preliminary investigation of the interaction between inc d and the ph domain of cert
TRANSCRIPT
Preliminary Investigation of the Interaction Between Inc D and the PH domain of CERT
Cole McMullin
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.
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
Golgi
ER
Inactive Active
PpSR PH
FFATMRSTART
PP2Cε
PKD, CKIγ2
Figure courtesy of Jennifer Prashek
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.
Inc D1-40 Hydrophobic region 90-141
Host Cell Cytosol NTCT
CTNT
Can Inc D acquire ceramide from a phosphoryalted CERT?
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
Methods
Ni NTA Affinity
Q Anion Exchange
Ni His Trap Affinity
Gel Filtration
TEV cleavage
AmpHis Gb1
Inc D peptide
E. coli
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
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
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
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
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
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
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
ConclusionNo binding detected by size
exclusionPulldown not conclusivePerhaps the full length protein is
needed.
Expression of full length IncD
M P S
Inc D31
21.5
14.46.5
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.
AcknowledgementsDr. YaoJennifer PrashekAlex TroxelSchool of Biological Sciences