methods for apoptotic assay. apoptosis a physiological mechanism to eliminate excess, damaged or...
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Methods for Apoptotic Assay
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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
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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
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Apoptotic Pathway
Death receptors
Membrane alteration
Protease cascade
Mitochondrial changes
DNA fragmentation
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INTRINSIC PATHWAY Extrinsic pathway
Caspase3
caspase7
Apoptosis
procaspase9
cytochromeC
APAF-1 Apoptosome
XIAP
caspase9 caspase8
Procaspse3
procaspase7
Procaspase 8
Death receptors
Death inducing signaling complex
Ad
ap
tor
pro
tein
s
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Death domain adaptor protein
TNF FASL Apo2/Trial
Death effector domain
The extrinsic death receptor pathway
Death receptors
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The intrinsic apoptotic pathway
Caspase activation
Apoptotic insult
apoptosome
Caspase dependent cell death
AIF
chromatin
nuclease
chromatin
nuclease
DNA fragmentation
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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
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Caspase family
Death Effector domain
Caspase recruitment Domain
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EGL1
CED-9
CED-4
CED-3
apoptosis
Nematodes
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mitochondria
Cell death
CED-9
Pro CED-3
CED-4
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
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BID, BIM
BCL-2
SMAC/DIABLO
IAP
APAF-1
caspase9
Caspase3,7
Cytochrome C/dATP
AIF
HTR2/OMI
EndoG
Apoptosis
Apoptotic stimulimammals
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Reaper
Hid
Grim
Sickle
DIap1
Apaf1
Dronc
Drice
Cellular target
Apoptosis
Fruitfly
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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)
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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
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Apoptotic cells
Engulfment of apoptotic cells
Phagocytic cells
DNA degradation
Phosphotidylserine
Cytoskeleton remodelingELMORAC1
Dock180
CED-5
CED-2CRKII
PSR
CD19
CD91
CD14
αvβintegrin,
αvβintegrin,
Scavenger receptor
CED-1CED-6 CED-7
Membrane blebbing
nematodes mammalians
Phosphotidylserine
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Decision to die,
Execution of death, caspase activation
Engulfment signal, LPC….
degradation
Apoptotic induction, caspase activation
Eat me signal,CD31/ PCAM-1
psp
s
Anexin I
Anexin I
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180 b.p.
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Increased low molecular weight DNA in apoptotic cells
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Terminal dideoxynucleotidyl transferase
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annexinV: Ca+2 dependent phospholipid binding protein, high affinity to PS
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Substrate cleavage
Caspase activation
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Apoptin 誘發細胞凋亡機制之探討
Mechanisms Involved in Apoptosis
induced by Apoptin Associated Proteins
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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)
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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)
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Noteborn et al, Cancer Res, Carcinogenesis,1995
p53 Apoptin
Apoptosis
Bcl-2
BAG-1
CrmA
ICE
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Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells
Danen-Van Oorschot et al, PNAS 1997
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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
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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
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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
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Purpose
To study the molecular mechanisms involved in Apoptin induced apoptosis
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Identification of Apoptin-Associated Proteins by Identification of Apoptin-Associated Proteins by
Yeast Two-Hybrid SystemYeast Two-Hybrid System
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APAP1
APAP2
Growth onSD/-Trp/-Leu/-His
-gal filter assay
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APAP3
APAP4
Growth onSD/-Trp/-Leu/-His
-gal filter assay
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APAP3 Identical to a Interferon induced IFP35
APAP2 An unknown gene with 6000b.p. with ten
ankyrin repeats
APAP4 Identical to Hippi/Hip-1 protein interactor
APAP1 Identical to DEDAF (Death Effector Domain-
Associated Factor) and human RYBP (Ring1
and YY1 Binding Protein)
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APAP3 Identical to a Interferon induced IFP35
APAP2 An unknown gene with 6000b.p. with ten
ankyrin repeats
APAP4 Identical to Hippi/Hip-1 protein interactor
APAP1 Identical to DEDAF (Death Effector Domain-
Associated Factor) and human RYBP (Ring1
and YY1 Binding Protein)
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Cloning and characterization of Apoptin associated
protein 1 ( APAP1)
PART1
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APAP1 is identical to DEDAF (Death Effector Domain-Associated Factor) and RYBP (Ring1 and YY1 Binding Protein)
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APAP1
226 a.a.
Peptide structure of APAP1
Zinc finger NLS NLS
KKEKK KKTKPK
CSVCTFRNSAEAFKCSIC
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Purpose
Demonstration of in vivo and in vitro binding of Apoptin and APAP1
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GST pull down of Apoptin and APAP1
pGEX/
Apoptin
pET32a(+)/
APAP1
E.coli transformation( BL-21)
IPTG induction
GST pull down
SDS PAGE
Western Blot( anti-His detection)
4oC sonication
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Apoptin associate with APAP1 in vitro
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Co-immunoprecipitation of Apoptin and APAP1
cell harvest, IP/ -GFP
DNA transfection
24hrs, 37oC
SDS-PAGE
Blot -Flag and -GFP
HeLa cells
GFP
pEGFPC2/
Apoptin
pRK5F/
APAP1 Flag
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Apoptin associate specifically with APAP1 in vivo
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Interaction domain mapping of Apoptin and APAP1
Small scale LiAC/ yeast transformation
Growth on SD-Trp-Leu-His medium
colony lift filter assay
pAS2-1/
Apoptin
BD
pACT/
APAP1 mutant
AD
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Cellular localization of Apoptin and APAP1
16 hrs after transfection
psRed c1/
APAP1
RFPGFP
pEGFPC2/
Apoptin
HEL cells HeLa cells
DNA transfection
cells fix and DAPI stain
Fluororescent microscope
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Apootin and APAP1 are colocalized at the nucleus region of HeLa cells
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Purpose:
Involvement of APAP1 in Apoptin induced Apoptosis
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Apoptotic Assay of Apoptin and APAP1
DNA co-transfection
48hrs, 37oC
GFPpEGFPC/
pRK5F/APAP1
Flag Flag
pRK5F/Apoptin
or
HEL cells HeLa cells
cells fix, PI stain and fluorescent microscope
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APAP1 expression level in human tumor cell lines
Human tumor & normal cell lines
total cell protein extraction
SDS PAGE
Blot - APAP1 antibody
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Normal cells Tumor cells
Higher APAP1 expression level in human tumor cell lines
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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.
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Lower APAP1 expression level in human tumor cell lines
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Apoptosis
APAP1
APAP4 APAP4
APAP1
AP
AP
1
AP
AP
1
AP
AP
4
Tumor cells
P
ApoptinP
PAPAP4
APAP1 AP
AP
1
AP
AP
1
AP
AP
1
APAP4
APAP4
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APAP1
APA
P4
APAP4
AP
AP
4
APAP4AP
AP
4 AP
AP
1
APAP1
APA
P4APAP4
AP
AP
4
APAP4AP
AP
4
Apo
ptin
AP
AP
1
Cell survival
Normal cells
Apoptin
Ap
op
tin
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Study and Characterization of APAP1/DEDAF in zebrafish development
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Peptide structure of Z-APAP1
zAPAP1 257 a.a.
Zinc finger
GFWDCS VCTFRNSAEA FKCSIC
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Actin
ZAPAP1
ZRYBP
PCR Amplification of APAP1 of zebrafish cDNA
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Tissue Scan of Zebrafish cDNA
ZAPAP1
Actin
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APAP1 siRNA increases the abnormal frequency of abnormal embryo
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