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Discovery of Genes Activated by the Mitochondrial Unfolded Protein Response (mtUPR) and Cognate Promoter Elements  [PDF]
Jonathan E. Aldridge, Tomohisa Horibe, Nicholas J. Hoogenraad
PLOS ONE , 2007, DOI: 10.1371/journal.pone.0000874
Abstract: In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebpβ gene is similarly activated and CHOP and C/EBPβ subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes. We found that these genes encoding mitochondrial proteases YME1L1 and MPPβ, import component Tim17A and enzymes NDUFB2, endonuclease G and thioredoxin 2, all contain a CHOP element in their promoters. In contrast, genes encoding mitochondrial proteins Afg3L2, Paraplegin, Lon and SAM 50, which do not have a CHOP element, were not up-regulated. Conversely, genes with CHOP elements encoding cytosolic proteins were not induced by the accumulation of unfolded proteins in mitochondria. These results indicate that mtUPR responsive genes appear to share a requirement for a CHOP element, but that this is not sufficient for the regulation of the mtUPR. A more detailed analysis of promoters of mtUPR responsive genes revealed at least two additional highly conserved, putative regulatory sites either side of the CHOP element, one a motif of 12 bp which lies 14 bp upstream of the CHOP site and another 9 bp element, 2 bp downstream of the CHOP site. Both of these additional elements are conserved in the promoters of 9 of the ten mtUPR responsive genes we have identified so far, the exception being the Cpn60/10 bidirectional promoter. Mutation of each of these elements substantially reduced the mtUPR responsiveness of the promoters suggesting that these elements coordinately regulate mtUPR.
2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2
Hao Liu, Chen Jiang, Christopher J Lavis, Amanda Croft, Li Dong, Hsin-Yi Tseng, Fan Yang, Kwang Tay, Peter Hersey, Xu Zhang
Molecular Cancer , 2009, DOI: 10.1186/1476-4598-8-122
Abstract: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1α and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1α, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells.These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1α/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.TNF-related apoptosis-inducing ligand (TRAIL) appears to be a promising candidate for cancer therapeutics because of its ability to preferentially induce apoptosis in malignant cells [1-3]. The potential significance of TRAIL as an anti-cancer agent has been supported by studies in animal models showing selective toxicity to human tumor xenografts but not normal tissues [4,5]. Induction of apoptosis by TRAIL is mediated by its interaction with two death domain containing receptors, TRAIL-R1 and -R2 [1-3]. This in turn orchestrates the assembly of the death-inducing signaling complex (DISC) that contains adapter components such as Fas associated death domain (FADD) that activates initiator caspases, caspase-8 and -10, leading eventually to activation of effector caspases such as caspase-3 and t
Sinulariolide Induced Hepatocellular Carcinoma Apoptosis through Activation of Mitochondrial-Related Apoptotic and PERK/eIF2α/ATF4/CHOP Pathway  [PDF]
Yi-Jen Chen,Jui-Hsin Su,Chia-Yu Tsao,Chun-Tzu Hung,Hsiang-Hao Chao,Jen-Jie Lin,Ming-Hui Liao,Zih-Yan Yang,Han Hisang Huang,Feng-Jen Tsai,Shun-Hsiang Weng,Yu-Jen Wu
Molecules , 2013, DOI: 10.3390/molecules180910146
Abstract: Sinulariolide, an active compound isolated from the cultured soft coral Sinularia flexibilis, has potent anti-microbial and anti-tumorigenesis effects towards melanoma and bladder cancer cells. In this study, we investigated the effects of sinulariolide on hepatocellular carcinoma (HCC) cell growth and protein expression. Sinulariolide suppressed the proliferation and colony formation of HCC HA22T cells in a dose-dependent manner and induced both early and late apoptosis according to flow cytometry, Annexin V/PI stain and TUNEL/DAPI stain analyses. A mechanistic analysis demonstrated that sinulariolide-induced apoptosis was activated through a mitochondria-related pathway, showing up-regulation of Bax, Bad and AIF, and down- regulation of Bcl-2, Bcl-xL, MCl-1 and p-Bad. Sinulariolide treatment led to loss of the mitochondrial membrane potential, release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and caspase-3. Sinulariolide-induced apoptosis was significantly blocked by the caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK. The increased expression of cleaved PARP also suggested that caspase-independent apoptotic pathway was involved. In the western blotting; the elevation of ER chaperones GRP78; GRP94; and CALR; as well as up-regulations of PERK/eIF2α/ATF4/CHOP; and diminished cell death with pre-treatment of eIF2α phosphatase inhibitor; salubrinal; implicated the involvement of ER stress-mediated PERK/eIF2α/ATF4/CHOP apoptotic pathway following sinulariolide treatment in hepatoma cells. The current study suggested sinulariolide-induced hepatoma cell cytotoxicity involved multiple apoptotic signal pathways. This may implicate that sinulariolide is a potential compound for the treatment of hepatocellular carcinoma.
HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization  [PDF]
John P. Norman, Seth W. Perry, Holly M. Reynolds, Michelle Kiebala, Karen L. De Mesy Bentley, Margarita Trejo, David J. Volsky, Sanjay B. Maggirwar, Stephen Dewhurst, Eliezer Masliah, Harris A. Gelbard
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003731
Abstract: Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.
Surveillance-Activated Defenses Block the ROS–Induced Mitochondrial Unfolded Protein Response  [PDF]
Eva D. Runkel,Shu Liu,Ralf Baumeister ,Ekkehard Schulze
PLOS Genetics , 2013, DOI: 10.1371/journal.pgen.1003346
Abstract: Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPRmt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPRmt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPRmt. Activation of the UPRmt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPRmt, suggesting that surveillance-activated defenses specifically inhibit the UPRmt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.
ER Stress-Inducible Factor CHOP Affects the Expression of Hepcidin by Modulating C/EBPalpha Activity  [PDF]
Susana J. Oliveira, Jorge P. Pinto, Gon?alo Picarote, Vera M. Costa, Félix Carvalho, Maria Rangel, Maria de Sousa, Sérgio F. de Almeida
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006618
Abstract: Endoplasmic reticulum (ER) stress induces a complex network of pathways collectively termed the unfolded protein response (UPR). The clarification of these pathways has linked the UPR to the regulation of several physiological processes. However, its crosstalk with cellular iron metabolism remains unclear, which prompted us to examine whether an UPR affects the expression of relevant iron-related genes. For that purpose, the HepG2 cell line was used as model and the UPR was activated by dithiothreitol (DTT) and homocysteine (Hcys). Here, we report that hepcidin, a liver secreted hormone that shepherds iron homeostasis, exhibits a biphasic pattern of expression following UPR activation: its levels decreased in an early stage and increased with the maintenance of the stress response. Furthermore, we show that immediately after stressing the ER, the stress-inducible transcription factor CHOP depletes C/EBPα protein pool, which may in turn impact on the activation of hepcidin transcription. In the later period of the UPR, CHOP levels decreased progressively, enhancing C/EBPα-binding to the hepcidin promoter. In addition, analysis of ferroportin and ferritin H revealed that the transcript levels of these iron-genes are increased by the UPR signaling pathways. Taken together, our findings suggest that the UPR can have a broad impact on the maintenance of cellular iron homeostasis.
Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages  [PDF]
Judith A. Smith ,Mike Khan,Diogo D. Magnani,Jerome S. Harms,Marina Durward,Girish K. Radhakrishnan,Yi-Ping Liu,Gary A. Splitter
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003785
Abstract: Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.
The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling  [PDF]
Catherine Trivigno,Theodor E. Haerry
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016799
Abstract: Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.
Determination of the melon chloroplast and mitochondrial genome sequences reveals that the largest reported mitochondrial genome in plants contains a significant amount of DNA having a nuclear origin
Luis Rodríguez-Moreno, Víctor M González, Andrej Benjak, M Carmen Martí, Pere Puigdomènech, Miguel A Aranda, Jordi Garcia-Mas
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-424
Abstract: The nucleotide sequences of the melon chloroplast and mitochondrial genomes were determined. The chloroplast genome (156,017 bp) included 132 genes, with 98 single-copy genes dispersed between the small (SSC) and large (LSC) single-copy regions and 17 duplicated genes in the inverted repeat regions (IRa and IRb). A comparison of the cucumber and melon chloroplast genomes showed differences in only approximately 5% of nucleotides, mainly due to short indels and SNPs. Additionally, 2.74 Mb of mitochondrial sequence, accounting for 95% of the estimated mitochondrial genome size, were assembled into five scaffolds and four additional unscaffolded contigs. An 84% of the mitochondrial genome is contained in a single scaffold. The gene-coding region accounted for 1.7% (45,926 bp) of the total sequence, including 51 protein-coding genes, 4 conserved ORFs, 3 rRNA genes and 24 tRNA genes. Despite the differences observed in the mitochondrial genome sizes of cucurbit species, Citrullus lanatus (379 kb), Cucurbita pepo (983 kb) and Cucumis melo (2,740 kb) share 120 kb of sequence, including the predicted protein-coding regions. Nevertheless, melon contained a high number of repetitive sequences and a high content of DNA of nuclear origin, which represented 42% and 47% of the total sequence, respectively.Whereas the size and gene organisation of chloroplast genomes are similar among the cucurbit species, mitochondrial genomes show a wide variety of sizes, with a non-conserved structure both in gene number and organisation, as well as in the features of the noncoding DNA. The transfer of nuclear DNA to the melon mitochondrial genome and the high proportion of repetitive DNA appear to explain the size of the largest mitochondrial genome reported so far.The melon (Cucumis melo L.) is an important vegetable crop grown in temperate, subtropical and tropical regions worldwide. The melon belongs to the Cucurbitaceae family, which also comprises other vegetable crops such as cucumber, w
The mosquito Aedes aegypti has a large genome size and high transposable element load but contains a low proportion of transposon-specific piRNAs
Peter Arensburger, Robert H Hice, Jennifer A Wright, Nancy L Craig, Peter W Atkinson
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-606
Abstract: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism.Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome, suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.Research into the genome-wide regulation of transposons in model organisms such as Drosophila melanogaster, zebrafish and mice has revealed the importance of two small RNA pathways for controlling their movement thereby preserving genome stability [1-8]. This is especially important considering the abundance and diversity of transposons in eukaryote genomes in which unregulated movement of active elements, or the non-autonomous sequences they can re-mobilize, would lead to insertion mutagenesis throughout the genome resulting in a decrease in genetic fitness. In D. melanogaster the Piwi-interacting RNAs (piRNAs) appear to function primarily in the regulation of transposons in both the germ line and in somatic tissues that envelope the ovaries althoug
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