Dysregulation of RyR Calcium Channel Causes the Onset of Mitochondrial Retrograde Signaling.

This study shows that multiple modes of mitochondrial stress generated by partial mtDNA depletion or cytochrome c oxidase disruption cause ryanodine receptor channel (RyR) dysregulation, which instigates the release of Ca2+ in the cytoplasm of C2C12 myoblasts and HCT116 carcinoma cells. We also observed a reciprocal downregulation of IP3R channel activity and reduced mitochondrial uptake of Ca2+. Ryanodine, an RyR antagonist, abrogated the mitochondrial stress-mediated increase in [Ca2+]c and the entire downstream signaling cascades of mitochondrial retrograde signaling. Interestingly, ryanodine also inhibited mitochondrial stress-induced invasive behavior in mtDNA-depleted C2C12 cells and HCT116 carcinoma cells. In addition, co-immunoprecipitation shows reduced FKBP12 protein binding to RyR channel proteins, suggesting the altered function of the Ca2+ channel. These results document how the endoplasmic reticulum-associated RyR channels, in combination with inhibition of the mitochondrial uniporter system, modulate cellular Ca2+ homeostasis and signaling under mitochondrial stress conditions.

Esophageal 3D organoids of MPV17-/- mouse model of mitochondrial DNA depletion show epithelial cell plasticity and telomere attrition.

Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with late-stage detection and poor prognosis. This emphasizes the need to identify new markers for early diagnosis and treatment. Altered mitochondrial genome (mtDNA) content in primary tumors correlates with poor patient prognosis. Here we used three-dimensional (3D) organoids of esophageal epithelial cells (EECs) from the MPV17-/- mouse model of mtDNA depletion to investigate the contribution of reduced mtDNA content in ESCC oncogenicity. To test if mtDNA defects are a contributing factor in ESCC, we used oncogenic stimuli such as ESCC carcinogen 4-nitroquinoline oxide (4-NQO) treatment, or expressing p53R175H oncogenic driver mutation. We observed that EECs and 3D-organoids with mtDNA depletion had cellular, morphological and genetic alterations typical of an oncogenic transition. Furthermore, mitochondrial dysfunction induced cellular transformation is accompanied by elevated mitochondrial fission protein, DRP1 and pharmacologic inhibition of mitochondrial fission by mDivi-1 in the MPV17-/- organoids reversed the phenotype to that of normal EEC organoids. Our studies show that mtDNA copy number depletion, activates a mitochondrial retrograde response, potentiates telomere defects, and increases the oncogenic susceptibility towards ESCC. Furthermore, mtDNA depletion driven cellular plasticity is mediated via altered mitochondrial fission-fusion dynamics.

Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

The mitochondria-to-nucleus retrograde signaling (MtRS) pathway aids in cellular adaptation to stress. We earlier reported that the Ca2+- and calcineurin-dependent MtRS induces macrophage differentiation to bone-resorbing osteoclasts. However, mechanisms through which macrophages sense and respond to cellular stress remain unclear. Here, we induced mitochondrial stress in macrophages by knockdown (KD) of subunits IVi1 or Vb of cytochrome c oxidase (CcO). Whereas both IVi1 and Vb KD impair CcO activity, IVi1 KD cells produced higher levels of cellular and mitochondrial reactive oxygen species with increased glycolysis. Additionally, IVi1 KD induced the activation of MtRS factors NF-κB, NFAT2, and C/EBPδ as well as inflammatory cytokines, NOS 2, increased phagocytic activity, and a greater osteoclast differentiation potential at suboptimal RANK-L concentrations. The osteoclastogenesis in IVi1 KD cells was reversed fully with an IL-6 inhibitor LMT-28, whereas there was minimal rescue of the enhanced phagocytosis in these cells. In agreement with our findings in cultured macrophages, primary bone marrow-derived macrophages from MPV17-/- mice, a model for mitochondrial dysfunction, also showed higher propensity for osteoclast formation. This is the first report showing that CcO dysfunction affects inflammatory pathways, phagocytic function, and osteoclastogenesis.-Angireddy, R., Kazmi, H. R., Srinivasan, S., Sun, L., Iqbal, J., Fuchs, S. Y., Guha, M., Kijima, T., Yuen, T., Zaidi, M., Avadhani, N. G. Cytochrome c oxidase dysfunction enhances phagocytic function and osteoclast formation in macrophages.

Correction to: HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

[This corrects the article DOI: 10.1038/celldisc.2016.45.].

Mitochondrial genome and functional defects in osteosarcoma are associated with their aggressive phenotype.

Osteosarcoma (OSA) is an aggressive mesenchymal tumor of the bone that affects children and occurs spontaneously in dogs. Human and canine OSA share similar clinical, biological and genetic features, which make dogs an excellent comparative model to investigate the etiology and pathogenesis of OSA. Mitochondrial (mt) defects have been reported in many different cancers including OSA, although it is not known whether these defects contribute to OSA progression and metastasis. Taking a comparative approach using canine OSA cell lines and tumor tissues we investigated the effects of mtDNA content and dysfunction on OSA biology. OSA tumor tissues had low mtDNA contents compared to the matched non-tumor tissues. We observed mitochondrial heterogeneity among the OSA cell lines and the most invasive cells expressing increased levels of OSA metastasis genes contained the highest amount of mitochondrial defects (reduced mtDNA copies, mt respiration, and expression of electron transport chain proteins). While mitochondria maintain a filamentous network in healthy cells, the mitochondrial morphology in OSA cells were mostly "donut shaped", typical of "stressed" mitochondria. Moreover the expression levels of mitochondrial retrograde signaling proteins Akt1, IGF1R, hnRNPA2 and NFkB correlated with the invasiveness of the OSA cells. Furthermore, we demonstrate the causal role of mitochondrial defects in inducing the invasive phenotype by Ethidium Bromide induced-mtDNA depletion in OSA cells. Our data suggest that defects in mitochondrial genome and function are prevalent in OSA and that lower mtDNA content is associated with higher tumor cell invasiveness. We propose that mt defects in OSA might serve as a prognostic biomarker and a target for therapeutic intervention in OSA patients.

hnRNPA2 mediated acetylation reduces telomere length in response to mitochondrial dysfunction.

Telomeres protect against chromosomal damage. Accelerated telomere loss has been associated with premature aging syndromes such as Werner's syndrome and Dyskeratosis Congenita, while, progressive telomere loss activates a DNA damage response leading to chromosomal instability, typically observed in cancer cells and senescent cells. Therefore, identifying mechanisms of telomere length maintenance is critical for understanding human pathologies. In this paper we demonstrate that mitochondrial dysfunction plays a causal role in telomere shortening. Furthermore, hnRNPA2, a mitochondrial stress responsive lysine acetyltransferase (KAT) acetylates telomere histone H4at lysine 8 of (H4K8) and this acetylation is associated with telomere attrition. Cells containing dysfunctional mitochondria have higher telomere H4K8 acetylation and shorter telomeres independent of cell proliferation rates. Ectopic expression of KAT mutant hnRNPA2 rescued telomere length possibly due to impaired H4K8 acetylation coupled with inability to activate telomerase expression. The phenotypic outcome of telomere shortening in immortalized cells included chromosomal instability (end-fusions) and telomerase activation, typical of an oncogenic transformation; while in non-telomerase expressing fibroblasts, mitochondrial dysfunction induced-telomere attrition resulted in senescence. Our findings provide a mechanistic association between dysfunctional mitochondria and telomere loss and therefore describe a novel epigenetic signal for telomere length maintenance.

Cigarette Smoke Toxins-Induced Mitochondrial Dysfunction and Pancreatitis Involves Aryl Hydrocarbon Receptor Mediated Cyp1 Gene Expression: Protective Effects of Resveratrol.

We previously reported that mitochondrial CYP1 enzymes participate in the metabolism of polycyclic aromatic hydrocarbons and other carcinogens leading to mitochondrial dysfunction. In this study, using Cyp1b1-/-, Cyp1a1/1a2-/-, and Cyp1a1/1a2/1b1-/- mice, we observed that cigarette and environmental toxins, namely benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induce pancreatic mitochondrial respiratory dysfunction and pancreatitis. Our results suggest that aryl hydrocarbon receptor (AhR) activation and resultant mitochondrial dysfunction are associated with pancreatic pathology. BaP treatment markedly inhibits pancreatic mitochondrial oxygen consumption rate (OCR), ADP-dependent OCR, and also maximal respiration, in wild-type mice but not in Cyp1a1/1a2-/- and Cyp1a1/1a2/1b1-/- mice. In addition, both BaP and TCDD treatment markedly affected mitochondrial complex IV activity, in addition to causing marked reduction in mitochondrial DNA content. Interestingly, the AhR antagonist resveratrol, attenuated BaP-induced mitochondrial respiratory defects in the pancreas, and reversed pancreatitis, both histologically and biochemically in wild-type mice. These results reveal a novel role for AhR- and AhR-regulated CYP1 enzymes in eliciting mitochondrial dysfunction and cigarette toxin-mediated pancreatic pathology. We propose that increased mitochondrial respiratory dysfunction and oxidative stress are involved in polycyclic aromatic hydrocarbon associated pancreatitis. Resveratrol, a chemo preventive agent and AhR antagonist, and CH-223191, a potent and specific AhR inhibitor, confer protection against BaP-induced mitochondrial dysfunction and pancreatic pathology.

Aggressive triple negative breast cancers have unique molecular signature on the basis of mitochondrial genetic and functional defects.

Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.

Paediatric Peripheral Primitive Neuroectodermal Tumour - A Clinico-Pathological Study from Southern India.

INTRODUCTION: Primitive Neuroectodermal Tumour (PNET)/Ewing Sarcomas (ES) are aggressive childhood malignancies with neuroectodermal differentiation. AIM: To study the clinical presentation, morphology, Immun-ohistochemistry (IHC), management and outcome of all the cases of paediatric pPNET/ES reported in our tertiary care centre over a period of six years. MATERIALS AND METHODS: This was a retrospective study conducted at Sri Ramachandra Medical College and Research Institute, Chennai, India. All biopsy proven cases of peripheral PNET/ES, in patients less than 18 years of age for a period of six years were included in this study. The corresponding clinical details regarding initial presentation, treatment and follow up were retrieved from the case files and analysed. Survival rate was calculated and Kaplan-Meier survival curve was plotted. RESULTS: We describe eleven cases of paediatric peripheral PNET/ES. The mean age at presentation was 94.08 (±58.27) months with a male/female ratio of 1.2:1. About 27.3% cases, all male with a mean age of 140 months at presentation, had distant metastasis during initial diagnosis. Biopsy showed small round blue cell morphology on light microscopy. IHC revealed strong membranous staining for CD99 in all cases. All children were treated with neo-adjuvant chemotherapy and then surgery, followed by radiotherapy if indicated. The cases were followed up for a mean duration of 20.82 months (ranging from one to 66 months). Nine children are doing well on follow up (81.8% survival rate). Two cases with metastasis at initial presentation died. Patients with metastatic disease exhibited a mean duration of survival of 9.66 (±7.24) months and those with localized disease exhibited a mean duration of survival of 25 (±22.88) months. CONCLUSION: Metastasis at diagnosis is the single most important factor affecting prognosis. This was reflected in the present study where cases with metastasis exhibited a short mean duration of survival when compared to localized disease. It is likely that many cases of PNET/ES were not accurately identified in the past as IHC plays a vital role in the diagnosis of these small round blue cell tumours. IHC in adjunct with molecular studies has improved diagnostic accuracy. Multidisciplinary management and good supportive care when the lesion is localized has lead to improved survival.

Mitochondrial LON protease-dependent degradation of cytochrome c oxidase subunits under hypoxia and myocardial ischemia.

The mitochondrial ATP dependent matrix protease, Lon, is involved in the maintenance of mitochondrial DNA nucleoids and degradation of abnormal or misfolded proteins. The Lon protease regulates mitochondrial Tfam (mitochondrial transcription factor A) level and thus modulates mitochondrial DNA (mtDNA) content. We have previously shown that hypoxic stress induces the PKA-dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IVi1, and Vb and a time-dependent reduction of these subunits in RAW 264.7 murine macrophages subjected to hypoxia and rabbit hearts subjected to ischemia/reperfusion. Here, we show that Lon is involved in the preferential turnover of phosphorylated CcO subunits under hypoxic/ischemic stress. Induction of Lon protease occurs at 6 to 12 h of hypoxia and this increase coincides with lower CcO subunit contents. Over-expression of flag-tagged wild type and phosphorylation site mutant Vb and IVi1 subunits (S40A and T52A, respectively) caused marked degradation of wild type protein under hypoxia while the mutant proteins were relatively resistant. Furthermore, the recombinant purified Lon protease degraded the phosphorylated IVi1 and Vb subunits, while the phosphorylation-site mutant proteins were resistant to degradation. 3D structural modeling shows that the phosphorylation sites are exposed to the matrix compartment, accessible to matrix PKA and Lon protease. Hypoxic stress did not alter CcO subunit levels in Lon depleted cells, confirming its role in CcO turnover. Our results therefore suggest that Lon preferentially degrades the phosphorylated subunits of CcO and plays a role in the regulation of CcO activity in hypoxia and ischemia/reperfusion injury.

Mitochondrial dysfunction and mitochondrial dynamics-The cancer connection.

Mitochondrial dysfunction is a hallmark of many diseases. The retrograde signaling initiated by dysfunctional mitochondria can bring about global changes in gene expression that alters cell morphology and function. Typically, this is attributed to disruption of important mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis and regulation of apoptosis. Recent studies showed that in addition to these factors, mitochondrial dynamics might play an important role in stress signaling. Normal mitochondria are highly dynamic organelles whose size, shape and network are controlled by cell physiology. Defective mitochondrial dynamics play important roles in human diseases. Mitochondrial DNA defects and defective mitochondrial function have been reported in many cancers. Recent studies show that increased mitochondrial fission is a pro-tumorigenic phenotype. In this paper, we have explored the current understanding of the role of mitochondrial dynamics in pathologies. We present new data on mitochondrial dynamics and dysfunction to illustrate a causal link between mitochondrial DNA defects, excessive fission, mitochondrial retrograde signaling and cancer progression. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression.

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies.

Desmoplastic Ameloblastoma Arising in a Dentigerous Cyst - A Case Report and Discussion.

Dentigerous cyst is a fairly common odontogenic cyst with an uncomplicated prognosis. However, reports of odontogenic and non-odontogenic tumors arising from the cysts have been published for many years. Therefore, the importance of thorough histopathological examination and regular follow-up cannot be overemphasized. Here is a case report of a possible development of desmoplastic ameloblastoma from a dentigerous cyst. This case is unique for two main reasons. Firstly, to our knowledge such a case has not been reported previously. Secondly, the tumor seemed to arise from the capsule and not from lining of the dentigerous cyst. Transformation of odontogenic rests into cysts is known to occur in the syndromic cases of odontogenic keratocysts (resulting in recurrent and daughter cysts) but not in dentigerous cysts. This case report may prompt greater interest in the role of the supposedly inactive odontogenic nests in the capsule of these cysts.

Mitochondrial respiratory defects promote the Warburg effect and cancer progression.

In the past decade mitochondria have emerged as an important cellular signaling hub controlling metabolism, epigenetics, and cell fate. Dysfunctional mitochondria initiate a retrograde nuclear response that influences the cellular reprograming observed in various human pathologies, including cancer. New data suggest that loss of cytochrome c oxidase function promotes the Warburg effect and upregulates several genes with roles in tumor development.

ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds.

A polymorphic mutation in the acetaldehyde dehydrogenase 2 (ALDH2) gene has been epidemiologically linked to the high susceptibility to esophageal carcinogenesis for individuals with alcohol use disorders. Mice subjected to alcohol drinking show increased oxidative stress and DNA adduct formation in esophageal epithelia where Aldh2 loss augments alcohol-induced genotoxic effects; however, it remains elusive as to how esophageal epithelial cells with dysfunctional Aldh2 cope with oxidative stress related to alcohol metabolism. Here, we investigated the role of autophagy in murine esophageal epithelial cells (keratinocytes) exposed to ethanol and acetaldehyde. We find that ethanol and acetaldehyde trigger oxidative stress via mitochondrial superoxide in esophageal keratinocytes. Aldh2-deficient cells appeared to be highly susceptible to ethanol- or acetaldehyde-mediated toxicity. Alcohol dehydrogenase-mediated acetaldehyde production was implicated in ethanol-induced cell injury in Aldh2 deficient cells as ethanol-induced oxidative stress and cell death was partially inhibited by 4-methylpyrazole. Acetaldehyde activated autophagy flux in esophageal keratinocytes where Aldh2 deficiency increased dependence on autophagy to cope with ethanol-induced acetaldehyde-mediated oxidative stress. Pharmacological inhibition of autophagy flux by chloroquine stabilized p62/SQSTM1, and increased basal and acetaldehyde-mediate oxidative stress in Aldh2 deficient cells as documented in monolayer culture as well as single-cell derived three-dimensional esophageal organoids, recapitulating a physiological esophageal epithelial proliferation-differentiation gradient. Our innovative approach indicates, for the first time, that autophagy may provide cytoprotection to esophageal epithelial cells responding to oxidative stress that is induced by ethanol and its major metabolite acetaldehyde. Defining autophagymediated cytoprotection against alcohol-induced genotoxicity in the context of Aldh2 deficiency, our study provides mechanistic insights into the tumor suppressor functions of ALDH2 and autophagy in alcohol-related esophageal carcinogenesis.

Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors.

Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi.

Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

Defects in cytochrome c oxidase expression induce a metabolic shift to glycolysis and carcinogenesis.

Mitochondrial metabolic dysfunction is often seen in cancers. This paper shows that the defect in a mitochondrial electron transport component, the cytochrome c oxidase (CcO), leads to increased glycolysis and carcinogenesis. Using whole genome microarray expression analysis we show that genetic silencing of the CcO subunit Cox4i1 in mouse C2C12 myoblasts resulted in metabolic shift to glycolysis, activated a retrograde stress signaling, and induced carcinogenesis. In the knockdown cells, the expression of Cox4i1 was less than 5% of the control and the expression of the irreversible glycolytic enzymes (Hk1, Pfkm and Pkm) increased two folds, facilitating metabolic shift to glycolysis. The expression of Ca (2+) sensitive Calcineurin (Ppp3ca) and the expression of PI3-kinase (Pik3r4 and Pik3cb) increased by two folds. This Ca (2+)/Calcineurin/PI3K retrograde stress signaling induced the up-regulation of many nuclear genes involved in tumor progression. Overall, we found 1047 genes with 2-folds expression change (with p-value less than 0.01) between the knockdown and the control, among which were 35 up-regulated genes in pathways in cancer (enrichment p-value less than 10(- 5)). Functional analysis revealed that the up-regulated genes in pathways in cancer were dominated by genes in signal transduction, regulation of transcription and PI3K signaling pathway. These results suggest that a defect in CcO complex initiates a retrograde signaling which can induce tumor progression. Physiological studies of these cells and esophageal tumors from human patients support these results. GEO accession number = GSE68525.

LocSigDB: a database of protein localization signals.

LocSigDB (http://genome.unmc.edu/LocSigDB/) is a manually curated database of experimental protein localization signals for eight distinct subcellular locations; primarily in a eukaryotic cell with brief coverage of bacterial proteins. Proteins must be localized at their appropriate subcellular compartment to perform their desired function. Mislocalization of proteins to unintended locations is a causative factor for many human diseases; therefore, collection of known sorting signals will help support many important areas of biomedical research. By performing an extensive literature study, we compiled a collection of 533 experimentally determined localization signals, along with the proteins that harbor such signals. Each signal in the LocSigDB is annotated with its localization, source, PubMed references and is linked to the proteins in UniProt database along with the organism information that contain the same amino acid pattern as the given signal. From LocSigDB webserver, users can download the whole database or browse/search for data using an intuitive query interface. To date, LocSigDB is the most comprehensive compendium of protein localization signals for eight distinct subcellular locations. Database URL: http://genome.unmc.edu/LocSigDB/

Targeting of splice variants of human cytochrome P450 2C8 (CYP2C8) to mitochondria and their role in arachidonic acid metabolism and respiratory dysfunction.

In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.