Dissertation > Excellent graduate degree dissertation topics show

Studies on the Endocytic Trafficking and furin Protease-induced Cleavage of Several Recombinant Pseudomonas Exotoxin Proteins in Hela Cells

Author: LiLiang
Tutor: ChenZhiZuo
School: Shantou University
Course: Biochemistry and Molecular Biology
Keywords: Pseudomonas exotoxin A fluorescent protein ADP-ribsylation FRET
CLC: Q93
Type: Master's thesis
Year: 2011
Downloads: 29
Quote: 0
Read: Download Dissertation

Abstract


Pseudomonas exotoxin A (PEA), produced by the Gram-negative bacterium Pseudomonas aeruginosa, is a toxic protein capable of inactivating the eukaryotic elongation factor 2 (EF-2) by transferring the adenosine diphosphate (ADP)-ribose group from nicotinamide adenine dinucleotide (NAD+) to EF-2. ADP-ribosylated EF-2 is inactive in participating in the ribosome-mediated peptide chain elongation, thus arresting the protein synthesis process in the intoxicated cells. Cells that are unable to synthesize essential proteins eventually die, although PEA is also known to intoxicate cells through other parallel processes, such as inducing apoptosis. The PEA-mediated cell intoxication is a complex cellular and molecular process. Numerous efforts and endeavors have been dedicated to exploring the targeted receptors on cytoplasm, the trafficking routes and processes, and the molecular mechanism of intoxication of PEA in the cell cytosol.In this work, we designed four different recombinant toxins fused with fluorescent proteins and studied their endocytic and intracellular trafficking processes using laser confocal microscopy and immunocytochemistry. The dynamics of toxin trafficking were quantitated in real times. The intracellular organelles visited by PEA during trafficking were identified. The cleavage efficiency of PEA to express toxicity was estimated. The results showed that medium acidification is not an important factor in affecting binding of PEAs to cytoplasmic receptors, internalization to the early endosomes, late endosomes, and trans-Golgi network. Modifying the C terminus by the signal sequence KDEL promoted PEA trafficking efficiency to endoplasmic reticulum (ER), indicating that PEA trafficking is mediated by the KDEL receptors retrieval system in ER.The domain III of PEA is a 38 KDa fragment possessing ADP-ribosylating activity, releasable after cleavage by furin in the domain II to express full toxicity. We constructed a fusion toxin eBFP-PE44-eGFP (BPG) to estimate the cleavage efficiency by furin-like proteases using the fluorescent resonance energy transferring (FRET) method. The cleavage efficiency of BPG at 2 hrs following endocytosis was approximately 5%. The cleavage process was mediated by low pH, consistent with the view that medium acidification could promote toxicity of PEA. Immunocytochemical labeling indicated that most of the activated domain III fragments were still located within ER after 3 hrs, indicating that only a small fraction of the internalized PEAs translocated to the cytosol and mediated ADP-ribosylation of EF-2.

Related Dissertations

  1. Identification of Genes Related to Virulence of Fusarium Graminearum and Biopaning of DON Toxin Specific Affinity Peptides,S432.4
  2. Construction and Application of the Transgenic Yeast Strain Expressing Metallothionein,Q78
  3. The Toxic Threshold by Biuret to Crops in the Compound Fertilizer and Research on the Degrading Bacterium of Biuret,S143
  4. Construction and Genetic Transofrmation of the Rice Chloroplast-specific Expression Vector,S511
  5. The Research on Fluorescence Spectrum Imaging and Photobleaching at Single Molecule Level,O657.3
  6. An Optimized Co-Culture Model of Mouse Blastocysts and Mouse Melanoma Cells,Q813
  7. Studies on the in Vivo Interaction between MD-2 and TLR4 Based on FRET,R459.7
  8. Fabrication and Properties Research of Novel Fluorescent Nanoparticles for Cupric Ion Detection,TB383.1
  9. Studies on the Synthesis of CD36 Transmembrane Peptides and Mechanism of Transmembrane Domain Interactions,R96
  10. Gold Nanoparticles Quenching Fluorescence Probe for Glucose Detection and Cell Imaging Based on Fluorescence Resonance Energy Transfer (fret),Q503
  11. Parasitism and Pathogenicity of Nematode-endoparasitic Hirsutella Spp. Against Heterodera Glycines and Caenorhabditis Elegans,S432.45
  12. Inverstigation of Sulfhydryl Oxidases and Their Probes,Q55
  13. Preparation and Characterization of Ratiometric Mercury Ion Sensor Based on FRET,TQ316.334
  14. Preparation and biological analysis of semiconductor nanomaterials,TB383.1
  15. Fermentation temperature on the biosynthesis of active,S482.28
  16. Human respiratory syncytial virus replicon functional micro preliminary study,R373
  17. The Analysis of GPD1 Promoter for the Key Enzyme of Glycerol Synthesis from Candida Glycerinogenes and Its Study in Tobacco,Q943.2
  18. Construction of Fusion Expression Plasmid PQE3-IL3-PE38KDEL,R392.1
  19. Construction of Plant Expression Vectors with Multi-Selectable Marker,Q943.2
  20. A promoter probe vector construction and low temperature inducible promoter,Q78
  21. Study on Gene Expression and Immune Response after the Immunization of Somatostatin DNA Vaccine in Mice,S852.4

CLC: > Biological Sciences > Microbiology
© 2012 www.DissertationTopic.Net  Mobile