Download Yeast Two Hybrid Screening: A new service at the DKFZ

Host-Pathogen Interaction
Networks
Sonja Blasche, M.Sc.
Genomics And Proteomics Core Facility
Group of Manfred Koegl
Genomics & Proteomics
Core Facilities
What is the Yeast Two Hybrid-System?
Page 2
• A genetic method to identify potential protein-
protein interactions. Works in yeast.
GAL4-DBD
Bait
PO I
Protein of Interest
• Widely used.
• Can be automated.
Dr. Manfred Koegl
Prey (library)
GAL4-AD
P O IB 1 !
A protein binding to POI!
Genomics & Proteomics
Core Facilities
Page 3
Y2H Data Quality
• Y2H data sets detect <25% of interactions
• Can we improve this?
N-
CN(library)
L4 - ADGAL4Prey
Preyprey
(library)
- AD
prey
N-terminal
N-terminal
N-terminal
(standard)
(20% of interactions
detected)
C-
N-
N-
CPrey
(library)
Prey
(library)
GAL4 - ADGAL4 - A
prey
prey
C-terminal
C-terminal
C-terminal
(additional interactions
detected?)
C-terminal vectors by Thorsten Stellberger (Group of Peter Uetz)
Dr. Manfred Koegl
C-
Genomics & Proteomics
Core Facilities
Page 4
N- and C-terminal Libraries
 Screens against Influenza and Bacterial Baits
Hits C-terminal library
Bacterial pathogen
Hits N-terminal library
18
4
Influenza virus
23
1
8
total
41
1
12
Dr. Manfred Koegl
Genomics & Proteomics
Core Facilities
Page 5
Overview
Pathogens:
Viruses:
• SARS Corona Virus
• Influenza
• Varicella Zoster Virus
Bacterial Pathogens:
• surface proteins of gram postive pathogens
• Yersinia enterocolitica (effector proteins)
in process:
• Enterohemorrhagic E. coli str. sakai
(effector proteins)
Dr. Manfred Koegl
SARS
SARS coronavirus
9
Genomics & Proteomics
Core Facilities
Host-pathogen
interactions of
Yersinia enterocolitica,
VZV and SARS
coronavirus
Page 6
8
7
6
5
4
3
2
1
0
apoptosis
cytoskeleton
regulation of
organization and transcription,
biogenesis
DNA-dependent
transcription
VZV
signal
protein kinase
small GTPase
regulator activity
I-kappaB
kinase/NFkappaB cascade
apoptosis
cytoskeleton
regulation of
organization and transcription,
biogenesis
DNA-dependent
transcription
Yee
signal
protein kinase
small GTPase
regulator activity
I-kappaB
kinase/NFkappaB cascade
apoptosis
cytoskeleton
regulation of
organization and transcription,
biogenesis
DNA-dependent
signal
transduction
protein kinase
cascade
small GTPase
regulator activity
I-kappaB
kinase/NFkappaB cascade h
factor complex
transduction
Varicella
zoster
viruscascade
9
8
7
6
5
4
3
2
1
0
factor complexenterocolitica
transduction
cascade
Yersinia
9
8
7
6
5
4
3
2
e
ca
sc
ad
B
or
er
eg
ula
t
te
in
de kina
se
n
an
sc
uc
tio
nf
ac
to
r
transcription
factor complex
tio
n
tio
an
sc
rip
yt
o
sk
ele
to
n
0
op
to
sis
Dr. Manfred Koegl
1
ap
Ekaterina von dall’Armi
Albrecht von Brunn
Jürgen Haas
Genomics & Proteomics
Core Facilities
Page 7
Enterohemorrhagic E. coli (EHEC)
In process:
• Clone effector proteome of EHEC str. sakai
• Screen against human libraries
• GO-term analysis
• Detailed examination of targeted
pathways
Similar strategies between pathogens in host cell
manipulation?
Dr. Manfred Koegl
Genomics & Proteomics
Core Facilities
Page 8
Outlook
Goals:
• Detect similarities in host cell manipulation by
viral and bacterial pathogens
• Identify important nodal points and check
eligibility as drug targets
• Discovery of novel drugs in collaboration with
the partners from AntiPathoGN
Dr. Manfred Koegl