Enforced lysosomal biogenesis rescues erythromycin- and clindamycin-induced mitochondria-mediated cell death in human cells.

Antibiotics are the front-line treatment against many bacterial infectious diseases in human. The excessive and long-term use of antibiotics in human cause several side effects. It is important to understand the underlying molecular mechanisms of action of antibiotics in the host cell to avoid the side effects due to the prevalent uses. In the current study, we investigated the crosstalk between mitochondria and lysosomes in the presence of widely used antibiotics: erythromycin (ERM) and clindamycin (CLDM), which target the 50S subunit of bacterial ribosomes. We report here that both ERM and CLDM induced caspase activation and cell death in several different human cell lines. The activity of the mitochondrial respiratory chain was compromised in the presence of ERM and CLDM leading to bioenergetic crisis and generation of reactive oxygen species. Antibiotics treatment impaired autophagy flux and lysosome numbers, resulting in decreased removal of damaged mitochondria through mitophagy, hence accumulation of defective mitochondria. We further show that over-expression of transcription factor EB (TFEB) increased the lysosome number, restored mitochondrial function and rescued ERM- and CLDM-induced cell death. These studies indicate that antibiotics alter mitochondria and lysosome interactions leading to apoptotsis and may develop a novel approach for targeting inter-organelle crosstalk to limit deleterious antibiotic-induced side effects.

hsa-miR-4485 regulates mitochondrial functions and inhibits the tumorigenicity of breast cancer cells.

The modulation of mitochondrial functions is important for maintaining cellular homeostasis. Mitochondria essentially depend on the import of RNAs and proteins encoded by the nuclear genome. MicroRNAs encoded in the nucleus can translocate to mitochondria and target the genome, affecting mitochondrial function. Here, we analyzed the role of miR-4485 in the regulation of mitochondrial functions. We showed that miR-4485 translocated to mitochondria where its levels varied in response to different stress conditions. A direct binding of miR-4485 to mitochondrial 16S rRNA was demonstrated. MiR-4485 regulated the processing of pre-rRNA at the 16S rRNA-ND1 junction and the translation of downstream transcripts. MiR-4485 modulated mitochondrial complex I activity, the production of ATP, ROS levels, caspase-3/7 activation, and apoptosis. Transfection of a miR-4485 mimic downregulated the expression of regulatory glycolytic pathway genes and reduced the clonogenic ability of breast cancer cells. Ectopic expression of miR-4485 in MDA-MB-231 breast carcinoma cells decreased the tumorigenicity in a nude mouse xenograft model. Furthermore, levels of both precursor and mature miR-4485 are decreased in tumor tissue of breast cancer patients. We conclude that the mitochondria-targeted miR-4485 may act as a tumor suppressor in breast carcinoma cells by negatively regulating mitochondrial RNA processing and mitochondrial functions.

TRIM4; a novel mitochondrial interacting RING E3 ligase, sensitizes the cells to hydrogen peroxide (H2O2) induced cell death.

The emerging evidences suggest that posttranslational modification of target protein by ubiquitin (Ub) not only regulate its turnover through ubiquitin proteasome system (UPS) but is a critical regulator of various signaling pathways. During ubiquitination, E3 ligase recognizes the target protein and determines the topology of ubiquitin chains. In current study, we studied the role of TRIM4, a member of the TRIM/RBCC protein family of RING E3 ligase, in regulation of hydrogen peroxide (H2O2) induced cell death. TRIM4 is expressed differentially in human tissues and expressed in most of the analyzed human cancer cell lines. The subcellular localization studies showed that TRIM4 forms distinct cytoplasmic speckle like structures which transiently interacts with mitochondria. The expression of TRIM4 induces mitochondrial aggregation and increased level of mitochondrial ROS in the presence of H2O2. It sensitizes the cells to H2O2 induced death whereas knockdown reversed the effect. TRIM4 potentiates the loss of mitochondrial transmembrane potential and cytochrome c release in the presence of H2O2. The analysis of TRIM4 interacting proteins showed its interaction with peroxiredoxin 1 (PRX1), including other proteins involved in regulation of mitochondrial and redox homeostasis. TRIM4 interaction with PRX1 is critical for the regulation of H2O2 induced cell death. Collectively, the evidences in the current study suggest the role of TRIM4 in regulation of oxidative stress induced cell death.

NLRX1 acts as tumor suppressor by regulating TNF-α induced apoptosis and metabolism in cancer cells.

Chronic inflammation in tumor microenvironment plays an important role at different stages of tumor development. The specific mechanisms of the association and its role in providing a survival advantage to the tumor cells are not well understood. Mitochondria are emerging as a central platform for the assembly of signaling complexes regulating inflammatory pathways, including the activation of type-I IFN and NF-κB. These complexes in turn may affect metabolic functions of mitochondria and promote tumorigenesis. NLRX1, a mitochondrial NOD-like receptor protein, regulate inflammatory pathways, however its role in regulation of cross talk of cell death and metabolism and its implication in tumorigenesis is not well understood. Here we demonstrate that NLRX1 sensitizes cells to TNF-α induced cell death by activating Caspase-8. In the presence of TNF-α, NLRX1 and active subunits of Caspase-8 are preferentially localized to mitochondria and regulate the mitochondrial ROS generation. NLRX1 regulates mitochondrial Complex I and Complex III activities to maintain ATP levels in the presence of TNF-α. The expression of NLRX1 compromises clonogenicity, anchorage-independent growth, migration of cancer cells in vitro and suppresses tumorigenicity in vivo in nude mice. We conclude that NLRX1 acts as a potential tumor suppressor by regulating the TNF-α induced cell death and metabolism.

TNF-α regulates miRNA targeting mitochondrial complex-I and induces cell death in dopaminergic cells.

Parkinson's disease (PD) is a complex neurological disorder of the elderly population and majorly shows the selective loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) region of the brain. The mechanisms leading to increased cell death of DAergic neurons are not well understood. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine is elevated in blood, CSF and striatum region of the brain in PD patients. The increased level of TNF-α and its role in pathogenesis of PD are not well understood. In the current study, we investigated the role of TNF-α in the regulation of cell death and miRNA mediated mitochondrial functions using, DAergic cell line, SH-SY5Y (model of dopaminergic neuron degeneration akin to PD). The cells treated with low dose of TNF-α for prolonged period induce cell death which was rescued in the presence of zVAD.fmk, a caspase inhibitor and N-acetyl-cysteine (NAC), an antioxidant. TNF-α alters mitochondrial complex-I activity, decreases adenosine triphosphate (ATP) levels, increases reactive oxygen species levels and mitochondrial turnover through autophagy. TNF-α differentially regulates miRNA expression involved in pathogenesis of PD. Bioinformatics analysis revealed that the putative targets of altered miRNA included both pro/anti apoptotic genes and subunits of mitochondrial complex. The cells treated with TNF-α showed decreased level of nuclear encoded transcript of mitochondrial complexes, the target of miRNA. To our knowledge, the evidences in the current study demonstrated that TNF-α is a potential regulator of miRNAs which may regulate mitochondrial functions and neuronal cell death, having important implication in pathogenesis of PD.

Side effects of antibiotics during bacterial infection: mitochondria, the main target in host cell.

Antibiotics are frontline therapy against microbial infectious diseases. Many antibiotics are known to cause several side effects in humans. Ribosomal RNA (rRNA) is the main target of antibiotics that inhibit protein synthesis. According to the endosymbiont theory, mitochondrion is of bacterial origin and their molecular and structural components of the protein expression system are almost similar. It has been observed that the rate of mutations in mitochondrial rRNA is higher as compared to that of nuclear rRNA. The presence of these mutations may mimic prokaryotic rRNA structure and bind to antibiotics targeted to ribosomes of bacteria. Mitochondrial functions are compromised hence may be one of the major causes of side effects observed during antibiotic therapy. The current review had summarized the studies on the role of antibiotics on mitochondrial functions and its relevance to the observed side effects in physiological and pathological conditions.

Antiviral signaling protein MITA acts as a tumor suppressor in breast cancer by regulating NF-κB induced cell death.

Emerging evidences suggest that chronic inflammation is one of the major causes of tumorigenesis. The role of inflammation in regulation of breast cancer progression is not well established. Recently Mediator of IRF3 Activation (MITA) protein has been identified that regulates NF-κB and IFN pathways. Role of MITA in the context of inflammation and cancer progression has not been investigated. In the current report, we studied the role of MITA in the regulation of cross talk between cell death and inflammation in breast cancer cells. The expression of MITA was significantly lower on in estrogen receptor (ER) positive breast cancer cells than ER negative cells. Similarly, it was significantly down regulated in tumor tissue as compared to the normal tissue. The overexpression of MITA in MCF-7 and T47D decreases the cell proliferation and increases the cell death by activation of caspases. MITA positively regulates NF-κB transcription factor, which is essential for MITA induced cell death. The activation of NF-κB induces TNF-α production which further sensitizes MITA induced cell death by activation of death receptor pathway through capsase-8. MITA expression decreases the colony forming units and migration ability of MCF-7 cells. Thus, our finding suggests that MITA acts as a tumor suppressor which is down regulated during tumorigenesis providing survival advantage to tumor cell.

TRIM13 regulates caspase-8 ubiquitination, translocation to autophagosomes and activation during ER stress induced cell death.

The emerging evidences suggest that endoplasmic (ER) stress is involved in onset of many pathological conditions like cancer and neurodegeneration. The persistent ER stress results in misfolded protein aggregates, which are degraded through the process of autophagy or lead to cell death through activation of caspases. The regulation of crosstalk of autophagy and cell death during ER stress is emerging. Ubiquitination plays regulatory role in crosstalk of autophagy and cell death. In the current study, we describe the role of TRIM13, RING E3 ubiquitin ligase, in regulation of ER stress induced cell death. The expression of TRIM13 sensitizes cells to ER stress induced death. TRIM13 induced autophagy is essential for ER stress induced caspase activation and cell death. TRIM13 induces K63 linked poly-ubiquitination of caspase-8, which results in its stabilization and activation during ER stress. TRIM13 regulates translocation of caspase-8 to autophagosome and its fusion with lysosome during ER stress. This study first time demonstrated the role of TRIM13 as novel regulator of caspase-8 activation and cell death during ER stress.

Nucleo-cytoplasmic trafficking of TRIM8, a novel oncogene, is involved in positive regulation of TNF induced NF-κB pathway.

TNF induced nuclear factor kappa B (NF-κB) is one of the central signaling pathways that plays a critical role in carcinogenesis and inflammatory diseases. Post-translational modification through ubiquitin plays important role in the regulation of this pathway. In the current study, we investigated the role of TRIM8, member of RING family ubiquitin ligase in regulation of NF-κB pathway. We observed that TRIM8 positively regulates TNF induced NF-κB pathway. Different domains of TRIM8 showed discrete functions at the different steps in regulation of TNF induced NF-κB pathway. Ubiquitin ligase activity of TRIM8 is essential for regulation of NF-κB activation in both cytoplasm as well as nucleus. TRIM8 negates PIAS3 mediated negative repression of NF-κB at p65 by inducing translocation of PIAS3 from nucleus to cytoplasm as well as its turnover. TNF induces translocation of TRIM8 from nucleus to cytoplasm, which positively regulates NF-κB. The cytoplasmic translocation of TRIM8 is essential for TNF induced NF-κB but not for p65 mediated NF-κB regulation. TRIM8 also enhanced the clonogenic and migration ability of cells by modulating NF-κB. The further study will help to understand the role of TRIM8 in inflammation and cancer.

Systematic analysis of small RNAs associated with human mitochondria by deep sequencing: detailed analysis of mitochondrial associated miRNA.

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.

The leucine aminopeptidase of Staphylococcus aureus is secreted and contributes to biofilm formation.

BACKGROUND: Staphylococcus aureus has emerged as a major drug-resistant pathogen in hospital- and community-acquired infections. Leucine aminopeptidase (LAP) is known to be essential for survival of the bacteria; however the LAP of S. aureus has not been extensively characterized. In this study, we report a detailed characterization of the S. aureus LAP. METHODS: LAP from S. aureus was cloned, purified, and further biochemically characterized. The expression of LAP was analyzed by Western blotting. Growth and biofilm formation were analyzed spectrophotometrically. RESULTS: LAP was cloned from S. aureus and expressed as a 55 kDa protein, whereas the molecular weight of the native protein is approximately 600 kDa. LAP showed amidolytic activity against l-leucine p-nitroanilide. Optimal activity was observed at pH 8.5 and 37°C with a V(max) of 2500µmol/min/mg protein. LAP enzymatic activity was inhibited by ion chelators and enhanced by divalent metal ions, specifically Ni. LAP is secreted by laboratory as well as clinical strains. Bestatin, an inhibitor of LAP, inhibits S. aureus growth and biofilm formation. CONCLUSIONS: To our knowledge, this is the first detailed characterization of LAP from S. aureus and suggests its importance in survival and pathogenesis.

TRIM13 regulates ER stress induced autophagy and clonogenic ability of the cells.

Autophagy is one of the cellular adaptive processes that provide protection against many pathological conditions like infection, cancer, neurodegeneration, and aging. Recent evidences suggest that ubiquitination plays an important role in degradation of proteins or defective organelle either through proteasome or autophagy. In this study, we describe the role of TRIM13, ER resident ubiquitin E3 ligase in induction of autophagy and its role during ER stress. The ectopic expression of TRIM13 in HEK-293 cells induces autophagy. Domain mapping showed that coiled-coil (CC) domain is required for induction of autophagy. TRIM13 is stabilized during ER stress, interacts with p62/SQSTM1 and co-localizes with DFCP1. TRIM13 regulates initiation of autophagy during ER stress and decreases the clonogenic ability of the cells. This study for the first time demonstrates the role of TRIM13 in induction of autophagy which may play an important role in regulation of ER stress and may act as tumor suppressor.

Clinical isolates of Vibrio fluvialis from Kolkata, India, obtained during 2006: plasmids, the qnr gene and a mutation in gyrase A as mechanisms of multidrug resistance.

Resistance profiles and their correlation with genetic factors were investigated in 12 isolates of Vibrio fluvialis obtained from hospitalized patients in Kolkata, India, in 2006. All the strains displayed drug resistance with varying antibiograms. However, resistance to ampicillin and neomycin was common to all of them. Three isolates harboured plasmids carrying drug-resistance genes that could be transferred to recipient strains by conjugation and transformation. PCR results indicated the absence of class 1 integrons and SXT elements in these isolates. A mutation in gyrase A (serine 83→isoleucine) and the presence of the qnrVC-like [corrected] gene were found to contribute towards quinolone resistance. In the 12 isolates, the qnrVC-like [corrected] gene was associated only with two plasmid-bearing isolates, L10734 and L9978, which displayed resistance to quinolones. The gene was transferable during transformation and conjugation, indicating that it was plasmid-borne. Taken together, these data indicate that plasmids, the qnrVC-like [corrected] gene and a mutation in gyrase A were responsible for the observed drug resistance in these strains. To the best of our knowledge, this is the first report of the presence of the qnrVC-like [corrected] allele in V. fluvialis isolates from India.