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Proteomic analysis of the Stress response in insects

Proteomic analysis of the Stress response in insects. Rodney Hull. Stress. “Stress is a condition evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche ” – ( Korsloot 2004)

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Proteomic analysis of the Stress response in insects

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  1. Proteomic analysis of the Stress response in insects Rodney Hull

  2. Stress • “Stress is a condition evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche” – (Korsloot 2004) • Not permanent and it results in a specific set of molecular responses that should counteract it and increase the chances of the individual’s survival. • Stress response pathways should therefore ensure the organisms survival, while reducing the age related genetic variation to a minimum

  3. Insects have evolved to occupy every biological niche other than those of the Polar Regions and deep marine environments. In terms of species number insects represent the largest class in the animal kingdom, with about 1-3 million species currently described and many as yet undiscovered species. Stress DNA damage Infection Drosophila melanogaster Euoniticellus intermedius

  4. DNA Damage. Replication fork stalls Drosophila Genotoxic stress Camptothecin, DSBs, UV, Genotoxic stress (DSBs) ATR MEI-41 DmATM CHK1, CHK2 ATM GRPS, MNK p53 Mdm2 Pro-apoptotic signals ? p53 p53P p53P (functional orthologues of Diablo/Smac) Bax, Nox, Puma Rpr, hid, skl DIABLO/Smac & cytochrome c DIAPI (inhibitor of caspases) Caspases IAPs caspases apoptosis apoptosis

  5. 2D gel electrophoresis of D. melanogaster crude protein extract before and after exposure to camptothecin.

  6. Oxidative metabolism Predominantly enzymes involved in glycolysis and the citric acid cycle Significant up-regulation of ATP synthase components- increase in ATP usage. Apoptotic stimuli -increase in cellular ATP levels . Energy is required for the orderly progression of apoptosis and for DNA damage repair

  7. Triphosphateisomerase (TPI) levels decreases during the recovery from camptothecin treatment.   • The TPI mutant D. melanogasterwasted away - motor impairment • vacuolar neuropathology • severely reduced lifespan. • Similiar pathologies are found in human TPI deficiency. • Resemble the side effects of camptothecin treatment. • The inhibition of TPI by camptothecin treatment may provide an explanation for some of these side effects.

  8. Protein folding DNA damage results in the generation of free radicals- Expression of proteins involved in de-toxification and anti-oxidant activities increases following camptothecin treatment Glutathione S transferases & superoxide dismutase significantly up-regulated The expression of the iron binding protein ferritin also increases significantly (may protect the cell against ROS. The expression of the nitrogen metabolising enzyme Glutamine synthetase increases significantly -enzyme is prone to damage by oxidative stress.

  9. Cytoskeletal and Other proteins The levels of Mitochondrial processing peptidase (MPP) also increase following camptothecin exposure. The expression levels of the 20S proteosome, which is involved in the degradation of damaged, unneeded or mis-folded proteins, is also significantly increased following camptothecin exposure. Yolk protein is known to act as an antioxidant in queen bees improving their lifespan Regulated by hormones- Hormone fluctuation?

  10. 0.2 0.18 0.16 0.14 0.12 Absorbance 0.1 0.08 0.06 0.04 0.02 0 0 50 100 150 200 250 300 350 400 Time (seconds) Camptothecin treated Untreated A: GST activity assay GST amount detected by 2Dpage GST activity detected by enzyme assay Camptothecin treated Untreated B: The relative activities and amounts of GST as determined by enzyme assays and 2D PAGE GST enzyme assay for camptothecin and untreated fly extracts to confirm optical density changes do reflect changes in protein amount. • The camptothecin treated samples had an activity of 0.15 μmol/min/ml • Untreated sample had an activity 0.051 μmol/min/ml. • The camptothecin treated flies therefore had threefold higher activities of GST. • 2D page showed a 6 fold higher amount of GST present in camptothecin treated flies.

  11. Snama • Identified from a promoter trap mutagenesis screen for apoptotic genes • SNAMA is an RBBP6 orthologue • Consists of a ubiquitin-like domain, a RING-finger like motif and p53- and Rb1- binding domains. • The exact role of SNAMA is unknown. • DWNN shares 22% identity with ubiquitin but has an ubiquitin like fold . • Snama PA = 139 kDa. • Snama PB = 55kDa • Snama is expressed throughout development with its levels decreasing in the later embryonic stages. Double negative mutants of snama are not viable, • May regulate Dmp53 and retinoblastoma protein (RBF). • RBBP6 is known to enhance the activity of Mmd2 and to • interact with p53 in vertebrates Candidate for p53 • regulation in the invertebrate system perhaps without the • cooperation of Mdm2.

  12. Western blot analysis of Drosophila melanogaster treated with camptothecin, methyl pyruvate and both camptothecin and methyl pyruvate. Anti Dmp53 detects a 45 and 30 kDa band. • 43.7 kDa Dmp53-PA – • 36.1 kDa Dmp-53-PD Anti-Snama • 35 kDa band in all flies regardless of treatment • 40 kDa band in all flies regardless of treatment • 50 kDa is detected only when flies are exposed to methyl pyruvate.

  13. Marker Crude extract 10 ug DNA 8 ug DNA 6 ug DNA 4 ug DNA EMSA Promoter Region - Luciferase assay DNA biotin labelled Crude protein extract from Drosophila Incubate DNA and protein extract Check for shift in mobility compared to wild type (western blot) DNA-protein extracts purified with streptavidin resin Run elution on SDS gel Mass Spec of selected bands

  14. DREF (DNA replication-related element-binding factor) • The Drosophila DREF homo-dimer binds specifically to the DRE sequence (5'-TATCGATA) in the promoters of many DNA replication/ cell proliferation-related genes to activate their transcription • Ectopic expression of DREF induces abnormal DNA synthesis, apoptosis and failure to • Differentiate • Knockdown of DREF in vivo demonstrated its requirement for normal progression through the cell cycle • DNA replication, transcriptional regulation, cell cycle regulation, growth signal transduction and protein metabolism.

  15. Conclusion • Camptothecin exposure results in a glycolytic flux in normal cells • This metabolic shift is also different to that observed in cancer cells (Warburg hypothesis). • The differences could be exploited to reduce stress on normal cells during chemotherapy. • Methyl pyruvate in the diet (bypassing the glycolytic pathway) led to differential expression of Dmp53 and Snama and improvement in embryonic development • Possible use of Drosophila as a model system to study Camptothecin pharmacodynamics.

  16. Beauveria bassiana Euoniticellus intermedius

  17. Immune System in Drosophila and Coleoptera Most studies in TenebriomolitorTribolium castaneum and Holotrichiadiomphalia (GNBP3) (PGRP-SA) Serine protease cascade via an modular serine protease (MSP) followed by two types of CLIP domain serine proteases at pro-spaёtzle .

  18. 2D gel electrophoresis of E. intermedius hemolymph proteins In both treated and untreated beetles. In treated beetles only. In untreated beetles

  19. 2D gel electrophoresis of E. intermedius hemolymph proteins

  20. Serine proteases Mediate extracellular signaling activated by bacterial and fungal pathogens. Spots 2203 and 3304 yielded several fragments - sequences of proteins encoded by CL8Contig1 and CL20Contig1. P=0.0054 p=0.048 2203 3304 • An alignment of protein sequences encoded by these contigs with Psh, • HolotriciaPPAF-II.

  21. Fragment 5310 used to design primers for RT-PCR Clone alligned with a serine protease (cl15contig1). Not necessarily immune related Not necessarily equivalent molecule

  22. Pattern recognition receptors p= 0.0067 Apolipophorin III (apoLp-III) Lipid transport protein in the hemolymph. Important for insect immunity PRR that responds to b-1,3-glucanPAMPS found in fungi. ApoLp-IIIhas been shown to bind to bacterial and fungal cell wall components . Apolipophorin III 7203

  23. Other proteins p=0.776 Similiar to leucine rich repeat region of Toll 6 Transferrin – Iron binding protein with antimicrobial activity Toll 6410 p=0.036 Transferrin 6204

  24. Drosophila Tribolium E. intermedius Fungi β-glucan Fungi β-glucan Pattern recognition molecular patterns (PAMPS) Gram +ve bacteria Lys-type PGN Fungi β-glucan Virulence Factors GNBP3 (007956_ 1645_0 789_c_s) IDGF GNBP3 GNBP1 PGRP-SA PGRP-SD Extracellular recognition factors GNBP3 Apolipophorin III (6203) (Cl123Contig1) Persephone Persephone (3304) (CL23 contig1) Danger signals ModSP SP MSP Pro-MSP Persephone (CL23 contig1) Serine protease Cascade ? Serpin Grass Nec-1 (CL111Contig1) Serine protease cascade SAE Pro-SAE Spirit Spheroide Sphinx Pro-SPE Spatzle processing enzyme (Cl47Contig1) SPE Spatzle processing enzyme Pro-Spz Spz Pro-Spz Spz Pro-Spz Spz Cl238contig1 Transmembrane receptor Toll (CL673 contig1) Toll (CL673 contig1) Toll (6410) (Cl673contig1 Transferrin \Defensin? Antimicrobial peptides Antimicrobial peptides Effectors

  25. In beetles -Defensins -Coleoptericin and holotricin, acoleptins -A single cecropin has been identified from A. luxuriosaunconfirmed report in Eleodes -Alo-3 knottin type fold (present in plant antimicrobial peptides not insects) -Scarabaecin 8 cystines (beetle drosomycin?) Antimicrobial activity

  26. Antimicrobial activity. 1 M NaCleluant Positively charged proteins with pI> 7.5 Protein extract Hi S Cation exchange column C18hydrophobicity 5% CH4CN 80% CH4CN 40% CH4CN 60% CH4CN Protein flow through Negatively charged proteins pH 7.5 wash Protein s with pI <7.5 Reverse phase HPLC

  27. Inhibition assays • Anti-bacterial activity against Gram positive (strong activity) and Gram negative (weak activity) is present in hemolymph and whole body extract • Anti-bacterial activity is in the positively charged protein fraction • pI greater than 7.5 • Negatively charged => glycine rich • Tribolium castaneum Defensin pI= 9.3 Cecropin pI =9.21 • Activity is due to hydrophobic protein

  28. Inhibition assays Fungal infection – Antibacterial activity Hydrophobic positive protein samples were the most effective State of immune challenge no effect on the potency of the antimicrobial peptide. Why – because Dung is dirty (microbe rich environment)

  29. Inhibitory activity is a heat stable protein p=0.102 p=0.004 Liquid inhibition assays performed on Micrococcus luteus Proteinase treatment abolishes activity Heat stable up to 50o C Activity is due to a heat stable protein p= 0.002 p=0.0001

  30. Conclusions • Treatment of the beetles with fungus activates the toll signalling pathway- Aspects of the Toll signalling pathway seem to conserved in E. Intermedius • The poorly defined pathway characterized by PRR apolipophorin is also activated by fungal infection. • Antimicrobial peptide appears to be a Defensin

  31. Comparison between the proteomic response of D. melanogaster to DNA damage and E. intermedius to infection.

  32. No stress response pathway occurs in isolation.

  33. Conclusion Similar functional classes up-regulated in both responses. Cytoskeletal proteins Metabolism proteins significantly up-regulated in both stress responses. Much larger in response to DNA damage (glycolytic flux.) Developmental proteins have known functions in immunity Stress response-ROS

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