1. GAPDH, A WELL-KNOWN
GLYCOLYTIC ENZYME, MEDIATES
APOPTOSIS BY EPIGENETIC WAYS
Pei-Ju Chin
Molecular Genetics and Biochemistry Program
Department of Biology
Georgia State University
Atlanta, GA 30303
2. Programmed Cell Death
Under control
Irreversible
A mechanism to remove abnormal or
unhealthy cells
Embryogenesis
Infection
Damaged cells which cannot be
repaired
Morphological characteristics
Cell shrinkage, appearance of
apoptotic bodies
Chromatin condensation,
hyperpolarization of mitochondria,
increase of membrane permeability,
accumulation of reactive oxygen species
(ROS)
Courtesy of Philip Yau
Zoli et al. Breast Cancer Res. 7:R681
What is apoptosis?
3. Why do I choose yeast as my model system?
Saccharomyces cerevisiae
Budding yeast/Baker’s yeast
Homology
Homology with higher eukaryotes
Apoptosis signaling pathway
Epigenetic model
Histone modification
Chromatin conformation
Advantages
Annotated genome
Available mutant library (Euroscarf, ATCC)
Available GFP-tag library (Invitrogen)
Courtesy of Alan Wheals, University of Bath, UK
4. Environmental stresses trigger apoptosis by
accumulating ROS inside cells
Madeo F. et. al. 2004. Cur. Opin. Microbiol. 7:655-660
Metallic ion
5. Avery. Adv Appl Microbiol. 2001;49:111-42
Metals and other oxidant stressors such as H2O2 generate ROS
(reactive oxygen species – superoxide, peroxide, hydroxyl radicals)
Metal
Metal
O2
, H2O2, OHº
Metal binding molecules, vacuolar
sequestration etc.
Antioxidant
defenses
Membrane
damage
Membrane
damage
DNA
damageProtein
damage
Uptake Efflux
Metallic ion triggers apoptosis by accumulating
ROS inside yeast cells
6. Metals resulting in apoptosis by different mechanisms
Metals
Redox-active Redox-inactive
Directly generate
ROS
•Indirectly generate ROS
•Displace redox-active metals from enzymes
•Deplete antioxidant defenses
Cu, Cr, Fe
Cd, Pb, Hg
7. Why study cadmium ?
Widely used in industries
Electroplating
Anti-corrosion
Rechargeable batteries
Hybrid/Electric car
Painting
Yellow color given
Photo diode (CdS)
Photo drum
Solar cell
Improper disposal harms our
health
8. Why study Cd-induced toxicity/apoptosis?
Carried by zinc-binding proteins
Same oxidation state (+2)
Neurodegeneration disease(Danford et. al., 1982; Rieder et
al., 1983)
Parkinson’s Disease
Alzheimer's Disease
Nephrotoxicity
Cancer
Leukemia (Aleksandrowicz et. al., 1982)
Aging
Crohn’s Disease ( Penny et al., 1983)
9. A famous epi case resulted from cadmium contamination-
Itai-Itai (Pain-Pain) Disease
1945
Kakioma mine with zinc
ore
Waste was flushed to
Jinzu river
Weaken bone and joint
Kidney failure
Courtesy of Kanazawa Medical University, Japan and University of California, Santa Cruz, USA
11. There are three GAPDH isozymes in the budding yeast
TDH1 TDH2 TDH3
Location Chromosome X Chromosome VII Chromosome VII
Abundance ? Only in aging cells Major
12. TDH3 is induced and shown with different conformation
after Cd exposure
Shanmuganathan A., 2008
14. TDH3 nuclear translocalization is found in apoptotic
yeast cells
Before 30 uM Cd treatment
After 30 uM Cd treatment for 1 hour
Shanmuganathan A., 2008
15. GFP-TDH3 fused strain is non-apoptotic
Untreated 30 uM Cd treated
BY4741 wildtype
BY4741 GFP-TDH3
16. Overdriven pentose phosphate pathway is not found in
GPF-TDH3 strain after Cd treatment
0
10
20
30
40
50
60
70
0 5 15 30 60 90
relativeconcentration(uM/A)
exposure time (min)
GSH/GSSG in wt with 30uM Cd exposure
Average GSH-Old
Average GSSG-Old
0
10
20
30
40
50
60
70
0 5 15 30 60 90
concentration(uM/A)
exposure time (min)
GSH/GSSG in TDH3-GFP with 30uM Cd exposure
NormalizedGSH
NormalizedGSSG
20. GAPDH, role beyond as a glycolytic enzyme
Energy production
Kinase activity(Kawamoto and Caswell, 1986)
Catalyzing tubulin polymerization into microtubules (Durrieu et al. 1987;
Muronetz et al. 1994)
Membrane fusion (Glaser and Gross,1995), Calcium-dependent fusogen
activity (Hessler et al., 1998)
Target of nitric oxide (Brune and Lapetina, 1996)
5’-UTR and 3’-UTR mRNA binding activity (Nagy and Rigby, 1995; Schultz
et al., 1996)
Nuclear protein that induces gene expression (Morgenegg et al., 1983)
Nuclear tRNA export protein (Singh and Green, 1993)
Uracil DNA glycosylase activity (Meyer-Siegler et al., 1991)
Ap4A-binding protein (Baxi and Vishwanatha, 1995)
Apoptosis (Ishitani et al., 1996; Sawa et al., 1997; Hara et al., 2005)
21. The role of GAPDH inside nucleus
GAPDH shows the binding activity to nucleic acid
Transcriptional Factors/DNA repair enzyme
RNA-based affinity DNA-based affinity
22. General questions in my proposal
What does GAPDH (TDH3) do inside the nucleus?
Does GAPDH participate in apoptosis by acting as a signaling
molecule rather than its glycolytic activity?
24. Does the protein interaction between SET and
GAPDH regulate the caspase-independent
apoptosis responses?
Specific Aim I
25. Rationale
GzmA Activates proapoptotic
protein
Procaspase (Fan et al., 2003)
DNase (Yamada et al., 2003)
SET, as a epigenetic modulator,
binds GzmA
↓GzmA activity
SET binds to GAPDH as well
Hypothesis
GzmA activity would be rescued by
sequestering SET protein with GAPDH
GzmA SET GAPDH
26. What will I test and how will I do?
I: In vitro GzmA activity assay
E. coli
GST-GzmA construct
E. coli
GST-SET construct
E. coli
GST-TDH3 construct
Induce and harvest protein, then purified by GST affinity column
GzmA
SET
BLT
GzmA
SET
TDH3
BLT
GzmA
TDH3
BLT
GzmA
BLT
Time
Activity
(%)
Time
Activity
(%)
Time
Activity
(%)
Time
Activity
(%)
27. What will I test and how will I do?
I: In vitro GzmA activity assay (Con’t)
Constant GzmA
Constant TDH3
Variable SET
SET Conc. (unit)
GzmA
Activity
Constant GzmA
Constant SET
Variable TDH3
TDH3 Conc. (unit)
GzmA
Activity
28. Complementation Test
Examination Group
Reference for basal expression
What will I test and how will I do?
II: In vivo GzmA activity assay
S. cerevisiae BY4741
with Cd treatment
Total protein extraction
S. cerevisiae BY4741
without Cd treatment
1. Western Blotting with Anti-GzmA
2. GzmA Activity Assay
GzmA activity/protein unit
in non- and apoptotic yeast cell
S. cerevisiae BY4741 ΔTDH3
with Cd treatment
S. cerevisiae BY4741 ΔTDH3
without Cd treatment
S. cerevisiae BY4741 ΔTDH3::pCM186-TDH3
without Cd treatment
S. cerevisiae BY4741 ΔTDH3::pCM186-TDH3
with Cd treatment
29. What if the result doesn’t fit my hypothesis?
Alternative hypothesis
Modification of TDH3 is necessary for binding with SET
GzmA
SET
X
In vitro
assay
TDH3
M
GzmA
SET
TDH3
M
X
S. cerevisiae BY4741 Wt
whole cell lysate
after Cd treatment
30. Pitfalls and comments for the proposed tests
Limitation of experimental method
I will use ΔTDH3 mutant and it is auxotrophic
Pyruvate supplement might be required
To tumble glycolytic flow
Non-specific digestion of BLT
Any kind of protease with Arg or Lys digestion activity
Measuring the background by using 3,4-dichloroisocoumarin
(3,4-DCI) as a GzmA-specific inhibitor
31. Conclusions and future directions
TDH3 would alter the activity of SET as a histone
methyltransferase (HAT) as well
To use ΔTDH3 may not be the best solution
To construct a glycolytic TDH3 protein without SET binding domain
32. Does GAPDH-mediated histone H2B expression
influenced by redox status exert the apoptotic
potential of a Cd-stressed yeast cell?
Specific Aim II
33. Rationale
GAPDH augments H2B expression
NAD as an enhancer
Oxidative environment in apoptotic cells
↑NAD/NADH ratio
Hypothesis
GAPDH exerts apoptosis response by
promoting H2B expression
H2B
GAPDH
TFs
H2B
Kinase
S10Ph
H2BNAD
34. What will I test and how will I do?
I: Does H2B augment apoptosis?
S. cerevisiae BY4741
ΔH2B
Un- or treated with Cd
S. cerevisiae BY4741
ΔH2B::pCM186-H2B
S. cerevisiae BY4741
ΔH2B::pCM186
S. cerevisiae BY4741
wildtype
Capillary electrophoresis
For measuring NAD/NADH
wildtype ΔH2B ΔH2B
pCM186
ΔH2B
pCM186-H2B
%
Apoptotic
cell
Propidium iodine
or DHR stain
Flow cytometer
35. Cell sorting
Apoptotic
1.34 X 106
Non-apoptotic
6.34 X 106
Liquid
phase
2OD ≒2 X 107 cells
Courtesy from MRC Flow Cytometry Core Facility, UK
BD FACS Aria II
Cell Sorter
What will I test and how will I do?
II: Does apoptotic cell preserve more TDH3-bound H2B
promoter?
Un- or treated with Cd
S. cerevisiae BY4741 wildtype
Propidium iodine
or DHR stain
36. Quantitative Chromatin Immunoprecipitation (qChIP)
Mikhail Spivakov and Amanga G. Fisher. 2007 Nat Rev
Genet 8: 263-271
Anti-TDH3 IgG
H2B promoter-specific primers
What will I test and how will I do?
II: Counting for TDH3-bound H2B promoter
37. What will I test and how will I do?
II: Counting for TDH3-bound H2B promoter
Cd- treated
Cd- untreated
38. Non-apoptotic
Apoptotic
What if the result doesn’t fit my hypothesis?
Alternative hypothesis
Histone code is more critical than the amount itself
S10Ph
H2B
H2B
H2B
H2B
S10Ph
H2B
Kinase
H2B
Western Blotting
by Anti-H2BS10Ph
+Cd Control
anti-β-act
anti-
H2BS10Ph
Wildtype
pCM186-H2B
Kinase
39. Pitfalls and comments for the proposed tests
Limitation of experimental method
I will use propidium iodine to label and sort cells.
Propidium iodine is a DNA chelating dye and may interfere the
antibody binding efficient in ChIP assay.
Only an indirect evidence for GAPDH as an oxidation
sensor is provided.
Quite difficult to manipulate redox status without causing any
artificial effects
Is apoptosis triggered by Cd treatment or the manipulation of redox
status?
We cannot mimic apoptosis in a test tube
In vitro test is not feasible
40. Conclusions and future directions
A argument of chromatin condensation and apoptosis does
exist
Which one is in up-stream?
Where does TDH3 bind in apoptotic yeast genome
ChIP assay by using Anti-TDH3
A descriptive study
To provide some cues for GAPDH as a epigenetic modulator
http://www.vincibiochem.it/AMChIP-IT.htm
41. General conclusions
I hereby raise the ideas to elucidate the role of GAPDH in
apoptotic yeast cells
To regulate caspase-independent apoptosis responses
As a oxidation sensor to enhance the chromatin condensation
GzmA SET GAPDH
H2B
GAPDH
TFs
H2B
Kinase
S10Ph
H2B
NAD
