??????????? ??? ??????? Protein

1
??????????? ??? ???????Protein protein
interactions

• ???? ????' ???? ??????
• ???? ???? ?????

2
??? ????!?

• ???? ???? ?? ??????? ?????????.
• ????????? ????????.
• ??????? ?????.
• ???? ???????? ???? ????? ??????.
• ????? ?????? ???? ?????? ??????? ?? ??????.

3
?? ??????

• ????? ?????? ????? ?????? ??? ??? ??
• ?????? - Immunoprecipitation.
• ????? ?????? ????? ? ????? ??????? ????? ????
  ????.
• ?????? ????? high throughput.
• ???? ???? ????? ???????, ????????? ????????????

4
??????? ?????

• ???? ????? ?????? ???????????.
• ?????? ?????.
• ???? ?? ???? ???? ?? ??????????? ????.
• ???? ?? ???? ???? ???? ?? ??????????? ????.

5
???? ??????

• ????? ?? ???????
• Legrain P et al. (2001) Protein--protein
  interaction maps a lead towards cellular
  functions, Trends Genet17(6)346-52.
• von Mering C. et al. (2002) Comparative
  assessment of large-scale data sets of
  protein-protein interactions. Nature.
  417(6887)399-403

6
Yeast 2 Hybrid Assay
Hybrid proteins
Fishing for proteins that interact with bait
domain
Lodish 5th 11-39
7
Transcriptional activation by hybrid proteins in
yeast
Lodish 5th 11-39
8
Lodish 5th 11-39
9
Matrix approach
sticky
Auto activator
Legrain P et al. (2001) Protein--protein
interaction maps a lead towards cellular
functions, Trends Genet
10
Matrix approach

• ?? ??????? ?????? ??????.
• ?????? ????????? ??? ??? ?????? ????? 841 ???
  692 ??? 141 ??????.
• Sticky preys
• Auto activators
• Pools ????? array

11
Screening of fragment libraries
Legrain P et al. (2001) Protein--protein
interaction maps a lead towards cellular
functions, Trends Genet
12
Screening of fragment libraries

• ??? ???? ???????? ??????.
• ????? ? interacting domain.
• ???? ???? ???? ????? ???? ??????.

13
?????? ???????

• False negative
• ????? ?????? ? PCR ( 13 ?? ????? ??? ?????).
• 90 ????????????? ??????? ??????.
• ? pools ???? ???? ?????? ????? ? array
• False positives
• ????? ???? ??? ?????? ????????.
• Sticky, auto activators.

14
?????? ??????
False negatives ???? ???? ???? ???? ?????? ????
False positives ???? ????? ??? ?????? ???????? ????
???? ????? ????? ????
15
Y2H

• In vivo
• ????? "????? ??????" ?????.
• ?? ???? ????? ??????? ??????.
• ? fusion protein ???? ?????? ???????????.
• ?? 2 ??????? ?????? ????? ??? ??? ????? ??
  ?????????? ???????.
• ?????? ??????.
• ?? ????? ????? ?????????.

16
Epitope Tagging
ANALYSIS OF PROTEINS AND PROTEOMES BY MASS
SPECTROMETRY Matthias Mann et al Annu. Rev.
Biochem. 2001.
17
Epitope Tagging MS (Mass Spectrometry)

• Internal check for consistency.
• ????????? ??????? ?????? ??????????.
• ???? ?? ????????? ????????? ?????? ???????.
• ?????? ???? ??????.
• ???? ?????? ???? ????? ???????.

18
MS vs. Y2H
Y2H MS
????? ??????? ????????? ?????
???? ????? "??????" ??????? ?????? ??????
????? "??????????" ?????? ?????? ?????? ???????
????? ????
19
TAP - Tandem Affinity Purification
A generic protein purification method for protein
complex characterization and proteome
exploration Guillaume Rigaut et al 1999 Nature
America
20
HMS-PC High-Throughput Mass-Spectrometric
Protein Complex Identification

• ????? ??? ?? ?????? - inducible overexpression

TAP HMS-PC
????? ????? ????? ????? ?????? ???????????
21
Correlated mRNA expression (Synexpression)

• ????? ????? mRNA ? ????? (clustering) ???? ?"?
  ??????.
• ????? ?????.
• ???? ???? ????? ??????????.
• ?? ????? ?? ?????? ??????.
• ????? ???? ??????????? ??????.

22
Genetic Interactions(Synthetic lethality)

• ??? ??????? ?? ??????? ????? ?? ??????? ???? ?
  ??? ?????? ????? ??????????.
• ????? ?? ???? ??? ??? ????? ???????.
• ?? ????? ?? ??? ????? ????.

23
In Silico Predictions Through Genome Analysis

• ??????????? ???????? ????? ??????.
• ??????? ?? ?????? ????? ??????????.
• ??????? ? fusion protein.
• ???? ????.
• ????? ???? ?????? ????? ????? ????.
• ?? ?????.
• ????? ??????? ?????? ??? ??????????.
• ?? ?? ??????? ???????????.

24
Comparative assessment of large-scale data sets
of protein-protein interactions

• von Mering C. et al. (2002)
• Nature

25
?? ???? ????? ?? ????

• ????? ??????? ????? ?????, ??????? ?????, ??????
  ??? ???? ????.
• ????? ??????? ????? ????? ???????.
• ?? 2,400 ???? 80,000 ??????????? ????? ?????
  ????? ???.
• ?????? ???? ?? ????? ??????.
• False positives.
• ??????? ???? ????? ?? ?????.

26
Counting interactions
27
????? ???????????? ?"? ?????? ????????
28
Transport and sensing
29
????? ?"? ???? ????? ???????
30
????? ?"? ????? ???
31
????? ?"? ????? ???
32
(No Transcript)
33
????? ???????

• ????? ????? ????? ????????????? ??????.
• ????? ?? ???? ? 50 false positives.
• ????? ????? ????? ?? ?????.
• ??????? ?????? ??????? ????? ???.

34
Towards a proteome-scale map of the human
protein-protein interaction network. Rual, J.
et al (2005). Nature 437

• ????? ??? ??????? ????

35
???? ??????

• ????? ?? ? Human ORFeome v1.1 -
• ???? 8,100 ORFs (????? ??? ?? ????? 22,000
  ???? ???????).
• ????? ??????????? ??? ?? ???- 8,100X8,100.
• ?????? ???? ?? ? 4,000 ??????????? ????? ?????,
  ?????? ????? ?2,750 ?????? .
• ????? ? Y2H ?????.
• ???? reproducibility ???? (55).
• ??? False positives - ???? ???????? ???? ?????
  ????? ????? ?????? ?????.
• Co-affinity purification GST pull down assay in
  human 293 cells.

36
(No Transcript)
37
?????
???? - ???? ???? - ?????
38
?????? ????????

• ??????? ?????? ?? ???? ????? ??????????.
• ??? ???????????? ?????? ????? upstream ???????
  ??????? ???? ???? ????? ????.
• ???? ????? ????. Gene Ontology terms.
• ??????????? ??? ??????? ??????? ????? ???
  ?????????.

39
??? ???????????? (????? ????)
???? - ???? ???? - ?????
40
????? ?"?????? ??????????"
???? - ????? ??? - ????
41
????????? ?? ????

• ???? ? iteractome ?? ?????.
• ???? ???????? ???? ????? ?????? Hubs.
• ??? ???? (No high clustering).
• Subgraphs complexes - - 102 ???? 172 ????
  ????? ???? ???? ???? ??? ?????? ???? (?? 10
  ?????????).

42
????? ?????

• ????? ?? ???? ???? ????????????? ??????? ????.
• ???? ???? ?????? ???????? ???????? ??? ????? ???
  ?? ??????? ????? ?? ???.
• ???? ????? ?????.

43
Proteome survey reveals modularity of the yeast
cell machinery

• Gavin AC et al. (2006)
• Nature.

44
(No Transcript)
45
?? ?????

• TAP
• 6,466 ORF - ?? ? ORF ??????? ????.
• ????? 73 ??????????? ??????? (MIPS ).
• 74 ?????? ????? ?????????? ????? ?? ????? ??
  ??????.
• ?? ??????? ??????? ???? ??????? ????? ????
  ?????.
• 64 ??????????? ??????? ???? ???? ????? ?????
  ???? ?? ????????? ??????.
• Socio affinity index ?????? ????? ?? ?????? ??
  ??? ??????? ?????? ?? ???. ????? ?? ???? ??????
  ??? ????? ???? ???? ????? ??? ???? ??????
  ???????????? ???????? ??????.

46
Core ???? ???? ??????????? Attachment ??????
?? ???? ????????????. Modules ???? ???????
????? ????. (?????? ??????? ?? 3.3 ?????, ??????
?? 1.6)
47
???????? ??????
?????? ?? ????? ????
????? ????? ? cell cycle
????? ? Y2H
48
??????? ????? ??????? ???????? ????? ???
49
Attachments ?????? ??????? ??????
50
??????? ?????? ??????? ????? ??????? ???????????
51
Core module cross talk
52
????? ??????? ??????????
53
????? ?????

• ????? ???? ???? ??? ?????? ?????? ?? ? cell cycle
  .
• ????? ???????????? ???????? ????? ??????
  ????????.
• ????? ?"?????" ??? ????? ???????? ?????? ??? ???
  ????? ??????? ????? "??????" ?????.
• ?? ???? ?????? ????? ?? ???????????? ??? ?????
  ?????????? ?????.

54
?????????? ?? ????
???? ? http//string.embl.de/ ??? ???? ??
??????????? www.ebi.ac.uk/intact
55
Attachments specify a particular function
56
??? ?????

• ????? ?? ????? ??? ????? ????????????, ???????.
• ????? ???? ?????? ??? ?? ???????.
• ???? ????? ????.
• ???? ?????.

57
??????

• Legrain P et al. (2001) Protein--protein
  interaction maps a lead towards cellular
  functions, Trends Genet17(6)346-52.
• von Mering C. et al. (2002) Comparative
  assessment of large-scale data sets of
  protein-protein interactions. Nature.
  417(6887)399-403.
• Gavin AC et al. (2006) Proteome survey reveals
  modularity of the yeast cell machinery. Nature.
• Rual, J. et al. (2005). Towards a proteome-scale
  map of the human protein-protein interaction
  network. Nature 437, 1173 - 1178