Download Apoptosis

Methods for Apoptotic Assay
Apoptosis
 A physiological mechanism to eliminate excess,
damaged or dangerous cells from an organism
without damaging surrounding cells and tissues
 Necessary for normal embryogenesis
 Maintenance of tissue homeostasis
Apoptotic morphology
 Membrane blebbing
 Aggregation of chromatin at the nuclear membrane
 Ends with fragmentation of cell into small bodies
 Begin with shrinking of cytoplasm and condensation
of nucleus
 Formation of apoptotic bodies
 Mitochondria become leaky due to pore formation
 involving proteins of the bcl-2 family
Apoptotic Pathway
Death receptors
 Membrane alteration
 Protease cascade
 Mitochondrial changes
DNA fragmentation
INTRINSIC PATHWAY
Extrinsic pathway
cytochromeC
APAF-1
Death receptors
Apoptosome
Death
inducing
signaling
complex
procaspase9
caspase9
caspase8
Procaspase 8
XIAP
Procaspse3
procaspase7
Caspase3
caspase7
Apoptosis
The extrinsic death receptor pathway
Death receptors
Death domain
adaptor protein
Death effector
domain
TNF
FASL
Apo2/Trial
The intrinsic apoptotic pathway
Apoptotic insult
AIF
nuclease
chromatin
Caspase activation
nuclease
apoptosome
Caspase dependent cell
death
chromatin
DNA fragmentation
Protease cascade
*Conserved domains for caspase interaction:
Death
Domain( DD) QACRG
Death Effector Domain( DED)– hydrophobic interaction
Caspase recruitment Domain( CARD)—electrostatic
interaction
* Amplify a suicide signal in the cell whose death pathways
have only weakly initiated
Caspase family
Death Effector domain
Caspase recruitment Domain
Nematodes
EGL1
CED-9
CED-4
CED-3
apoptosis
Pro CED-3
CED-9
CED-4
mitochondria
Cell
death
Active CED-3
βNAC
Icd-1( inhibitor of cell death -1))
Early embryo arrest
Accumulation of extra die cells
Extra die cells in nematode show normal survival
mammals
Apoptotic stimuli
AIF
BID, BIM
HTR2/OMI
EndoG
BCL-2
Cytochrome C/dATP
SMAC/DIABLO
APAF-1
caspase9
Caspase3,7
IAP
Apoptosis
Fruitfly
Reaper
Hid
Grim
Sickle
Apaf1
Dronc
DIap1
Drice
Cellular target
Apoptosis
Eat me signal
PS ( phosphotidylserine) displayed on the plasma membrane on the
dying cells
Engulfment receptors in mammals:
CD91, CD14, CD36, αvβintegrin, phosphotidyl serine receptor( PSR)
Cell Engulfment
ced1: engulfment receptor
ced-6: homologue to the mammalian PTB domain bearing
adaptor GULP
ced-2( CRKII)
ced5 ( DOCJ-180)
ced10( small GTP-ase Rac-1)
Cytoskeletal remodeling
Engulfment of apoptotic cells
nematodes
mammalians
Phagocytic cells
RAC1
Cytoskeleton remodeling
Membrane blebbing
Apoptotic cells
ELMO
Phosphotidylse
rine
Phosphotidyl
serine
CED-5
Dock180
CED-2
CRKII
PSR
CD19
CD91
DNA degradation
CD14
αvβintegrin,
Scavenger receptor
αvβintegrin,
CED-1
CED-6
CED-7
Decision to die,
Apoptotic induction, caspase activation
Execution of death, caspase activation
Engulfment signal,
LPC….
ps
Anexin I
Eat me
signal,CD31/
PCAM-1
degradation
Anexin I
180 b.p.
Increased low molecular weight DNA in apoptotic cells
Terminal dideoxynucleotidyl transferase
annexinV: Ca+2 dependent phospholipid binding protein, high
affinity to PS
Substrate
cleavage
Caspase
activation
Apoptin誘發細胞凋亡機制之探討
Mechanisms Involved in Apoptosis
induced by Apoptin Associated Proteins
Chicken Anemia Virus
diameter 23 - 25 nm
single-stranded circular DNA (2319 nt)
polycistronic mRNA
VP1 51.6 kDa
VP2 24 kDa
VP3 13.6 kDa (Apoptin)
CAV infection cause depletion of erythroblastoid and
lymphoid cell in young chicken
(Jerissen & Noteborn et al, J Viorogy,1992)
Apoptosis is responsible for the thymocyte depletion in
young chicken
(Jerissen et al, J Virology,1992)
VP3 protein of CAV is sufficient to induce apoptosis in
chicken mononuclear cells
( Zhung & Noteborn et al, Leukaemia1995)
p53
Apoptin
Bcl-2
BAG-1
CrmA
ICE
Apoptosis
Noteborn et al, Cancer Res, Carcinogenesis,1995
Apoptin induces apoptosis in human transformed and
malignant cells but not in normal cells
Danen-Van Oorschot et al, PNAS 1997
Apoptin activity and localization in human normal vs,
transformed/tumorgenic cells
Human cells
Apoptin
Apoptotic activity
localization
Normal primary
None
cytoplasmic
Tumor-derived/transformed
yes
nuclear
Noteborn etal, 1998, Mutation Research
The effect of transforming genes and UV-irradiation
on Apoptin induced apoptosis
Donors of fibroblast
Apoptin induced Apoptosis
Apoptin expression plus
none
SV40 largeT
UV
Healthy
no
yes
no
Cancer prone
no
yes
yes
Noteborn etal, 1998, Mutation Research
Apoptin –expressing adenoviral vector specifically kills
tumor cells of xenogenic HepG2 tumor cells in
Balb/Cnu/nu
Pietersen et al 1999 Gene Therapy
Apoptin induced apoptosis requires activation of
caspases in human tumor cells
Oorschot et al 2000 J Virology
A tumor specific kinase activity regulate the viral death
protein Apoptin
Rohn et al 2002 JBC
Apoptin induces tumor specific apoptosis as a globular
multimer
Leviveld et al 2002 JBC
Purpose
To study the molecular mechanisms involved in
Apoptin induced apoptosis
Identification of Apoptin-Associated Proteins by
Yeast Two-Hybrid System
Growth on
SD/-Trp/-Leu/-His
APAP1
APAP2
b-gal
filter assay
Growth on
SD/-Trp/-Leu/-His
APAP3
APAP4
b-gal
filter assay
APAP1 Identical to DEDAF (Death Effector DomainAssociated Factor) and human RYBP (Ring1
and YY1 Binding Protein)
APAP2 An unknown gene with 6000b.p. with ten
ankyrin repeats
APAP3 Identical to a Interferon induced IFP35
APAP4 Identical to Hippi/Hip-1 protein interactor
APAP1 Identical to DEDAF (Death Effector DomainAssociated Factor) and human RYBP (Ring1
and YY1 Binding Protein)
APAP2 An unknown gene with 6000b.p. with ten
ankyrin repeats
APAP3 Identical to a Interferon induced IFP35
APAP4 Identical to Hippi/Hip-1 protein interactor
PART1
Cloning and characterization of Apoptin associated
protein 1 ( APAP1)
APAP1 is identical to DEDAF (Death Effector DomainAssociated Factor) and RYBP (Ring1 and YY1 Binding
Protein)
Peptide structure of APAP1
APAP1
226 a.a.
Zinc finger
NLS
KKEKK
CSVCTFRNSAEAFKCSIC
NLS
KKTKPK
Purpose
Demonstration of in vivo and in vitro binding of Apoptin
and APAP1
GST pull down of Apoptin and APAP1
pGEX/
pET32a(+)/
Apoptin
APAP1
E.coli transformation( BL-21)
IPTG induction
4oC sonication
GST pull down
SDS PAGE
Western Blot( anti-His detection)
Apoptin associate with APAP1 in vitro
Co-immunoprecipitation of Apoptin and APAP1
GFP
pEGFPC2/
pRK5F/
Apoptin
APAP1
Flag
DNA transfection
HeLa cells
24hrs, 37oC
cell harvest, IP/ -GFP
SDS-PAGE
Blot -Flag and -GFP
Apoptin associate specifically with APAP1 in vivo
Interaction domain mapping of Apoptin and APAP1
BD
AD
pAS2-1/
pACT/
Apoptin
APAP1
mutant
Small scale LiAC/ yeast transformation
Growth on SD-Trp-Leu-His medium
colony lift filter assay
Cellular localization of Apoptin and APAP1
RFP
GFP
pEGFPC2/
Apoptin
psRed c1/
APAP1
DNA transfection
HEL cells
HeLa cells
16 hrs after transfection
cells fix and DAPI stain
Fluororescent microscope
Apootin and APAP1 are colocalized at the nucleus region of HeLa cells
Purpose:
Involvement of APAP1 in Apoptin induced Apoptosis
Apoptotic Assay of Apoptin and APAP1
or
GFP
pEGFPC/
pRK5F/
APAP1
Flag
pRK5F/
Apoptin
Flag
DNA co-transfection
HEL cells
HeLa cells
48hrs, 37oC
cells fix, PI stain and fluorescent microscope
APAP1 expression level in human tumor cell lines
Human tumor & normal cell lines
total cell protein extraction
SDS PAGE
Blot - APAP1 antibody
Normal cells Tumor cells
Higher APAP1 expression level in human tumor cell lines
summary
1. Apoptin associated specifically with APAP1 in
vitro and in vivo
2. Binding of Apoptin to APAP1 is localized to the region
of a.a. 1-59 of Apoptin
3. APAP1 and Apoptin are colocalized at the nucleus
region of HeLa cells
4. APAP1 induces apoptosis in both HeLa and HEL cells
5. Apoptin induced apoptosis is probably mediated by
APAP1 through DED domain
6. Higher APAP1 expression level were observed in
human tumor cell lines than normal cells.
Lower APAP1 expression level in human tumor cell lines
Tumor cells
Apoptin
Apoptosis
Normal cells
Cell survival
Study and Characterization of APAP1/DEDAF in
zebrafish development
Peptide structure of Z-APAP1
zAPAP1 257 a.a.
Zinc finger
GFWDCS VCTFRNSAEA FKCSIC
ZAPAP1
ZRYBP
Actin
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24
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27 s
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36
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PCR Amplification of APAP1 of zebrafish cDNA
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Tissue Scan of Zebrafish cDNA
ZAPAP1
Actin
APAP1 siRNA increases the abnormal frequency of abnormal embryo