Transcript 北京大学

具有泛素及Nedd8调节功能的病源
蛋白在巨噬细胞中的功能探究
陈龙
北京大学 生命科学学院
Long Chen School of Life-Science， Peking University
11/26/2011
目录
1
Background
2
Results
3
Conclusion and future work
4
Acknowledgements
Backgroud

Ubiquitin(泛素) and Nedd8
◦ Ubiquitin ——2004 Noble Price
DUB
(How Ub works（泛素化原理）,Modified from Jixin Cui)

Nedd8 is similar to Ubiquitin(cell cycle（细胞周期） progression and
cytoskeletal（细胞骨架） regulation)
Background

Cif and family protein(especially CHBP)
(Q. Yao
et al.,
PNAS
U.S.A.
106,
3716
(2009))
Bacterial Effector
Cif——Enteropathogenic E. coli（大肠杆菌）
CHBP——Burkholderia pseudomallei（鼻疽杆菌）
Background

Glutamine（谷氨酸）
Deamidation（脱氨基
作用） and
Dysfunction of
Ubiquitin/NEDD8
Induced by CHBP and
Cif
Ubiquitin
Difference between Cif and CHBP
Nedd8
Background

Macrophage（巨噬细胞）
(Macrophage,From Wikipedia)
(Immune System（免疫系统）, from Nature)
Questions we want to know
First
How could we deliver the protein into
the macrophage
Second
How do the different protein effect on
the macrophage
Finally
The reason for the influence on the
macrophage
Results

By osmotic（渗透压） lysis, the protein didn't show
great influence on the macrophage.The osmotic lysis
is not sensitive enough for this experiment.
400000
Fluorescence Intensity
350000
300000
250000
200000
150000
100000
50000
0
未处理
Yogendra Singh, et at,
JBC, 1989
mock
Lethal Factor
(ATP assay for osmotic lysis on P80 cell, 24h)
Results

PA(Protective Antigen)LF(Lethal Factor，致死因
子)n system. Preparation
for LFn-CHBP, LFn-CHBP
C/A, LFn-Cif, LFn-Cif C/A,
LFn and PA.
LFn
CHBP
LFn
CHBP C/S
LFn
Cif
LFn
Cif C/A
PA
PA-LF/EF system from anthrax bacteria（炭
疽杆菌） , from google
Results

LFn-CHBP leads the macrophage to cell
death while LFn-Cif doesn't effect the
macrophage seriously.
Macrophage P80
Mock
CHBP
Cif
Results
LFn-CHBP leads the macrophage to cell
death while LFn-Cif doesn't effect the
macrophage seriously.
120.00%
100.00%
80.00%
Flourescence Intersity

60.00%
40.00%
20.00%
0.00%
mock
CHBP
CHBP C/A
Cif
ATP assay(mock, PA+LFn-CHBP and
LFn-Cif, 24h.)
Cif C/A
Results
The poor influence of LFn-Cif is mainly due to
the failure to enter the macrophage.
Hela（海拉细胞）
Mock
PA+LFn-Cif
PA+LFn-CHBP
Results

Nedd8 is the key to the cell death caused by
the disease-causing protein.
TLR4 –KO i-BMM
HeLa
80
60
40
MLN4924
PYR-41
100
20
0
Mock
MLN4924
Mock
TLR4-KO i-BMM
MLN4924
HeLa
100
Cell survival (%)
TLR4-KO i-BMM
120
Cell survival (%)
Cell survival (%)
100
80
60
40
20
0
0.15
80
0.5
1.5
5
15
50
150
Inhibitor conc. (μM)
60
40
20
0
Mock
PYR-41
Mock
PYR-41
( Different effect from Ub E1 inhibitor（抑制剂） and Nedd8 E1 inhibitor on the
Hela, From Jixin Cui)
Conclusion and future work
Conclusion:
1,LFn-CHBP lead to macrophage cell death.
2,The osmotic lysis is not sensitive enough for this
test.
3,Nedd8 is the key to the cell death caused by the
disease-causing protein.
Future work:
1,Apoptosis（凋亡） or Necrosis（坏死）?
2,If we save the Nedd8, will the macrophage or
even other cell survive from these diseasecausing bacteria?
Acknowledgements

Chun-Tsung Scholarship and Prof.Tsung-Dao Lee
Dr. Feng Shao
Prof.Yi Rao
Dr. Jixin Cui (my graduate mentor)
Jiayi Wang， Yinan Gong，Liping Liu and other
labmembers.
 My friend， Mo Xu

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

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School of Life-Science, Peking University
NIBS（National Institute of Biological Sciences，
Beijing）