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)
•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
•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
Drosophila melanogaster
Infection
Euoniticellus intermedius
DNA Damage.
Genotoxic
stress
Camptothecin,
DSBs, UV,
Replication
fork stalls
Drosophila
Genotoxic
stress (DSBs)
ATR
MEI-41
DmATM
ATM
CHK1, CHK2
GRPS, MNK
Mdm2
p53
?
p53
Pro-apoptotic
signals
p53P
p53P
Bax, Nox, Puma
Rpr, hid, skl
(functional
orthologues of
Diablo/Smac)
DIABLO/Smac & cytochrome c
caspases
apoptosis
IAPs
Caspases
apoptosis
DIAPI
(inhibitor of
caspases)
2D gel electrophoresis of D. melanogaster crude
protein extract before and after exposure to
camptothecin.
Oxidative metabolism
Oxidative metabolism
bellwether
Dihydrolipoamide
dehydrogenase…
Arginine kinase,
ATP synthase beta subunit
Aconitase
Pyruvate kinase
lethal (1) G0030, isoform A
UGP, isoform A
Enolase, isoform A
triose phosphate isomerase
Chain D, Fructose-1,6Bisphosphate Aldolase
OD
•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
500
450
400
350
300
250
200
150
100
50
0
•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.
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.
140
120
100
80
40
20
Glutamine synthetase 2,
isoform C
transferrin precursor
ferritin 2 light chain homolog
Superoxide dismutase
Glutathione S transferase S1,
isoform C
ERp60, isoform B
Chaperonin, isoform C
0
Protein disulfide isomerase,
isoform A
OD
60
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?
450
400
350
OD
300
250
200
150
100
50
0
Yolk protein 1
Yolk protein 2Odorant-binding protein
Mitochondrial
99a
processing20S
peptidase
proteasome alpha subunit Actin 88F
flightin
GST enzyme assay for camptothecin and untreated
fly extracts to confirm optical density changes do
reflect changes in protein amount.
0.2
The camptothecin treated
samples had an activity of
0.15 μmol/min/ml
• Untreated sample had an
activity 0.051
μmol/min/ml.
0.18
0.16
0.14
Absorbance
•
0.12
0.1
0.08
0.06
0.04
0.02
0
0
50
100
150
200
250
300
350
400
Time (seconds)
Camptothecin treated
• 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.
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
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.
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.
•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
4 ug DNA
6 ug DNA
8 ug DNA
•DNA biotin labelled
10 ug DNA
assay
Crude extract
•Promoter Region - Luciferase
Marker
EMSA
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.
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.
Beauveria bassiana
Euoniticellus intermedius
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-spaёtzle .
2D gel electrophoresis of E. intermedius hemolymph
proteins
pH3
pH10
pH3
pH10
80
70
60
50
40
30
25
20
Uninfected
In both treated and untreated beetles.
beetles
Fungal infection
In treated beetles only.
In untreated
Fungal infected
Uninfected
GOBP/Yolk protein/Knockdown/Katanin(9003)
Cryptochrome/ G protein alpha 73B (8402)
Sox 2 (8306)
Chemosensory protein 15/IDGF/Chitin/Silk protein(7303)
Metallopeptidase/IDGF/Coagulin(7301)
Proteosome component/Photolyase(7210)
Serine protein kinase/GK13718(7203)
Serine protease/Kakapo(7106)
Serine protein kinase/GK13718(6410)
Sphinx/Sex lethal(6204)
Serine protein kinase/GK13718/Cation transporting peptide
58Dc(6203)
Helicase/Nucleosporin(6005)
Dipeptidyl peptidase/Spellchecker(5406)
Gelsolin(5312)
Peptidase C19/Gelsolin(5310)
Nanchung/CAD/Helicase(5102)
GOBP(4002)
GOBP(4001)
Unidentified(3403)
Metallocarboxypeptidase/Bent/Persephone/Csl4(3304)
PEZ/GOBP(3004)
GOBP(3002)
Serine protease/Bent(2203)
GOBP (2102)
PEZ/ GOBP (101)
OD
2D gel electrophoresis of E. intermedius
hemolymph proteins
4
3.5
3
2.5
2
1.5
1
0.5
0
Serine proteases
•Mediate extracellular
signaling activated by
0.2
0.2
P=0.0054
p=0.048
bacterial and fungal
0.1
0.1
0
0
pathogens.
•Spots 2203 and 3304
yielded several fragments -
2203
3304
sequences of proteins
encoded by CL8Contig1
and CL20Contig1.
•An alignment of protein sequences encoded by these contigs with Psh,
Holotricia PPAF-II.
•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
91
19
gi|263191484|Tribolium castaneum
gi|86279283|Tenebrio molitor
5310
6
gi|166919235|Costelytra zealandica
3
55
gi|193527454|Chironomus riparius
gi|289191335|Nasonia vitripennis
9
31
gi|48095159|Apis mellifera
gi|146150460|Eriocheir sinensis
22
37
gi|73544890|Blattella germanica
gi|262088908|Ctenocephalides felis
gi|24643597|Drosophila melanogaster
gi|157113794|Aedes aegypti
Pattern recognition receptors
•Apolipophorin III (apoLp-III)
•Lipid transport protein in the
hemolymph.
3
p= 0.0067
1.5
•Important for insect immunity
0
•PRR that responds to b-1,3glucan PAMPS found in fungi.
•ApoLp-IIIhas been shown to
bind to bacterial and fungal cell
wall components .
Apolipophorin III 7203
Other proteins
1
p=0.776
•Similiar to leucine rich
repeat region of Toll 6
0.5
0
Toll 6410
•Transferrin – Iron
binding protein with
antimicrobial activity
1.5
p=0.036
1
0.5
0
Transferrin 6204
Drosophila
Fungi
β-glucan
Pattern recognition molecular
patterns (PAMPS)
Tribolium
Gram +ve bacteria
Lys-type PGN
Virulence Factors
E. intermedius
Fungi
β-glucan
Fungi
β-glucan
GNBP3
(007956_
1645_0
789_c_s)
Apolipophorin III
(6203) (Cl123Contig1)
IDGF
Extracellular recognition
factors
GNBP3
GNBP1
PGRP-SA
PGRP-SD
GNBP3
Persephone
Persephone
(3304)
(CL23 contig1)
Danger signals
ModSP
SP
MSP
Persephone
(CL23 contig1)
Serine protease cascade
Pro-MSP
Serine protease
Cascade ?
Serpin
Nec-1
(CL111Contig1)
Grass
SAE
Spirit
Sphinx
Pro-SAE
Spheroide
Pro-SPE
SPE
Pro-Spz
Spatzle processing enzyme
Spz
Pro-Spz
Spz
Spatzle processing enzyme
(Cl47Contig1)
Pro-Spz
Spz
Cl238contig1
Transmembrane receptor
Effectors
Toll (CL673 contig1)
Toll (CL673 contig1)
Antimicrobial peptides
Antimicrobial peptides
Toll (6410) (Cl673contig1
Transferrin
\Defensin?
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?)
Antimicrobial activity.
1 M NaCl eluant
Positively charged
proteins with pI> 7.5
Protein extract
Hi S Cation exchange column
C18 hydrophobicity
5%
CH4CN
pH 7.5
wash
Protein s with
pI <7.5
60%
CH4CN
100
90
Reverse
phase
HPLC
80
Acetonitrile conentration (%)
Protein flow
through
Negatively
charged
proteins
40%
CH4CN
80%
CH4CN
70
60
50
40
30
20
10
0
0
5
10
15
20
25
Time (minutes)
30
35
40
45
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
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)
Inhibitory activity is a heat stable protein
•Liquid inhibition assays
performed on Micrococcus
120
p=0.102
100
p=0.004
80
luteus
•Proteinase treatment
abolishes activity
• Heat stable up to 50 o C
60
p= 0.002
40
p=0.0001
20
0
Untreated Proteinase Untreated Proteinase Ampicillin
positive K treated negative
K
control
control
• Activity is due to a heat
120
stable protein
100
80
60
40
20
0
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
Comparison between the proteomic response of D.
melanogaster to DNA damage and E. intermedius to
infection.
No stress response pathway occurs in isolation.
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