Genome-Wide Analysis of Effectors of Peroxisome Biogenesis

Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for β-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1

Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin

The development of novel targeted therapies has become an important research focus for lung cancer treatment. Our previous study has shown leptomycin B (LMB) significantly inhibited proliferation of lung cancer cells; however, p53 wild type lung cancer cells were resistant to LMB. Therefore, the objective of this study was to develop and evaluate a novel therapeutic strategy to sensitize LMB-resistant lung cancer cells by combining LMB and doxorubicin (DOX). Among the different treatment regimens, pretreatment with DOX (pre-DOX) and subsequent treatment with LMB to A549 cells significantly decreased the 50% inhibitory concentration (IC50) as compared to that of LMB alone (4.4 nM vs. 10.6 nM, P<0.05). Analysis of cell cycle and apoptosis by flow cytometry further confirmed the cytotoxic data. To investigate molecular mechanisms for this drug combination effects, p53 pathways were analyzed by Western blot, and nuclear proteome was evaluated by two dimensional-difference gel electrophoresis (2D-DIGE) and mass spectrometry. In comparison with control groups, the levels of p53, phospho-p53 (ser15), and p21 proteins were significantly increased while phospho-p53 (Thr55) and survivin were significantly decreased after treatments of pre-DOX and LMB (P<0.05). The 2D-DIGE/MS analysis identified that sequestosome 1 (SQSTM1/p62) had a significant increase in pre-DOX and LMB-treated cells (P<0.05). In conclusion, our results suggest that drug-resistant lung cancer cells with p53 wild type could be sensitized to cell death by scheduled combination treatment of DOX and LMB through activating and restoring p53 as well as potentially other signaling pathway(s) involving sequestosome 1