GAPDH, a well-known glycolytic enzyme, mediates

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

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?

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

Environmental stresses trigger apoptosis by

accumulating ROS inside cells

Madeo F. et. al. 2004. Cur. Opin. Microbiol. 7:655-660

Metallic ion

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

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, FeCd, Pb, Hg

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

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)

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

0.1

1

10

100

AC

S1

AC

S2

AD

H1

AD

H2

AD

H3

AD

H4

AD

H5

ALD

2

ALD

3

ALD

3

ALD

4

ALD

5

ALD

6

ARO

10

CD

C19

CTF1

9

EN

O1

EN

O1

EN

O2

FBA

1

FBP1

GA

L10

GLK

1

GPM

1

GPM

2

GPM

3

HXK1

HXK2

LAT1

LPD

1

PD

A1

PD

B1

PD

C1

PD

C5

PD

C6

PD

I1

PFK

1

PFK

2

PG

I1

PG

K1

PG

M1

PG

M2

PY

K2

SFA

1

SN

O4

TD

H1

TD

H1

TD

H3

TH

I3

TPI1

Amrita Fold-30

Peiju Fold-30

Amrita Fold-60

Peiju Fold-60

The expression level of glycolysis gluconeogenesis-associated

genes are induced after Cd treatment

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

TDH3 is induced and shown with different conformation

after Cd exposure

Shanmuganathan A., 2008

-Cd +Cd

Wildtype

ΔTDH3

S. cerevisiae BY4741 ΔTDH3 strain is non-apoptotic

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

GFP-TDH3 fused strain is non-apoptotic

Untreated 30 uM Cd treated

BY4741 wildtype

BY4741 GFP-TDH3

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

rela

tiv

e c

once

ntr

ation

(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

conce

ntr

ation (

uM

/A)

exposure time (min)

GSH/GSSG in TDH3-GFP with 30uM Cd exposure

NormalizedGSH

NormalizedGSSG

GFP-fused TDH3 preserves its glycolytic activity as wildtype

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0 10 20 30 40 50 60 70 80 90 100110120

WT 0+4

WT 3+1

GFP 0+4

GFP 3+1

GAPDH ActivityRela

tive

Act

ivity

Time (min)

GAPDH (TDH3, in this case) contributes to

apoptosis responses by acting as a signaling

molecule rather than its glycolytic function

Clue

GAPDH, role as a glycolytic enzyme

PDB ID:3GPD

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 fusogenactivity (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)

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

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?

GAPDHS-NO-

GAPDH

GAPDH

Siah1Proapoptotic protein

activation

Siah1

O

GAPDH

H

T

TSiah1

GAPDH

Cyt C AIF

VDAC1

SET

Cdk

β 1GAPDH

GzmA

H2B

GAPDH

TFs

H2B

Kinase

S10Ph

H2B

Cell cycle

retarded

Cytosol

Mitochondria

Nucleus

?

SET GAPDH

NA

D

Does the protein interaction between SET and

GAPDH regulate the caspase-independent

apoptosis responses?

Specific Aim I

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

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

(%)


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

Complementation Test

Examination Group

Reference for basal expression


II: In vivo GzmA activity assay

S. cerevisiae BY4741

with Cd treatment

Total protein extraction


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


S. cerevisiae BY4741 ΔTDH3::pCM186-TDH3


S. cerevisiae BY4741 ΔTDH3::pCM186-TDH3

with Cd treatment

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

TDH3M

GzmA

SET

TDH3M

X

S. cerevisiae BY4741 Wt

whole cell lysate

after Cd treatment

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

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

Does GAPDH-mediated histone H2B expression

influenced by redox status exert the apoptotic

potential of a Cd-stressed yeast cell?

Specific Aim II

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

H2B

NA

D


I: Does H2B augment apoptosis?


ΔH2B

Un- or treated with Cd


ΔH2B::pCM186-H2B


ΔH2B::pCM186


wildtype

Capillary electrophoresis

For measuring NAD/NADH

wildtype ΔH2B ΔH2B

pCM186

ΔH2B

pCM186-H2B

%Apoptotic

cell

Propidium iodine

or DHR stain

Flow cytometer

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


II: Does apoptotic cell preserve more TDH3-bound H2B

promoter?

Un- or treated with Cd

S. cerevisiae BY4741 wildtype

Propidium iodine

or DHR stain

Quantitative Chromatin Immunoprecipitation (qChIP)

Mikhail Spivakov and Amanga G. Fisher. 2007 Nat Rev

Genet 8: 263-271

Anti-TDH3 IgG

H2B promoter-specific primers


II: Counting for TDH3-bound H2B promoter


II: Counting for TDH3-bound H2B promoter

Cd- treatedCd- untreated

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

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

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

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

NA

D

Questions or comments?

pCM189-

Vector shuttling between prokaryotes and eukaryotes

Chromosome is highly compacted by histone

Histone code modification decides the chromatin

conformation

Histone code controls the gene expression in pre-

transcriptional level

Brian D. Strahl and C. David Allis. Nature 403, 41-45

Documents

GAPDH, a well-known glycolytic enzyme, mediates