Preliminary Investigation of the Interaction Between Inc D and the PH domain of CERT
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Transcript of Preliminary Investigation of the Interaction Between Inc D and the PH domain of CERT
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Preliminary Investigation of the Interaction Between Inc D and the PH domain of CERT
Cole McMullin
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BackgroundChlamydia is a genus of obligate intracellular
pathogens.Chlamydia trachomatis: responsible for the STD
Chlamydia, genital and ocular infections. May lead to infertility, ectopic pregnancy, PID. Commonly asymptomatic. Facilitates the transmission of HIV.
Chlamydia pneumoniae: respiratory pathogen responsible for approx. 10% of pneumonia cases worldwide.
Chlamydia psitacci: Parrot fever or Ornithosis. Symptoms commonly similar to flu or pneumonia in humans.
Antibiotic resistance and chronic infections may necessitate finding alternative treatments.
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PH STARTMR CC24 119
132 150 321 327
360 598
SR FFAT
Golgi Targeting
Regulation ? ER Targeting Lipid Transfer
Activity
Figure courtesy of Jennifer Prashek
CERT
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Golgi
ER
Inactive Active
PpSR PH
FFATMRSTART
PP2Cε
PKD, CKIγ2
Figure courtesy of Jennifer Prashek
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Inc D – PH domain interaction
Derre I. The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to Chlamydia-Inclusion Membrane Contact Sites. In: Swiss R, editor. e1002092 ed. Plos Pathogens2011.
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Inc D1-40 Hydrophobic region 90-141
Host Cell Cytosol NTCT
CTNT
Can Inc D acquire ceramide from a phosphoryalted CERT?
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Hypothesis: Inc D interacts with the PH domain via its positively charged regions at the NT and CT NT
CT
Design
Approach:• Use size exclusion experiments to determine if the two proteins interact.• Incubate Inc D peptides with PH domain:
1. CT and PH2. NT and PH
•Run the mixture through gel filtration. • SDS-PAGE to detect co-elution
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Methods
Ni NTA Affinity
Q Anion Exchange
Ni His Trap Affinity
Gel Filtration
TEV cleavage
AmpHis Gb1
Inc D peptide
E. coli
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Size Exclusion Experiment of CT- IncD and PH mixture
Inc D 92-141
hCERT 20-122
Inc D 92-141
hCERT 20-122
M 3 4 13 14 15 28 30 32 33
31
21.514.46.5
2015Dec4 IncD 92 to 141 and PH 20 to 122 24mlSD75001:10_UV 2015Dec4 IncD 92 to 141 and PH 20 to 122 24mlSD75001:10_Cond 2015Dec4 IncD 92 to 141 and PH 20 to 122 24mlSD75001:10_Conc 2015Dec4 IncD 92 to 141 and PH 20 to 122 24mlSD75001:10_Fractions
0.0
10.0
20.0
30.0
40.0
mAU
0.0 5.0 10.0 15.0 20.0 ml1 2 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 Waste
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Size Exclusion Experiment Inc D NT and PH
Inc D 1-36
hCERT 20-122
hCERT 20-122
2016 Feb 5 24mlSD75001 IncD 1 to 36 and PH 20 to 122:10_UV 2016 Feb 5 24mlSD75001 IncD 1 to 36 and PH 20 to 122:10_Cond 2016 Feb 5 24mlSD75001 IncD 1 to 36 and PH 20 to 122:10_Conc 2016 Feb 5 24mlSD75001 IncD 1 to 36 and PH 20 to 122:10_Fractions
0
20
40
60
80
100
120
140
160
mAU
0.0 5.0 10.0 15.0 20.0 ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Waste
Inc D 1-36
M 14 24 25 26 27 28 29 30 31 32 33
31
21.514.46.5
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Pulldown SchematicNi
Ni
Ni
His
His
His
NiAdd His-tagged
peptide
Add PH domain
Wash out unbound PH with buffer
Zeng, Xue (2014). Pulldown Assay: a technique to confirm interactions or to identify new interactions between proteins
Wash out unbound peptide with buffer
Elute His tagged peptide off of column
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Ni Pulldown of His Gb1 Inc D 1-36 and 92-141 with hCERT 20-122
M NT CT PH W1 W4 W1 W4 bd E1 E2 E3 bd
31
21.5
14.46.5
His tag + Ni beads
Addn. of PH
Inc D NT
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Ni Pulldown His Gb1 Inc D 1-36 with hCERT 20-122
NT PH M W1 W4 W1 W4 bds E1 E2 E3 bds
31
21.5
14.4
6.5
His tag + Ni beads
Addn. of PH
Inc D NT
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Ni pulldown His Gb1 Inc D 92-141 and hCERT 20-122
CT PH W1 W4 W1 W4 bd - M E1 E2 E3 bd
31
21.5
14.46.5
His tag + Ni beads
Addn. of PH
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Ni Pulldown His Gb1 and hCERT 20-122
His Gb1 PH W1 W4 W1 W4 bd E1 E2 E3 bd M
31
21.5
14.46.5
His tag + Ni beads
Addn. of PH
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ConclusionNo binding detected by size
exclusionPulldown not conclusivePerhaps the full length protein is
needed.
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Expression of full length IncD
M P S
Inc D31
21.5
14.46.5
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Future WorkNuclear magnetic resonance to detect
bindingIncubation of Inc D peptides and hCERT
PH domain in the presence of liposomes.The use of detergents during cell lysis to
purify a soluble full length Inc D.Pulldown assays using a different tag
(GST, FLAG, Myc).X-ray crystal structures, binding affinity
assays.
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AcknowledgementsDr. YaoJennifer PrashekAlex TroxelSchool of Biological Sciences