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Proteomic analysis of the Stress response in insects Rodney Hull

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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 individuals survival. Stress response pathways should therefore ensure the organisms survival, while reducing the age related genetic variation to a minimum Stress

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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 InfectionDNA damage Drosophila melanogaster Euoniticellus intermedius

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

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

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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

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Triphosphate isomerase (TPI) levels decreases during the recovery from camptothecin treatment. The TPI mutant D. melanogaster wasted 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.

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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.

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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?

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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 mol/min/ml Untreated sample had an activity 0.051 mol/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. B: The relative activities and amounts of GST as determined by enzyme assays and 2D PAGE GST amount detected by 2DpageGST activity detected by enzyme assay Camptothecin treatedUntreated A: GST activity assay 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 050100150200250300350400 Time (seconds) Absorbance Camptothecin treatedUntreated

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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.

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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. Western blot analysis of Drosophila melanogaster treated with camptothecin, methyl pyruvate and both camptothecin and methyl pyruvate.

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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 Marker Crude extract 10 ug DNA 8 ug DNA 6 ug DNA 4 ug DNA

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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.

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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.

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Beauveria bassiana Euoniticellus intermedius

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Immune System in Drosophila and Coleoptera Most studies in Tenebrio molitor Tribolium castaneum and Holotrichia diomphalia (GNBP3) (PGRP-SA) Serine protease cascade via an modular serine protease (MSP) followed by two types of CLIP domain serine proteases at pro-spatzle.

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

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2D gel electrophoresis of E. intermedius hemolymph proteins

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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. 33042203 p=0.048P=0.0054 An alignment of protein sequences encoded by these contigs with Psh, Holotricia PPAF-II.

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Fragment 5310 used to design primers for RT-PCR Clone alligned with a serine protease (cl15 contig1). Not necessarily immune related Not necessarily equivalent molecule

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

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

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

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

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Antimicrobial activity. Hi S Cation exchange column Protein extract Protein flow through Negatively charged proteins pH 7.5 wash Protein s with pI 7.5 C18 hydrophobicity 5% CH 4 CN 80% CH 4 CN 40% CH 4 CN 60% CH 4 CN Reverse phase HPLC

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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 Inhibition assays

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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)

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

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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

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

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No stress response pathway occurs in isolation.

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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