ANALYSIS OF CELLULAR RESPONSES TO HEAVY METAL-INDUCED STRESS IN Saccharomyces

cerevisiae

Pei-Ju Chin

Molecular Genetics and Biochemistry ProgramDepartment of Biology

Georgia State UniversityAtlanta, GA 30303

Research Interest in Houghton Lab

Model Organism: Saccharomyces cerevisiae (baker’s yeast)

Cellular response with heavy metal treatments Chromium (Cr), Cadmium (Cd) and Copper (Cu)

Oxidative Stress

Metal Exposure

Apoptosis Autophagy

Cell Fate Decision

Heavy Metals- Beneficial but Harmful

Widely used in industries

Electroplating

Anti-corrosion

Rechargeable batteries

Hybrid/Electric car

Painting

Yellow color given

Photo diode (CdS)

Photo drum

Light sensor in smart phone

Improper disposal harms the public health

Itai-Itai (Pain-Pain) Disease-Pandemic, Accidental Cadmium Exposure

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

Yeast Is Chosen as the Model System for the Simplicity and Homology with Higher Eukaryotes

Advantages

Easy to handle and manipulate

Annotated genome

Available mutant library

Available GFP-tag library

Homology

Homology with higher eukaryotes

Apoptosis and autophagy pathway is well-investigated Chin , PJ and Houghton, JE. Unpublished data

Why is Yeast Chosen as the Model System?

Braun RJ (2012) Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Frontiers in oncology 2:182.

Environmental Stress Triggers Apoptotic Responses by Accumulating ROS Inside Cells

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

Metallicion

Metal-induced ROS Generation- Direct and Indirect

Metals

Redox-active Redox-inactive

Directly generate

ROS

•Indirectly generate ROS

•Metal replacement from metalloenzymes

•Deplete antioxidant defenses

Cu, Cr, Fe Cd, Pb, Hg

Fenton ReactionCu2+ + H2O2 →Cu3+ + OH·+ OH−

Hanas, JS. And Gunn CG. Nucleic Acids Res. 1996, 24, 924-930

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)

Highly Reactive ROS Oxidizes Cellular Components

Metal-induced Response in S. cerevisiae-Acute and lethal

• 30 μ M Cd(NO3)2• 8mM Cu(NO3)2

Seedingculture

100mLYEPD

O/N

incubation

2OD600

1 hour

DIC

PI

Merge0.1

1

10

BM

H1

BM

H2

CD

C25

DN

M1

ER

F2

FIS

1

HX

T17

LTE

1

PH

B1

PH

B2

RT

G2

SH

R5

Fo

ld c

ha

ng

es

Cd-30 minsCd-60 mins

0.1

1

10

AS

F1

BIR

1C

DC

48

FU

N34

HX

T17

IFM

1K

AP

120M

CA

1O

AF

1R

SM

23R

TT

107

SN

L1

ST

M1

SV

F1

SX

M1

UT

H1

WW

M1

YO

P1

Fo

ld c

ha

ng

es

Cd-30 mins

Cd-60 mins

24~48 hours

Cd 1+3Cu 1+3

Post-incubate

3 hours

Wash

Yeast Genome Pathway Analysis, 2006

The Presence of Reduced-glutathione (GSH) Indicates Cell Suffering from Oxidative Stress

0.1

1

10

100

BU

D9

FB

A1

FB

P1

GN

D1

GN

D2

PF

K1

PF

K2

PG

I1P

GM

1P

GM

2P

RS

1P

RS

2P

RS

3P

RS

4P

RS

5R

BK

1R

KI1

RP

E1

SO

L1

SO

L3

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L4

SP

C1

TA

L1

TK

L1

TK

L2

TK

L2

VH

S1

ZW

F1

Fo

ld c

han

ges

Cd-30 mins Cd-60 mins

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0 5 15 30 60 90

Rel

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e co

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atio

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uM

/A)

Exposure time (min)

GSH (Reduced)GSSG (Oxidized)

Antioxidant Defense Mechanism upon Cadmium Exposure

ZWF1 and GND1 are induced

GSH/GSSG increases during Cd exposure

1

10

GND1 ZWF1

Fo

ld C

ha

ng

es

Cd-30 mins

Cd-60 mins

Post-Exposure Cell Fate Decision

Survival Route Antioxidant Defense Mechanism (eg. GSH) Autophagy

Elimination of harmful or damaged molecules

Suicide Route Damage is far beyond recoverable Altruistic, restrain the dissemination of damage to

entire population Programmed Cell Death/Type-I Cell Death Autophagy-associated cell death/Type-II Cell Death

Apoptosis-A Suicide Program with Tight Regulation

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)

Caspase-dependent Apoptosis

Courtesy of Philip Yau

Zoli et al. Breast Cancer Res. 7:R681

Classification of Cysteine-dependent Asparate-directed Proteases (Caspases)

MacKenzie, S.H. and Clark A.C. Death by Caspase Dimerzation. Protein Dimerization and Oligomerization in Biology. ISBN: 978-1-4614-3228-9. Landes Bioscience

Yeast Caspase 1 (Yca1p), a Metacaspase with Caspase 3-like activity

Wong, AH et. al. 2012. J Biol Chem 287: 29251-29259

Like its executioner orthologues, Yca1 is inactivated until the pro-domain is cleaved

Cell-Death

Pro

Cell-Death

Pro

ApoptosisCascade

Heavy Metals Such as Cadmium Results in the Accumulation of ROS and the Activation of Yca1p, an Only-known Caspase in Yeast

30 μM Cd 0+4 hrs 30 μM Cd 1+3 hrs

DHR

SR_FLICA

Nargund A. et al. 2008. Apoptosis 13: 811-821

Yca1p appears to have DEVD like activity.

Zvad-fmk Proves a Second Caspase-like Activity

IETD and DEVD activity

Nargund, A. Unpublished data

Pan-CaspaseInhibitor

Cysteine proteases in yeast

1. RIM13 – calpain like protease

2. ATG4 – involved in autophagy3. YCA1 – known function - yeast caspase4. SNO4

5. HSP33

6. HSP32

7. ESP1 – known function-acts as separase8. OTU2

Candidates for Second Caspase

Atg4p, second caspase-like protease invloved in Cd –induced apoptosis

IETD and DEVD activity

ATG4 shows very little IETD like activity in Cd treated YCA1Δ

Defensive Mechanisms before Triggering PCD

Antioxidant Defense Mechanism Superoxide dismutase (SOD), peroxidase, reduced glutathione

(GSH)…

Lysosomal/Ubiquitinylation Proteasome System (UPS) Clean up misfolded, damaged or harmful proteins

Stress granule (SG) formation Stalled translational pre-initiation complex (eIF2α, eIF4G,

PABP…)

Quick response after cell has recovered

Autophagy Degrade damaged organelles (eg. Mitochondria)

Autophagy (mitophagy) can lead to PCD as well (autophagic cell death/Type-II cell death)

Autophagy (Self-Eating) –Cellular Homeostasis Mechanism

Klionsky DJ et al. 2007. Autophagy 3:5 413-416

Are autophagic initiators required for apoptotic response in S. cerevisiae?

Aim I.

0.1

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100A

TG

1

AT

G2

AT

G3

AT

G4

AT

G5

AT

G6

AT

G7

AT

G8

AT

G9

AT

G10

AT

G11

AT

G12

AT

G13

AT

G14

AT

G15

AT

G16

AT

G17

AT

G18

AT

G19

AT

G2

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G2

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G31

AT

G32

AT

G33

Fo

ld C

ha

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es n

orm

alize

d b

y u

ntre

ate

d

Cd 30 mins

Cd 60 mins

Autophagic Genes Are Induced upon Cadmium Exposure in S. cerevisiae

1

10Fo

ld C

ha

ng

es N

orm

alize

d b

y u

ntre

ate

dCd 30 mins

Cd 60 mins

J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86

(Atg6)

Atg4p, Atg6p and Atg8p are Involved in the

Maturation of Autophagosome Biosynthesis

J. D. Lunemann, and C. Munz. Cell Death Differ (2009) 16, 79-86

Are the Autophagy Initiators required for Yca1p cleavage?

Chin, P.J. and Houghton, J.E. Unpublished data

Are the Autophagy Initiators required for Apoptotic Response in Yeast upon Metal Exposure?

Chin, P.J. and Houghton, J.E. Unpublished data

Population of propidium iodine (PI) positive

Summary of Aim I

Atg4p contributes to Caspase 8-like activity (Nargund A. Unpublished data)

Autophagic initiators: Atg4p, Atg6p and Atg8p are required for Yca1p-dependent apoptosis upon metal exposure

Does autophagosome apparatus facilitate Yca1p activation?

Aim II.

Yca1p Is Insoluble in vivo

Natural property per se (unlikely) E. coli-expressed His-tagged Yca1p is soluble

Be sequestered in vacuoles

[NP-40]

Yca1p is Trans-localized to Acidic Vacuoles upon Cadmium Treatment

DIC Yca1-GFP

MDC(Acidic vacuoles including

autophagosome )

Crude Isolation of Autophagosome

To investigate the presence of Yca1p in autophagosome

Yca1-GFP strain instead of wild type was used Anti-Yca1 WB requires specialized

lysis condition (detergent)

Anti-GFP was used for WB

Copper, instead of cadmium was used Yca1-GFP strain reacts poorly with

cadmium treatment

Ficoll15%

Ficoll8%

Ficoll4%

Ficoll0%

Autophagosome

Cell debrisInsoluble Fraction

Yca1p-GFP was Absent from Debris Sedimentation after Cu Treatment

25%

50%

100%

Fic

oll

0-4

%

Fic

oll

4-8

%

Sed

imen

tati

on

Yca

1-G

FP

ex

pre

ssio

n le

vel

Cu 0+4

Cu 1+3

(GAPDH-GFP)

Summary of Aim II.

Yca1p-GFP was present in autophagosome in both Cu-untreated or treated cells Autophagosome vacuoles sequester

the activity of Yca1p

Autophagosome facilitates the process of Yca1p

Yca1p-GFP was absent from the cell debris fractionation after Cu treatment Solubility of Yca1-GFP is increased

after Cu treatment

Accessible for initiators/self-cleavage process

Autophagosome

Yca1p

Cu/Cd

Soluble

insoluble

Is autophagy a life-saver or kiler in yeast undergoing heavy metal-induced oxidative stress ?

Aim III.

Intercommunication of autophagy and apoptosis in higher eukaryotic cells-Autophagic proteins as cytoprotector

Autophagy induces cytoprotection in neuron cells by removing aggregated huntingtin (Hara T. et. al. Nature 441:885-889)

Atg6 helps to resolve DNA-damaged foci (Mathew R. et. al. Genes Dev:21 1367-1381)

C. elegans lives longer in caloric restriction condition while autophagy plays an essential part (Jia K. et. al. Autophagy 3:597-599 )

Life span is prolonged in yeast and delay of chronological aging by rapamycin (Alvers et al. Autophagy 5:847-849)

ROS reduces the activity of Atg4 to de-lipidize Atg8-PE,therefore facilitates autophagy (Scherz-Shouval R. et. al. EMBO J 26:1749-1760)

Intercommunication of autophagy and apoptosis in higher eukaryotic cells-Autophagic proteins facilitate apoptosis

Atg12 cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to Bcl-2 and causes apoptosis (Rubinstein AD et. al. Mol Cell 44:698-709)

Atg4D cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to mitochondria, release cytochrome C and causes apoptosis (Virginie et. al. Autophagy 5: 1057-1059)

Atg6 cleaved by caspase-3, which leads to the exposure of BH3 domain that binds to mitochondria, release cytochrome C and causes apoptosis while defects its role to conjugate Atg5-Atg12(Luo S et. al. Cell Death Differ 17:268-277)

Autophagy selectively eliminates catalase and causes the accumulation of ROS (Yu L et. Al. PNAS 103:452-4957)

Distinguish pathways exist in S. cerevisiae

No Bcl-2.

No potent caspase-3 cleavage site of autophagic proteins

No BH3 domain in autophagic proteins

Autophagy, A Life-saver or Killer?

Debates do exist

Cell type-dependent Cells from different tissue or genotype

Autophagy inducer/inhibitor -dependent Rapamycin, 3-MA, bafilomycin A1

Stress type-dependent Hypoxia, starvation, ROS, misfold protein accumulation

Time/Occasion-dependent When to introduce autophagy and apoptosis

What if the role of autophagy is decided by the stage it is introduced?

Rapamycin Induces Autophagic Response by Inhibiting the Hyper-phosphorylation of Atg13p Caused by TORC1

Rapamycin-induced Autophagy is Demonstrated by the Increased Atg8-PE to Atg8 Ratio in Dose-independent Manner

Kiel Jan A. K. W. 2010. Phil. Trans R. Soc. 365: 819-830

[rapa]

Treatment Schema

Independent Events Autophagy only

Apoptosis only

Two-Stage Events Autophagy->Apoptosis

vice versa

Overlapping Events Treatment A-D

The Duplicitous Role of Autophagy in S. cerevisiae

Rescue Introduced prior to Cd

High autophagic activity

Kill Introduced after Cd

Basal autophagic Activity

One activity impedes another

Autophagy Facilitates Cd-induced Apoptotic Response-Type-II Cell Death

All PI has MDC Type-II CD

Not all MDC has PI Survival?

Summary of Aim III.

Duplicitous role of autophagy Autophagy prior to Cd-induced apoptosis: Protective

Autophagy follows Cd-induced apoptosis: Destructive

The trend of apoptosis and autophagy activity is mutually exclusive

Is the appearance of granularity after rapamycininduction a useful marker for monitoring autophagicflow?

Aim IV.

Rapamycin

Untreated Cd Untreated Cd

Side Scatter Detector (SSC)

5.6% 1.3%

Heterogeneity Serves as a Potential Marker for Monitoring Rapamycin–induced Autophagy in S. cerevisiae

S. cerevisiae BY4741 wt

Rapamycin Wash Cd Treatment

The Core Principle: Detection of Cellular Size (Volume) and Complexity (Granularity)

Courtesy of RIC Facility, Brigham Young University, MRC Clinical Sciences Centre, and iGEM 2010 website Sysmex.com

TEM Atg8(LC3) tagging orAcid vacuole staining

Transform/Turnoverof Atg8p by WB

Direct evidence of autophagophore formation

Most descriptive

Time-saving Easy to perform Dynamic (Toxicity of dyes)

Direct evidence of autophagic flux

Golden standard

Time consumingSophisticated facilities/well-trained personnel requiredAdequate matrix to avoid statistic biasNon-dynamic

Non-specific result of acid organelle labelingArtificial effects of GFPAdequate matrix to avoid statistic bias

Time consumingLabor intensiveNon-dynamic

Takeshige, A., et. Al. J. Cell. Biol. 119:301-311. Mizushima, N. Int . J. Biochem. Cell. Biol. 36:2491-2502. Mizushima, N., Yoshimori, T. Autophagy 3:542-545.

The Evaluation of Methodologies Measuring Autophagic Activities

[rapamycin] XY-Plot HistogramFSC HistogramSSC

0.0

1.0

2.0

4.0

8.0

FACS Profile Cell volume

Complexity

WT yeast cell responses to rapamycin

Cell Volume

Co

mp

lexity

The Augmentation of Q2 Population was Found in Wild Type upon Rapamycin Treatment

wtWild type atg4Δ

atg6Δ atg8Δ

The Character of Q2 Augmentation was Absent in Autophagic Mutants upon Rapamycin Treatment

Q2 Population Represents Autophagic Population

Wt was treated by 4 μg rapamycin for 2 hours

Q3 and Q2 population was sorted by FACS Aria

Sample was lysed and analyzed by WB

Cell Volume

Co

mp

lexity

Courtesy of NIA/IRP Flow Cytometry Unit

The Dynamic Monitoring of Autophagic Activities by Cytometry-based Method

Wild type, 4 μg/mL rapamycin treatment

The cells were sampled every 15 minutes

Cell Volume

Co

mp

lexity

Evaluation of the Performance between Immunoblotand Cytometry-based Methods in Routine Practice

Crude Protein Extraction/Quantification (1 hour)

SDS-PAGE (2 hours)

Atg8-probed Immunoblot (8 hours)

Sample Collection

Data Acquisition

Apoptotic Staining (optional)(1 minutes~2 hours)

Flow Cytometry

Summary

The fluctuation of Q2 population dynamically reflects the autophagic potential S. cerevisiae tends to increase its cell size and complexity upon

rapamycin treatment, which phenomenon was not observed in autophagic mutant

Q2 has higher Atg8-PE to Atg8 ratio than Q3

Pros of the cytometry-based technique Staining is not required Quick, easy and dynamic Cells are viable for downstream analysis

Cons of the cytometry-based technique Other cellular activities than autophagy which increase Q2 may

interfere the result. The specific staining may be required for excluding other activities Well-established control is required

General Discussions and Conclusions

The entire autophagy pathway are necessary for Yca1p processing in S. cerevisiae

Autophagic initiators are required for Yca1 activation

Solubility of Yca1p determines its accessibility

For the role of autophagic responses in apoptosis, the stage may be critical in S. cerevisiae

Early: autophagy helps cell to survive

Late (autophagic cell death, Type II Cell Death): autophagy further facilitates apoptosis

Granularity is a useful marker for monitoring autophagic flow

Thorough experimental design needs to be practiced

Clearance of damaged organelles

Cell recovered and survived

Anti-apoptotic Autophagy

Autophagy Initialization(Atg4p, Atg6p, Atg8p)

Apoptosis(Type I Cell Death)

Apoptosis Autophagy ApoptosisAutophagy

High autophagy flux(Atg8-PE/Atg8)

Yca1 Pro

Yca1 Pro

Yca1 Pro

Insoluble

soluble

Autophagosome

Acknowledgement Dissertation Committee

Dr. John E. Houghton Dr. Susanna F. Greer Dr. Irene T. Weber

Lab Alumni Dr. Anupama Shanmuganathan Dr. Amrita Nargund Rupa Koduru Abhishikta Madireddy Chelsea Hagan Yi Peng

Core Facility Debby Walthall Sonya R. Young Dr. Hyuk-Kyu Seoh Ping Jiang Gemeia Cameron

Biology Department Dr. Phang C. Tai Dr. Zehava Eichenbaum LaTesha M. Warren

Friends@Metro Atlanta Area

Funding Support NIH (GM579450) Georgia Research Alliance Molecular Basis of Disease, Georgia State University

Family Ping-Hwei Chin Chiu-Lan Huang Su-Ying Chin Hsuan Liu