Uniform nomenclature for the protein transport machinery of the mitochondrial membranes

[no abstract avaiable

Discovery of Genes Activated by the Mitochondrial Unfolded Protein Response (mtUPR) and Cognate Promoter Elements

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

The Chop Gene Contains an Element for the Positive Regulation of the Mitochondrial Unfolded Protein Response

We have previously reported on the discovery of a mitochondrial specific unfolded protein response (mtUPR) in mammalian cells, in which the accumulation of unfolded protein within the mitochondrial matrix results in the transcriptional activation of nuclear genes encoding mitochondrial stress proteins such as chaperonin 60, chaperonin 10, mtDnaJ, and ClpP, but not those encoding stress proteins of the endoplasmic reticulum (ER) or the cytosol. Analysis of the chaperonin 60/10 bidirectional promoter showed that the CHOP element was required for the mtUPR and that the transcription of the chop gene is activated by mtUPR. In order to investigate the role of CHOP in the mtUPR, we carried out a deletion analysis of the chop promoter. This revealed that the transcriptional activation of the chop gene by mtUPR is through an AP-1 (activator protein-1) element. This site lies alongside an ERSE element through which chop transcription is activated in response to the ER stress response (erUPR). Thus CHOP can be induced separately in response to 2 different stress response pathways. We also discuss the potential signal pathway between mitochondria and the nucleus for the mtUPR

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors

Identification of mtUPR response element in <i>chop</i> promoter.

<p>(A) and (B): <i>Chop</i> is transcriptionally activated by mtUPR. COS-7 cells co-transfected with empty vector or OTCΔ were assayed for GFP (A) or luciferase (B) 32 h after transfection. (C): identification of an mtUPR element in the <i>chop</i> promoter was determined by a deletion analysis as shown. Deletions are shown as distance (bp) from the <i>chop</i> transcription start site. The fold activation of the promoter constructs in cells transfected with OTCΔ (slash bars) are compared with vector controls (open bars) as relative luciferase (RLU) activity. RLU activity of the promoter constructs in cells treated with or without 2 µg/ml tunicamycin (TM), to produce erUPR is shown as a control. Data represents the mean±SEM from experiments performed in triplicate.</p

<i>Chop</i> and <i>c/ebp</i>β promoters contain AP-1 sites and are inducible by mtUPR.

<p>(A) Nucleotide sequence alignment of the mammalian <i>chop</i> promoters (−278 to −222) from human, bovine, mouse and rat. Bold letters show the highly conserved bases of the AP-1 site and the asterisks show the highly conserved sequence surrounding the AP-1 site in <i>chop</i> promoters. The position of the putative novel element of N 30 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000835#pone.0000835-Xie1" target="_blank">[18]</a> is shown in the box. (B): Nucleotide sequences of mammalian <i>c/ebp</i>β promoter region around AP-1 site (Human, Mouse, and Rat) is compared with the <i>chop</i> promoter sequence (−278 to −233). The numbers refer to the distance from transcription initiation site of human <i>chop</i> or human, mouse, and Rat <i>c/ebp</i>β. The asterisks indicate the conserved nucleotides around the AP-1 site. (C): C/EBPβ expression in response to mtUPR. Extracts from cells transfected with vector or OTCΔ were subjected to western blotting and probed with antibodies against C/EBPβ and tubulin as control and show that C/EBPβ, like CHOP is induced by expression of OTCΔ.</p