Redox regulation, Oxidative Stress and Selenoproteins

Redox and Protein Folding

Danyelle M. Townsend May 18 – 22, 2015

Charleston, SC

Endoplasmic Reticulum (ER)���Albert Claude 1945���

Lace work structure”

Rough ER

•  Protein synthesis: Secreted,

membrane bound, digestive enzymes, hormones and antibodies

•  Translocon docking sites for ribosome

•  GSH/ GSSG ~10:1

Smooth ER:

•  Lipid / phospholipid and

steroid synthesis

•  Calcium ion storage

Sarcoplasmic ER:

Calcium storage: major role in excitation / contraction coupling

Demand exceeds capacity ……..

Conditions

Probable effects

temperature shifts

(e.g., heat-shock)

protein denaturation

protein aggregation

heavy metals, arsenite, iodoacetamide

reaction with sulfhydryl goups, conformational changes in protein

anoxia, hydrogen peroxide, superoxide ions, free radicals

oxygen toxicity, free radical fragmentation of proteins

proteasome inhibitors

inhibition of proteolysis

amytal, azide, dinitrophenol, ionophores

antimycin

inhibition of oxidative phosphorylation, changes in redox state,

covalent modification of proteins

hydroxylamine

cleavage of asparagine-glycine bonds

ethanol

translation errors

amino acid analogs, puromycin

abnormal proteins

Agents/Treatments that induce ER stress response

Grek and Townsend (2013): ER stress and Cancer Biology:

Misfolded protein"

Retrograde transport "through the translocon "

Ubiquitination"

Degradation in the"26S proteasome"

ERAD"

Sec proteins and ER export

Adapted from www.arthritis-research.com

Zhang, K. et al. J. Biol. Chem. 2004

Three UPR transducers and one master regulator GRP78 / BiP: Glucose related protein / Binding Immunoglobin Protein

Zhang, K. et al. J. Biol. Chem. 2004;279:25935-25938

UPR signaling mediated by PERK protein kinase RNA-like endoplasmic reticulum kinase

Zhang, K. et al. J. Biol. Chem. 2004

UPR signaling mediated by IRE1 and ATF6

Nature Medicine: Iwawaki et al., (2004) 10:1014

Transgenic mouse model for monitoring endoplasmic reticulum stress in vivo

Wek et al., Nature Medicine (2010) 16: 374-376

www.anti-agingfirewalls.com

Duration and amplitude of ER stress determines cell fate

Unfolded and misfolded proteins magically appear?

H2N

CH2

COOH

S.

GSH

NO

GSNO

N2O3

SSG

H2N

CH2

COOH

SH SNO

H2N

CH2

COOH

SHS-glutathionylation

GSH / Trx

GSH/ Grx / Srx

SH

SOH

ROS RNS

ROS RNS

Sulfhydryl

Cysteinyl radical

Sulfenic acid

S-nitrosylation

J. Uys, P. Mulholand, D.M. Townsend (2014) Molecular Pharmacology

GSTP S-glutathionylates ER resident proteins

Mass Spectrometry Identification of S-glutathionylated ER proteins

Protein ID MW (kDa)

Calnexin 97

Calreticulin 60

Endoplasmin 94

SERCA2A 114

GRP78, Bip 78

Protein disulfide isomerase (A5) 59

GSTP is a resident Endoplasmic Reticulum (ER) protein

BIP

GSTP

Merge - TRIO

DAPI - nuclear

Calnexin

DAPI - nuclear

GSTP

Merge - TRIO

GSTP

Calnexin

GAPDH

Gstp1/p2 + - + - + -

ER Cyto Nuc

Histone H3

SERCA2

GSTP is a resident ER Protein

GSTP regulates ER Calcium homeostasis

0%

20%

40%

60%

80%

100%

120%

0 500 1000 1500

ER

Cal

cium

hom

eost

asis

(%

of c

ontr

ol)

Time (second)

Gstp1/p2+/+ Gstp1/p2-/-

Thapsigargin Gstp1/p2+/+

Gstp1/p2-/-

GSTP interacts with ER protein

and enhances S-glutathionylation

*

* *

0

20

40

60

80

100

120

Rel

ativ

e Pr

-SSG

leve

l

WT Gstp1/p2-/-

GSTP BIP

PDI

GSTP

Calnexin

GSTP

Calreticulin GSTP

IP: Calnexin

IP: BIP

IP: Calreticulin

IP: PDI

Total IP IP IgG

Disulfiram (10µM)

- - + -

Gstp1/p2+/+

Gstp1/p2-/-

Thapsigargin (nM)

Velcade (nM)

Gstp1/p2+/+ 121.7 23.6

Gstp1/p2-/- 16.5 3.0

ratio 7.4 7.9

Thapsigargin and Velcade are more toxic to Gstp1/p2-/- BMDDCs

Viability (IC50 value)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375108/

ER dysfunction in liver disease

J Pharmacol Exp Ther. 2013 Jan;344(1):286-94. doi: 10.1124/jpet.112.199067. Epub 2012 Oct 23.���Mouse liver protein sulfhydryl depletion after acetaminophen exposure.���

Yang X, Greenhaw J, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Davis K, Salminen WF.���Source���

Division of Systems Biology, National Center for Toxicological Research, Jefferson, Arkansas, USA.

Protein – SH depleted regions correlated with areas exhibiting histopathologic injury

NOT GLOBAL DECREASE IN LIVER

J Pharmacol Exp Ther. 2012 Feb;340(2):360-8. doi: 10.1124/jpet.111.187948. Epub 2011 Nov 1.���

Changes in mouse liver protein glutathionylation after acetaminophen exposure. ���

Yang X, Greenhaw J, Ali A, Shi Q, Roberts DW, Hinson JA, Muskhelishvili L, Beger R, Pence LM, Ando Y, Sun J, Davis K, Salminen WF.���

1h = Increased P-SSG

24h = Decreased P-SSG at site of necrosis

PROTEIN OXIDATION 10x > APAP-Conjugation

Increased resistance to acetaminophen hepatotoxicity���in mice lacking glutathione S-transferase Pi���Colin J. Henderson*, C. Roland Wolf* Neil Kitteringham,Helen Powell §, Diana Otto*, and B. Kevin

Park ������

NAPQI + GSH Detox (NOT related to GSTP)

NAPQI + Protein adduct equivalent

GSTP +/+ decreased GSH/GSSG

** Toxic role of GSTP downstream of protein conjugation of NADPQI

Decreased S-glutathionylation of BiP in GSTP1P2 knockout mouse

liver

GSTp

GAPDH

BiP

112213_SCW_II_141_Glut_BIP_FirstZT #16877 RT: 94.18 AV: 1 NL: 1.91E4F: ITMS + c NSI d Full ms2 847.87@cid35.00 [220.00-1710.00]

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

x5 x5

34DLGTTYSC(glut)VGVFK46

MH+ = 1694.74 Da

M+2H+ = 847.87 Da

y2

294.16

y4

450.23

b5-H2O

470.22

b4

387.23

y5

549.34

(y11-129)2+

669.46

y112+

733.98

y3

393.26

(M+2H-129-H2O)2+

774.56

(M+2H-129)2+

783.53

y6-129-H2O

810.65

(M+2H-H2O-NH3)2+

829.60

y7-129

915.44

y6

957.50

y7

1044.46

y8-129

1078.50

y8

1207.56

y9-129

1179.58

y9

1308.57

b9

1245.51

b11-129

1272.50

y11-129

1337.61

b11

1401.63

y11

1466.72

b12

1548.49

b9-129

1116.51

BiP is s-glutathionylated on C41 and C420

BiP

PSSG

Merge

HEPG2 Cells pre-treated with NAC (and treated with 15mM APAP for 16hrs) have

28% increase in GSH ���RESULTS:

0

20

40

60

80

100

120

140

160

Relative GSH concentration

* *

BiP-SSG is concurrent with APAP induced liver toxicity

•  BiP: GSTpi dissociates when under ER stress

•  GSTpi KO mice have 42% less SSG-BiP than GSTpi WT mice

•  NAC treatment increases GSH / decreases BiP-SSG and reduces apoptosis

Protein Disulfide Isomerase

•  55 kDa Homodimer •  14 PDI isoforms in ER •  Two – UPRE in promotor

–  (Kox = 1mM)

•  Widely distributed across eukaryotic tissues –  mM Concentrations –  1% total pr. Content of cells –  Decreased PDI leads to protein aggregation (ie Anti-

chaperone)

Tian et al., 2006: Cell: 14(1): 61-73

SH SH

S-----S

Disulfide Formation

Sulfydryl

Disulfide

PDI

S-S

PDI

SH SH

Disulfide Isomerizations

S-------S

S S

S S

S------S

PDI

SH SH

PABA/NO - - + + +

IP:α-PDI + - - + +

IP:IgG - + + - -

DTT

- - - - +

WB:PSSG

WB:PDI

250

105

75

50

PDI-SSG is concurrent with UPR activation

Townsend et al., Mol Pharm 2006

Townsend et al., Cancer Research 2009

Xiong et al., I J Cell Biology 2012

Grek et al., ER stress and Cancer 2014

PDI-SSG Alters Structure / Function

0

20

40

60

80

100

120

0 25 50 100Per

cent

Act

ivity

μM PABA/NO

Wavelength, nm

250 300 350 400 450 500 550

Em

ission, au

-20

0

20

40

60

80

100

120

GSTSG GST Intact

Wavelength, nm

180 200 220 240 260 280

Molecular ellipticity, C

D units

-40

-20

0

20

40

60

80

GST INTACTGSTSG

Circular Dichroism Intrinsic Fluorescence

W38

C47

WB:P-JNK

WB:JNK1/2

WB: P-eIF2

WB: eIF2

WB: P-PERK

WB: PERK

25 uM PABA/NO 0.45 uM TG

0 2 4 6 12 24 4 12 24 (h)

WB:Chop

WB:Actin

0 2 4 6 24 (h)

WB:Bip

WB:LC3

20 uM PABA/NO

UPR-Induced Cell Death

Generation of S-glutathionylation refractory PDI (conversion of basic a.a. adjacent to catalytic cysteines to neutral)

WCGHCK

a domain

WCGHCK

a’ domain PDI-WT

WCGFCL WCGFCL PDI-FLFL

Histidine (H)

Lysine (K)

Phenylalanine

(F)

Leucine

(L)

Alanine

(A)

PDIFLFL is refractory to S-glutathionylation

WB: PDI

WB: P-SSG

In Vitro

In Cells

PABA/NO PDIWT

0 25 50 100

PDIFLFL

0 25 50 100 PDIWT PDIFLFL

- + - +

Rotenone

FLAG- PDIWT

FLAG- PDIFLFL

+ - + PABA/NO - +

Ctr

IP: FLAG

WB: PDI-PSSG

WB: PDI

WB: P-SSG

WB: PDI

Rotenone (µM) 0 250 500 1000 100

Rotenone (uM)

Mea

n O

.D.

0

2

4

6

0

5

10

0

2

4

P

DI

mR

NA

fold

incr

ease

-  + - +

PDIWT PDIFLFL

-  + - +

PDIWT PDIFLFL

-  + - + PDIWT PDIFLFL

B

IP

mR

NA

fold

incr

ease

CH

OP

mR

NA

fold

incr

ease

S-glutathionylation refractory PDI

1)   Blunts the toxic effects of rotenone

2)   Diminishes activation of the UPR in

PC12 neuronal cells

Ueharal et al., Nature (2006) 441, 513-518

S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration

Takashi Uehara1,4, Tomohiro Nakamura1, Dongdong Yao1, Zhong-Qing Shi1, Zezong Gu1, Yuliang Ma2, Eliezer Masliah3, Yasuyuki Nomura4 and Stuart A. Lipton1,3

Grek, C and Townsend, D.M. (2013)

PDI is a chaperone for ERα What are the consequences of S-

glutahtionylation of PDI on ERα stability and transcriptional activation?

PDI: Chaperone for Estrogen Receptor alpha

Xiong, Y et al, Int J Cell Biol. 2012

ERα: hormone binding transcription factor Ligand binding domain DNA binding domain

DNA activation

Ross Weatherman: 2006: Nature Chemical Biology, 2, 175-176

Lin et al. Genome Biology (2004: 5:R6

PSSG

PDI

0 0.5 PABA/NO (h) 2

75

50

75

50

IP: PDI

75

50

0 0.5 PABA/NO (h) 2

IP: ERα

PDI

ERα

75

50

PDI-SSG blunts chaperone activity with ERα

Xiong et al., 2011. IJCB

ERα

Actin

MG132

PS341

DMSO

PABA/NO

+

+

+

+

+

+

+

-

+

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

0

50

100

150

200

0 2 4 8 (h)

ERα

Actin

A

C

B

Rela

tive

(%)

ERα

mRN

A

0

1

2

3

0 2h 8h

n=3

ERβ

RNS induce proteasomal degradation of ERα

Xiong, Y et al, Int J Cell Biol. 2012

0 2 4 8 (h)

E2 (20 nM)

ERα

Actin

ERβ

p21WAF1/CIP1

c-Myc

cyclin D1

0 2 4 8 (h)

PABA/NO (20 µM)

ERα

Actin

ERβ

p21WAF1/CIP1

cyclin D1

Cyclin D1 mRNA and protein were down-regulated by anti-estrogen ICI182780 (Faslodex). ICI182780 also increased the expression of the CDK inhibitor p21WAF1/CIP1 (Watts CK, et al. 1995)

PDI-SSG has anti-estrogen effects

Grek, C and Townsend, D.M. (2013)

ER stress is worth stressing over… ������

Parkinson’s

A

G

E

Huntington’s

Alzheimer's

Heart Disease

ER stress has been linked to the following medical conditions:

Acknowledgements •  Townsend Lab

–  Ying Xiong, Ph.D. –  Zhiwei Ye, Ph.D. –  Jordan Frederick, Ph.D. –  Lin He –  Tiffany Ancrum

•  Yefim Manevich, Ph.D.

•  Joachim Uys, Ph.D. •  Novelos Therapeutics, Inc.

Christopher Pazoles, Ph.D. Harry Palmin, M.B.A.

NIH Funding: NIEHS: R56 ES017453

NIGM: GM103542-02

•  Kenneth Tew, Ph.D., D.Sc. –  Jie Zhang, Ph.D. –  Robert Bowers, Ph.D. –  Christina Grek, Ph.D. –  Yian Chen, Ph.D.

•  National Cancer Institute Larry Keefer, Ph.D.

Joseph Saavedra, Ph.D. Mahdol University, Thailand Chonticha Saisawang, Ph.D. Albert Ketterman, Ph.D.