 ± 0.53, and P2: 4.44 ± 0.41. There is a decreased expression of AIF on intravenous glutamine administration, followed by a decrease in apoptosis in the podocyte. In conclusion, glutamine administration might represent the treatment of nephrotoxic-induced cisplatin.

Methionine and Tryptophan Play Different Modulatory Roles in the European Seabass (Dicentrarchus labrax) Innate Immune Response and Apoptosis Signaling-An In Vitro Study.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1β and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

Minimization of apoptosis-inducing CPP-Bim peptide

Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

Intrinsic and Extrinsic Cell Apoptotic Pathways in Patients with HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis: A Systematic Review.

We aimed to verify the influence of intrinsic and extrinsic cell apoptotic pathways on the inhibition of cellular apoptosis in patients with tropical spastic paralysis/myelopathy related to human T cell lymphotropic virus type 1. The databases accessed were PubMed, Scopus, Science Direct, and Web of Science. Neither the time of publishing nor the language of the articles was limited. The descriptors used for this systematic literature review were: Tropical Paraparesis, Proto-Oncogenic Protein C, Bcl-2, Bcl-X Protein, Bax protein, Fas ligand (FasL) protein, Fas receptor, TNF-related apoptosis-inducing ligand and Fas-associated protein with death domain (FADD)-like apoptosis regulating. The search resulted in 546 articles from which 9 articles were selected for analysis; ranging from serum levels of Bcl-2, Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) measured by enzyme-linked immunosorbent assay and the levels of cellular expression of Bcl-2 and Bcl-xL the TCD4+ lymphocytes accessed by western blot. Most studies accessed either gene expression or polymorphism of Fas, FasL, and TRAIL in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas one study used flow cytometry and fluorescence to determine Fas expression. Increased Bcl-xL expression inhibited T lymphocyte apoptosis, whereas Bcl-2, serum levels, and cellular expression did not influence T lymphocyte apoptosis and serum levels of Fas were significantly higher and associated with markers of leukocyte activation in patients with HAM/TSP. In addition, Fas polymorphism (FAS-670AA) was associated with higher proviral load. There is a need for additional research on this issue since the number of patients was small and the studies presented higher heterogeneity.

Biallelic mutations in the death domain of PIDD1 impair caspase-2 activation and are associated with intellectual disability

PIDD1 encodes p53-Induced Death Domain protein 1, which acts as a sensor surveilling centrosome numbers and p53 activity in mammalian cells. Early results also suggest a role in DNA damage response where PIDD1 may act as a cell-fate switch, through interaction with RIP1 and NEMO/IKKg, activating NF-κB signaling for survival, or as an apoptosis-inducing protein by activating caspase-2. Biallelic truncating mutations in CRADD—the protein bridging PIDD1 and caspase-2—have been reported in intellectual disability (ID), and in a form of lissencephaly. Here, we identified five families with ID from Iran, Pakistan, and India, with four different biallelic mutations in PIDD1, all disrupting the Death Domain (DD), through which PIDD1 interacts with CRADD or RIP1. Nonsense mutations Gln863* and Arg637* directly disrupt the DD, as does a missense mutation, Arg815Trp. A homozygous splice mutation in the fifth family is predicted to disrupt splicing upstream of the DD, as confirmed using an exon trap. In HEK293 cells, we show that both Gln863* and Arg815Trp mutants fail to co-localize with CRADD, leading to its aggregation and mis-localization, and fail to co-precipitate CRADD. Using genome-edited cell lines, we show that these three PIDD1 mutations all cause loss of PIDDosome function. Pidd1 null mice show decreased anxiety, but no motor abnormalities. Together this indicates that PIDD1 mutations in humans may cause ID (and possibly lissencephaly) either through gain of function or secondarily, due to altered scaffolding properties, while complete loss of PIDD1, as modeled in mice, may be well tolerated or is compensated for.

Paclitaxel Priming of TRAIL Expressing Mesenchymal Stromal Cells (MSCs-TRAIL) Increases Antitumor Efficacy of Their Secretome.

Adipose tissue derived MSCs engineered with the tumor necrosis factor-related apoptosis-inducing ligand protein (MSCs-TRAIL) have a significant anticancer activity. MSCs, without any genetic modifications, exposed to high doses of chemotherapeutic agents are able to uptake the drug and release it in amount affecting tumor proliferation. The purpose of this study was to verify the ability of MSCs-TRAIL to uptake and release paclitaxel (PTX) by providing an increased antitumor efficacy. MSCs and MSCs-TRAIL were tested for their sensitivity to Paclitaxel (PTX) by MTT assay and the cells were loaded with PTX according to a standardized procedure. The secretome was analysed by HPLC for the presence of PTX, microarray assay for soluble TRAIL (s-TRAIL) and tested for in vitro anticancer activity. MSCs-TRAIL were resistant to PTX and able to incorporate and then release the drug. The secretion of s-TRAIL by PTX loaded MSCs-TRAIL was not inhibited and the PTX delivery together with s-TRAIL secretion resulted into an increased antitumor efficacy of cell secretoma as tested in vitro on human pancreatic carcinoma (CFPAC-1) and glioblastoma (U87-MG). Our result is the first demonstration of the possible merging of two new MSCs therapy approaches based on genetic manipulation and drug delivery. If confirmed in vivo, this could potentiate the efficacy of MSCs-TRAIL and strongly contribute to reduce the toxicity due to the systemic treatment of PTX.

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from L-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from L-pipecolic acid with a concentration of 90.3mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on L-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. KEY POINTS: • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine.

Chemo-preventive effect of crocin against experimentally-induced hepatocarcinogenesis via regulation of apoptotic and Nrf2 signaling pathways

The results of the current study investigated the chemo-preventive effect of crocin against hepatocarcinogenesis in rats with particular focus on the evaluation of the modulatory impact of crocin on apoptotic and nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathways. Thioacetamide (TAA) (200 mg/kg, I.P.) was used for experimental induction of hepatocarcinogenesis in rats. Crocin administration significantly attenuated TAA-induced cancerous lesions with concomitant attenuation of impaired liver functions. This was associated with significant enhancement in hepatic Nrf2 and heme oxygenase-1 (HO-1) expression with parallel suppression in Keap-1 expression. Inline, crocin induced a significant improvement in hepatic oxidative status with enhanced antioxidant batteries. Crocin administration significantly suppressed the hepatic content of c-Jun N-terminal kinase (c-JNK) with significant upregulation in TNF-related apoptosis-inducing ligand (TRAIL) and caspase-8 protein expression as well as p53 gene expression; biomarkers of apoptosis. Moreover, hepatic expression of the apoptotic BAX significantly increased and the anti-apoptotic Bcl-2 significantly decreased in the liver specimen; biomarkers of intrinsic apoptosis. In conclusion; crocin attenuates experimentally induced hepato-carcinogenesis via modulation of oxidative/apoptotic signaling. Namely, crocin induced hepatic expression of Nrf2 with downstream modulation of endogenous HO-1 and Keap-1 signaling with modulation of various key players of apoptosis including; c-JNK, p53, TRAIL, caspase-8, BAX, and Bcl-2.

Supercharged eGFP-TRAIL Decorated NETs to Ensnare and Kill Disseminated Tumor Cells

BackgroundNETosis is an innate immune response elicited by activated neutrophils to fight microbial infections. Activated neutrophils release DNA fibers decorated with anti-microbial proteins called neutrophil extracellular traps (NETs) into the extracellular space to trap and kill surrounding microbes.MethodsHere, we show that tumor-derived IL-8 released by cancer cells also activates the release of NETs. Until now, there have been no existing technologies that leverage NETs as an anti-tumor drug delivery vehicle. In this study, we demonstrate the re-engineering of neutrophils to express an apoptosis-inducing chimeric protein, supercharged eGFP-TRAIL, on NETs that can ensnare and kill tumor cells while retaining their anti-microbial capabilities.ResultsWe observed significant TRAIL-induced apoptosis in tumor cells captured by TRAIL-decorated NETs.ConclusionsThis work demonstrates NETs as a promising technology to deliver protein in response to local cytokine signals.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

An Engineered Gene Nanovehicle Developed for Smart Gene Therapy to Selectively Inhibit Smooth Muscle Cells: An In Vitro Study.

In-stent restenosis is a serious concern for patients treated through the stenting procedure, although this can be solved using drug-eluting stents and/or drug-eluting balloon catheters. However, the chemical agents released from the drug-eluting layer for inhibiting smooth muscle cell (SMC) migration are inevitably associated with damage to vascular endothelial cell (ECs). The present in vitro study used a distinct strategy, in which a smart gene (phEGR1-PKCδ, an engineered plasmid consists of an SMC-specific promoter (human early growth response 1, hEGR1 promoter) ligated with a gene encoding apoptosis-inducing protein (protein kinase C-delta, PKCδ) was incorporated into a novel gene vehicle (Au cluster-incorporated polyethylenimine/carboxymethyl hexanoyl chitosan, PEI-Au/CHC) to form the PEI-Au/CHC/phEGR1-PKCδ complex, which was proposed for the selective inhibition of SMC proliferation. It was found that the cell viability of SMCs receiving the PEI-Au/CHC/phEGR1-PKCδ complex under simulated inflammation conditions was significantly lower than that of the ECs receiving the same treatment. In addition, the PEI-Au/CHC/phEGR1-PKCδ complex did not demonstrate an inhibitory effect on EC proliferation and migration under simulated inflammation conditions. Finally, the PEI-Au/CHC/phEGR1-PKCδ complexes coated onto a balloon catheter used in percutaneous transluminal coronary angioplasty (PTCA) could be transferred to both the ECs and the SMC layer of Sprague Dawley (SD) rat aortas ex vivo. These preliminary in vitro results suggest that the newly developed approach proposed in the present study might be a potential treatment for reducing the incidence rate of in-stent restenosis and late thrombosis in the future.

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function.

Flavoprotein oxidoreductases are members of a large protein family of specialized dehydrogenases, which include type II NADH dehydrogenase, pyridine nucleotide-disulphide oxidoreductases, ferredoxin-NAD+ reductases, NADH oxidases, and NADH peroxidases, playing a crucial role in the metabolism of several prokaryotes and eukaryotes. Although several studies have been performed on single members or protein subgroups of flavoprotein oxidoreductases, a comprehensive analysis on structure–function relationships among the different members and subgroups of this great dehydrogenase family is still missing. Here, we present a structural comparative analysis showing that the investigated flavoprotein oxidoreductases have a highly similar overall structure, although the investigated dehydrogenases are quite different in functional annotations and global amino acid composition. The different functional annotation is ascribed to their participation in species-specific metabolic pathways based on the same biochemical reaction, i.e., the oxidation of specific cofactors, like NADH and FADH2. Notably, the performed comparative analysis sheds light on conserved sequence features that reflect very similar oxidation mechanisms, conserved among flavoprotein oxidoreductases belonging to phylogenetically distant species, as the bacterial type II NADH dehydrogenases and the mammalian apoptosis-inducing factor protein, until now retained as unique protein entities in Bacteria/Fungi or Animals, respectively. Furthermore, the presented computational analyses will allow consideration of FAD/NADH oxidoreductases as a possible target of new small molecules to be used as modulators of mitochondrial respiration for patients affected by rare diseases or cancer showing mitochondrial dysfunction, or antibiotics for treating bacterial/fungal/protista infections.

Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage

Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.

P5397Mesenchymal stem cells derived from bone marrow promotes cardiomyocytes survival under hypoxia through exosomal miR-210

Abstract Mesenchymal stem cells derived from bone marrow promotes cardiomyocytes survival under hypoxia through exosomal miR-210 Background A paracrine effect was regarded as the key mechanism involved in the MSC (mesenchymal stem cell)-based treatment for myocardial infarction. In our pilot experiments, hypoxia remarkably promotes MSC to paracrine exosomal miR-210, which could significantly enhance the cardiomyocytes survival in hypoxic incubation, suggesting that exosomal miR-210 played critical roles in the favorable paracrine effect of MSC on cardiomyocytes. Purpose The aim of this study was to investigate the important mechanism by which MSCs promote the tolerance of cardiomyocytes to hypoxia by secreting exosomal miR-210. Methods and results The exosomes were isolated from MSCs conditioned medium through ultracentrifugation, and we detected that miR-210 was the most abundant in MSC-exosome and increased most prominently in the hypoxia. The extracted exosomes were prepared for conditioned medium and the effect on myocardial protection was examined. The viability of control group was much better than the cardiomyocytes treated with hypoxia, but it was further increased in the presence of MSC-exosome, however, measurement was significantly lower in cardiomyocytes in hypoxia with exosomes derived from MSCs treated with GW4869. Subsequently, the co-localization of miR-210 with exosome-specific surface markers CD81 and CD63 were observed by immunofluorescence technique. Continuous magnetic live cell imaging was used to observe the uptake of exosome by cardiomyocytes, and fluorescence localization was used to observe the localization of miR-210 with Cy3 fluorescence in cardiomyocytes. Then, we demonstrated that MSCs exosomal miR-210 exerts the cardioprotective effect by regulating the AIFM3 (apoptosis-inducing factor mitochondria-associated protein 3), and we directly overexpressed miRNA-210 in cardiomyocytes and the results showed that the regulatory activity of the intake of exosomal miR-210 was consistent with that of the biological exosomal miR-210. Finally, we verified the protective effect on the ischemic myocardium by constructing rat myocardial infarction models. The level of apoptosis was detected at 1 week after myocardial infarction. The left ventricular ejection fraction and ventricular remodeling were measured at 4 weeks. In vivo, we demonstrated that explanted miR-210 from transplanted MSCs significantly reduced myocardial necrosis and apoptosis induced by ischemia and improved cardiac function and myocardial remodeling. Conclusion Here, we show that the exosomal miR-210 secreted by MSCs significantly increase the viability of cardiomyocytes and cardiac function. These findings suggest that exosomal miR-210 is a key effector that mediates the protection against hypoxia. Acknowledgement/Funding National Natural Science Foundation of China (Grant Nos. 81470467)

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication.

Apoptosis-inducing protein of 56 kDa (AIP56) is a major virulence factor of Photobacterium damselae subsp. piscicida, a gram-negative pathogen that infects warm water fish species worldwide and causes serious economic losses in aquacultures. AIP56 is a single-chain AB toxin composed by two domains connected by an unstructured linker peptide flanked by two cysteine residues that form a disulphide bond. The A domain comprises a zinc-metalloprotease moiety that cleaves the NF-kB p65, and the B domain is involved in binding and internalisation of the toxin into susceptible cells. Previous experiments suggested that disruption of AIP56 disulphide bond partially compromised toxicity, but conclusive evidences supporting the importance of that bond in intoxication were lacking. Here, we show that although the disulphide bond of AIP56 is dispensable for receptor recognition, endocytosis, and membrane interaction, it needs to be intact for efficient translocation of the toxin into the cytosol. We also show that the host cell thioredoxin reductase-thioredoxin system is involved in AIP56 intoxication by reducing the disulphide bond of the toxin at the cytosol. The present study contributes to a better understanding of the molecular mechanisms operating during AIP56 intoxication and reveals common features shared with other AB toxins.

RNase L Induces Expression of A Novel Serine/Threonine Protein Kinase, DRAK1, to Promote Apoptosis.

Apoptosis of virus-infected cells is an effective antiviral mechanism in addition to interferon induction to establish antiviral state to restrict virus spread. The interferon-inducible 2′–5′ oligoadenylate synthetase/RNase L pathway results in activation of RNase L in response to double stranded RNA and cleaves diverse RNA substrates to amplify interferon induction and promote apoptosis. Here we show that RNase L induces expression of Death-associated protein kinase-Related Apoptosis-inducing protein Kinase 1 (DRAK1), a member of the death-associated protein kinase family and interferon-signaling pathway is required for induction. Overexpression of DRAK1 triggers apoptosis in the absence of RNase L activation by activating c-Jun N-terminal kinase (JNK), translocation of BCL2 Associated X (Bax) to the mitochondria accompanied by cytochrome C release and loss of mitochondrial membrane potential promoting cleavage of caspase 3 and Poly(ADP-Ribose) Polymerase 1 (PARP). Inhibitors of JNK and caspase 3 promote survival of DRAK1 overexpressing cells demonstrating an important role of JNK signaling pathway in DRAK1-mediated apoptosis. DRAK1 mutant proteins that lack kinase activity or nuclear localization fail to induce apoptosis highlighting the importance of cellular localization and kinase function in promoting cell death. Our studies identify DRAK1 as a mediator of RNase L-induced apoptosis.