Conclusions and Perspectives - 蛋白質半胱胺酸氧化修飾之偵測與亞磺酸還原酶抗氧化角色之研究

In this study, we focused on protein cysteine oxidation. First we utilized diamide as a

tool to approach thiol-specific oxidation, treating HeLa cells with high-dose diamide for

short time to survey its property of stimulating protein thiolation and effect on

antioxidant responses. Next we applied this diamide-induced thiolation method to figure

out the antioxidant role of sulfiredoxin. Besides its well-known function of reducing

hyperoxidized peroxiredoxin, by utilizing SRXN1 KO HAP-1 cell and SRXN1

over-expressed HEK cell as models, we found that sulfirdoxin might prevent cells from

diamide-induced protein thiolations, indicating a more essential role of sulfiredoxin, and

indicated there was possibly a compensatory activation of other antioxidant systems to

compromise sulfiredoxin’s function. Meanwhile, about thiol-modification detections,

we ameliorated the PEG-switch method, finding the optimal labeling condition of

PEG-maleimide, and applied this method to estimate protein redox status quantitatively.

Based on the labeling method, we proposed a scoring system that reflected the redox

status, and utilizing this method on investigating in cellulo oxidation models such as

insulin-triggered PTP1B oxidation.

In perspective, we modified the diamide treatment, and utilizing it to indicate an

unreported protective function of sulfiredoxin under thiol-specific attacks, implicating a

more essential role in antioxidant response. Additionally, we proposed a quantitative

display method of protein redox status, including the optimal tagging condition,

calculation and a scoring system. This method was focusing on cysteine oxidation,

could be operated easily and fast, and the most importantly, it could describe redox

status in a quantitative and comparable manner.

Figures

Figure 1. Diamide induced reversible thiolations in HeLa cells

(A) Diamide structure. (Picture credited to Sigma Aldrich website).

(B) Experimental pipeline of resin-assisted capture (RAC). Cells treated with diamide

were lysasted and harvested. Free cysteine thiol was alkylated by 100 mM IAA

followed by ammonium sulfate precipitation. Oxidized cysteine residue was reduced by

100 mM DTE, and the nascent exposure thiol was captured by thiolpropyl sepharose

with disulfide formation, so that pulled down oxidized proteins. Input and elution

fractions of RAC were analyzed with SDS-PAGE and Coomassie blue staining.

applied to RAC.

(D) HeLa cells were treated with 5 mM diamide for 5 min, and then washed in fresh

medium and cultured for 5 – 60 min for recovering. Cell lysates were subjected to RAC.

Ctrl: control group, 5D: 5 mM diamide treatment for 5 min, R5 – R60: recovering for 5

– 60 min.

Figure 2. Thiolated targets of diamide treatment

HeLa cells were treated with 5 mM diamide for 5 min, recovered for 30 min, and

then analyzed with RAC. The input, flow-through (FT) and elution fractions of RAC

were analyzed by immunoblotting. Hsp90: heat shock protein 90, PKM1/2: pyruvate

kinase M 1/2, Prx: peroxiredoxin. Ctrl: control group, 5D: 5 mM diamide treatment for

5 min, 5DR: recovered for 30 min.

Figure 3. Diamide affected redox status of glutathione, NADH and NADPH

HeLa cells were treated with 5 mM diamide for 5 min, and then extracted metabolites

from cultured cell by 66% acetonitrile. Samples were applied to LC-MS analysis to

verified metabolites including ADP, ATP, glutathione (GSH), glutathione disulfide

(GSSG), NADH, NAD+, and NADPH, and NADP+. The LC peaks were integrated by

Data Analysis® software, and normalized with the measurement of RAC control group.

The statistical significance between control and diamide-treated groups (3 repeats) was

examined by one-tail t-test. *: p-value < 0.05. **: p-value < 0.01.

Figure 4. Diamide triggered Nrf2 translocation and downstream gene expressions

(A) HeLa cells were treated with 5 mM diamide for 5 min and recovered for 30 min.

Cells were harvested and objected to fractionation. Nrf2 was probed by immunoblotting,

while GAPDH and Histone H3 were utilized as cytosolic and nuclear markers. Noted

that the predicted molecular weight of Nrf2 is 72 kD for cytosolic and 120 kD for

nuclear form according to Abcam® datasheet

http://www.abcam.com/nrf2-antibody-ab137550.html#description_images_10).

(B) Diamide-treated and recovered cells were applied to immunofluorescence of Nrf2

(green), while DAPI (blue) was used to point out cell nucleus. Nrf2 translocated into

nucleus after diamide treatment.

The transcription of Nrf2 downstream gene SOD2, NQO1 and GSTP1 were analyzed

by RT-PCR, whileβ-actin was used as internal control.

Figure 5. Knockout of SRXN1 led to truncation of sulfiredoxin

(A) SRXN1 in hyploid HAP-1 cells were edited by Crispr-Cas9 system, causing 1bp

adenosine insertion in exon 2. The parental and SRXN1-KO cells were supplied by

Horizon®. For confirming the insertion, genomic DNA was extracted from both cells as

template, and a 200 bp fragment containing the edited site was amplified by PCR and

subjected to sequencing.

(B) The insertion resulted in a frame shift while translation, leading to a truncation of

sulfiredoxin from I79 residue, which supposed to lose the active site of sulfiredoxin

(G98-R101). Protein structures, molecular weights and pI value were predicted by

ExPasy tool (https://swissmodel.expasy.org/).

Figure 6. Diamide-induced thiolations increased in SRXN1 KO HAP-1 cell

(A) Parental and SRXN1-KO HAP-1 cells were treated with 1 and 5 mM diamide for 5

min, and allowed to MTT assay. The results were normalized by the measurements of

control groups. The differences between parental and KO cells were analyzed by

one-tail t-test. *: p-value < 0.05.

(B) Parental and SRXN1 KO HAP-1 cells were treated with 5 mM diamide for 5 min

and elution fractions were analyzed by SDS-PAGE. Ctrl: control group; 5D: 5 mM

diamide treatment for 5 min; 5DR: recovering for 30 min.

cell model, including Hsp90, pyruvate kinase (PKM1/2), aldolase, and GAPDH, were

also confirmed in HAP-1 cell.

Figure 7. Thiolation of antioxidant enzymes increased in SRXN1 KO HAP-1 cell

Identified antioxidant enzymes in RAC samples, including peroxiredoxin (Prx),

thioredoxin 2 (Trx2), glutathione reductase (GR), were analyzed by immunoblotting,

while GAPDH was used as loading control. Ctrl: control group; 5D: 5 mM diamide

treatment for 5 min; 5DR: recovery for 30 min.

Figure 8. Diamide-induced protein glutathionylation and glutathione oxidation increased in SRXN1 KO cell

(A) Lysate of diamide-treated HAP-1 cells were analyzed by anti-glutathione (GSH)

immunoblotting for examing protein glutathionylation levels. β-ME: samples that

treated with β-mercaptoethanol in SDS-PAGE. The arrow indicates the glutathionylated

proteins, which able to be reduced by β-ME.

(B) The metabolite was extracted from HAP-1 cells by 66% acetonitrile, and the

samples were allowed to LC-MS analysis. The LC peak of oxidized glutathione (GSSG)

was integrated by Data Analysis® software, and was normalized by the measurement of

control group. The differences among groups, including diamide-treated (DA) and

Figure 9. Activity of thioredoxin reductase increased in SRXN1 KO cell

(A) Lysates of diamide-treated and recovered HAP-1 cells were allowed to anti-Prx

immunoblotting. Diamide treatment induced Prx dimerization in SRXN-1 KO cell,

which was reducible by adding β-mercaptoethanol.

(B) Lysate of parental and SRXN1-KO HAP-1 cells were subjected to immunoblotting

for examining the expression level of antioxidative enzymes, including thioredoxin 1

(Trx1), thioredoxin 2 (Trx2), thioredoxin reductase (TRR) and peroxiredoxin (Prx).

30 sec., reading = 20. Measurements were normalized by protein amount (quantitated

by BCA quantification), and the differences between parental and SXN1-KO cells were

analyzed by one-tail t-test. **: p-value < 0.01.

Figure 10. Diamide induced Srx dimerization in SRXN1 over-expressed HEK cell

(A) FLAG-SRXN1 overexpressing HEK cells were treated with 5 mM diamide for 5

min, and examined by anti-Srx immunoblotting with or without β-mercaptoethanol

(GAPDH as loading control). Dimerization was observed in diamide-treated group,

which could be reduced by β-mercaptoethanol.

(B) Diamide-treated SRXN1 overexpressing HEK cells were analyzed by anti-FLAG

immunoblotting.

binding partners by immunoblotting, whereas there were no signals of peroxiredoxin

(Prx), thioredoxin 1 (Trx1), and thioredoxin 2 (Trx2) be observed.

(D) Sulfredoxin immunoprecipitated by anti-Srx was examined by anti-FLAG and

anti-GSH. Dimerization increased after diamide treatment and could be reduced by

β-mercaptoethanol, whereas no glutathionylation was observed.

Figure 11. Sulfiredoxin dimerization was found in mitochondria

(A) FLAG-SRXN1 overexpressing HEK cells were subjected to fractionation. The

cytosolic and mitochondrial fractions were examined by anti-Srx immunoblotting, with

or without β-mercaptoethanol. SOD2 and GAPDH were used as mitochondrial and

cytosolic marker.

(B) FLAG-SRXN1 overexpressed HEK cell was treated with H2O2 0 - 10 mM for 10

min, and examined by anti-Srx immunoblotting with or without β-mercaptoethanol.

Figure 12. Hypothetic mechanism of sulfiredoxin in diamide-induced oxidation

In hypothesis, sulfiredoxin is sensitive to mitochondrial H2O2 stress in general. While

treated with diamide, cytosolic sulfiredoxin dimerizes for scavenging the oxidative

stress. When SRXN1 KO, both Trx and GSH systems become more active to

compensate the function of sulfiredoxin.

Fig. 13. Reaction of PEG-maleimide with protein cysteine residues causing mobility shift on SDS-PAGE

(A) Cultured cells were lysed and precipitated with ammonium sulfate. The pellet was

dissolved with lysis buffer, adding 1 mg/mL PEG-maleimide for tagging at 37°C for 30

minutes and then quenched with 2x sample buffer containing 4% β-mercaptoethanol

immediately. The sample was applied to SDS-PAGE and immunoblotting.

(B) In principal, exposed cysteine residues with low pKa can be labeled with

PEG-maleimide, thus resulting in mobility shift on SDS-PAGE. When protein is

PEG-maleimide. The oxidative status of specific protein can be detected and quantified

by calculating the ratio of shifted to nonshifted signals.

Fig. 14. Protein labeling efficiency with PEG-maleimide, PEG-vinyl sulfone

(A) HeLa cell lysate was incubated with PEG-maleimide at concentrations of 0, 0.01,

0.1, 1 mg/mL at 37 °C for 30 min, and followed by immunoblotting with

(B) Cell lysate prepared from different cell lines was incubated with PEG-maleimide at

concentrations of 0 - 1 mg/mL at 37 °C for 30 min, and followed by immunoblotting

with anti-peroxiredoxin.

The pellet was dissolved by lysis buffer containing PEG-maleimide at concentrations of

0 - 1 mg/mL, and incubated at 37 °C for 30 min, followed by immunoblotting with

anti-peroxiredoxin.

(D) HeLa cell lysate was precipitated with ammonium sulfate, and dissolved the pellet

with PEG-vinyl sulfone at concentrations of 0 – 1 mg/mL, incubating at 37 °C for 30

min followed by immunoblotting with anti-peroxiredoxin.

Note: There are four cysteine residues in human Prx1.

Fig. 15. Targets of PEG-maleimide labeling

HeLa cell lysate was incubated with m-PEG-maleimide at concentration of 0 -1

mg/mL at 37 °C for 30 min and followed by immunoblotting.

(A) Caspase 3 and caspase 9, which containing low-pKa cysteine residues, could be

labeled by PEG-maleimide and caused a band shift.

Note: there are 7 cysteine residues in procaspase 3 and 13 in procaspase 9.

(B) Examples of positive shifted signal following PEG-maleimide treatment. HDAC6:

histone deacetylase 6; FA syn: fatty acid synthase; Bcl-2: apoptosis regulator Bcl-2;

nm23: nucleoside diphosphate kinase A; PTP1B: protein tyrosine phosphate 1 B.

glutathione reductase; CamKII; calmodulin-dependent protein kinase II; NFκB: nuclear

factor κB; DTYMK: deoxythymidylate kinase.

Fig. 16. Detection and scoring of in vitro oxidized proteins

HeLa cell lysate that in vitro treated with H2O2 10 mM for 1 h was precipitated

immediately with ammonium sulfate. The pellet was dissolved with lysis buffer

containing 1 mg/mL PEG-maleimide, incubated at 37 °C for 30 min, and examined by

immunoblotting. Peroxiredoxin (Prx), prohibitin (PHB), and heat shock protein 27

(Hsp27) could be labeled by PEG-maleimide and caused band shift. Meanwhile, there

was no shifted signals in samples with H2O2 treatment, supposed that the active cysteine

residues were completely blocked with oxidative modifications.

(A) Immunoblot of Prx.

(B) Immunoblot of Hsp27. There is only one cysteine residue in Hsp 27.

followed the formula: Redox!score! = !f0!×!2⁰! + !f1!×!2¹!+!. . . +!fn!×!2ⁿ.

Fig. 17. Quantitation of in cellulo H

O

-induced Prx oxidation in HEK cell

(A) HEK cells were treated with H2O2 at concentration of 0, 1, 10 mM for 10 min in

was dissolved and incubated with PEG-maleimide for labeling (1 mg/mL, 37 °C, 30

min). Samples were examined by immunoblotting with anti-peroxiredoxin,

(B) HEK cells were treated with H2O2 at concentration of 0, 1, 10 mM for 10 min in

cellulo, than lysed with lysis buffer containing PEG-maleimide for labeling (1 mg/mL,

37 °C, 30 min). Samples were examined by immunoblotting with anti-peroxiredoxin.

integrated by Quanti-Scan for quantitation. The percentage of shifted and nonshifted

signals, and the ratio of shifted to nonshifted signals were shown as the bar graphs. The

ratio of shifted 2 to nonshifted were significantly increasing, whereas shifted 3 was

totally disappeared after H2O2 treatment.

Statistic analysis that comparing the results of 1 and 10 mM to 0 mM in 3-repeat data

was done with one-tail t-test. *: p-value < 0.05; **: p-value < 0.01.

Fig. 18. Quantitation of in cellulo H

O

-induced oxidation of Hsp27 in HEK cell

(A) HEK cells were treated with H2O2 at concentration of 0, 1, 10 mM for 10 min in

cellulo, and the lysate was precipitated with ammonium sulfate immediately. The pellet

was dissolved and incubated with PEG-maleimide for labeling (1 mg/mL, 37 °C, 30

min). Samples were examined by immunoblotting with anti-Hsp27.

(B) HEK cells were treated with H2O2 at concentration of 0, 1, 10 mM for 10 min in

cellulo, than lysed with lysis buffer containing PEG-maleimide for labeling (1

mg/mL, 37 °C, 30 min). Samples were examined by immunoblotting with anti-Hsp27.

Quanti-Scan for quantitation. The percentage of shifted and nonshifted signals, and the

ratio of shifted to nonshifted signals were shown as the bar graphs. Statistic analysis of

3-repeat data was done with one-tail t test. *: p-value < 0.05.

Fig. 19. Quantitation of insulin-induced oxidized PTP1B in HeLa cells

(A) HeLa cells were treated with 10 ng/mL insulin for 0, 5 and 10 min, and the lysate

containing 1 mg/ml PEG-maleimide, and incubated at 37 °C for 30 min. Samples were

examined by immunoblottng using anti-PTP1B antibody. Arrow 0: nonshifted; 1:

shifted band (with 1 m-PEG tag); P: shifted band which tagged in several cysteine

residues so that with a high molecular weight.

(B) HeLa cells were treated with 10 ng/mL insulin for 0, 5 and 10 min. The cells were

immediately lysed with lysis buffer containing 1 mg/ml PEG-maleimide. The cell lysate

was incubated at 37 °C for 30 min and then examined by immunoblottng using

anti-PTP1B antibody. Arrow 0: nonshifted; 1-2: shifted bands.

The percentage of shifted and nonshifted signals and the ratio of shifted to nonshifted

were shown as bar graph. Statistics analysis of 2-repeat data was done with one-tail t

test. *: p-value <0.05; **: p-value < 0.01.

Note: There are 10 cysteine residues in human PTP1B.

Tables

Accession Description Mass Score

(Parental)

Score (KO)

ESTD_HUMAN S-formylglutathione hydrolase OS=Homo sapiens GN=ESD

PE=1 SV=2 31442 317 294

GPX7_HUMAN Glutathione peroxidase 7 OS=Homo sapiens GN=GPX7 PE=1

SV=1 20983 36 -

GSHR_HUMAN Glutathione reductase, mitochondrial OS=Homo sapiens

GN=GSR PE=1 SV=2 56221 51 132

GSTK1_HUMAN Glutathione S-transferase kappa 1 OS=Homo sapiens

GN=GSTK1 PE=1 SV=3 25480 24 -

GSTO1_HUMAN Glutathione S-transferase omega-1 OS=Homo sapiens

GN=GSTO1 PE=1 SV=2 27548 84 48

GSTP1_HUMAN Glutathione S-transferase P OS=Homo sapiens GN=GSTP1

PE=1 SV=2 23341 954 763

PRDX1_HUMAN Peroxiredoxin-1 OS=Homo sapiens GN=PRDX1 PE=1 SV=1 22096 761 345 PRDX2_HUMAN Peroxiredoxin-2 OS=Homo sapiens GN=PRDX2 PE=1 SV=5 21878 456 220

PRDX3_HUMAN Thioredoxin-dependent peroxide reductase, mitochondrial

OS=Homo sapiens GN=PRDX3 PE=1 SV=3 27675 110 37 PRDX4_HUMAN Peroxiredoxin-4 OS=Homo sapiens GN=PRDX4 PE=1 SV=1 30521 158 90

PRDX5_HUMAN Peroxiredoxin-5, mitochondrial OS=Homo sapiens GN=PRDX5

PE=1 SV=4 22073 150 51

PRDX6_HUMAN Peroxiredoxin-6 OS=Homo sapiens GN=PRDX6 PE=1 SV=3 25019 361 172

TMX1_HUMAN Thioredoxin-related transmembrane protein 1 OS=Homo

sapiens GN=TMX1 PE=1 SV=1 31771 204 168

TRXR1_HUMAN Thioredoxin reductase 1, cytoplasmic OS=Homo sapiens

GN=TXNRD1 PE=1 SV=3 70862 87 30

TXND5_HUMAN Thioredoxin domain-containing protein 5 OS=Homo sapiens

GN=TXNDC5 PE=1 SV=2 47599 71 135

Table 1. Diamide-induced thiolated antioxidant enzymes in HAP-1 cell

RAC elution fractions of diamide-treated partental and SRXN1 KO HAP-1 cells were

subjected to LC-MS for protein identification. Antioxidant enzymes that were identified

are listed here. The complete dataset is listed in Appendix Table S1.

Accession Description Mass Score ACTG_HUMAN Actin, cytoplasmic 2 OS=Homo sapiens GN=ACTG1 PE=1 SV=1 41766 348 EF2_HUMAN Elongation factor 2 OS=Homo sapiens GN=EEF2 PE=1 SV=4 95277 166

HS90A_HUMAN Heat shock protein HSP 90-alpha OS=Homo sapiens GN=HSP90AA1

PE=1 SV=5 84607 145

H2A1B_HUMAN Histone H2A type 1-B/E OS=Homo sapiens GN=HIST1H2AB PE=1

SV=2 14127 137

H2B1J_HUMAN Histone H2B type 1-J OS=Homo sapiens GN=HIST1H2BJ PE=1 SV=3 13896 120 S10A9_HUMAN Protein S100-A9 OS=Homo sapiens GN=S100A9 PE=1 SV=1 13234 115

H2B1B_HUMAN Histone H2B type 1-B OS=Homo sapiens GN=HIST1H2BB PE=1

SV=2 13942 114

TCPQ_HUMAN T-complex protein 1 subunit theta OS=Homo sapiens GN=CCT8 PE=1

SV=4 59583 107

DESP_HUMAN Desmoplakin OS=Homo sapiens GN=DSP PE=1 SV=3 331569 85 CASPE_HUMAN Caspase-14 OS=Homo sapiens GN=CASP14 PE=1 SV=2 27662 84

PUR6_HUMAN Multifunctional protein ADE2 OS=Homo sapiens GN=PAICS PE=1

SV=3 47049 82

ACLY_HUMAN ATP-citrate synthase OS=Homo sapiens GN=ACLY PE=1 SV=3 120762 79

TGM1_HUMAN Protein-glutamine gamma-glutamyltransferase K OS=Homo sapiens

GN=TGM1 PE=1 SV=4 89730 75

PLAK_HUMAN Junction plakoglobin OS=Homo sapiens GN=JUP PE=1 SV=3 81693 66

TCPE_HUMAN T-complex protein 1 subunit epsilon OS=Homo sapiens GN=CCT5

PE=1 SV=1 59633 63

EF1G_HUMAN Elongation factor 1-gamma OS=Homo sapiens GN=EEF1G PE=1

SV=3 50087 57

ANXA2_HUMAN Annexin A2 OS=Homo sapiens GN=ANXA2 PE=1 SV=2 38580 51 S10A8_HUMAN Protein S100-A8 OS=Homo sapiens GN=S100A8 PE=1 SV=1 10828 49

KPRP_HUMAN Keratinocyte proline-rich protein OS=Homo sapiens GN=KPRP PE=1

SV=1 64093 47

TCPD_HUMAN T-complex protein 1 subunit delta OS=Homo sapiens GN=CCT4 PE=1

SV=4 57888 46

RS16_HUMAN 40S ribosomal protein S16 OS=Homo sapiens GN=RPS16 PE=1 SV=2 16435 45 1433E_HUMAN 14-3-3 protein epsilon OS=Homo sapiens GN=YWHAE PE=1 SV=1 29155 43

PPIA_HUMAN Peptidyl-prolyl cis-trans isomerase A OS=Homo sapiens GN=PPIA

PE=1 SV=2 18001 43

LYSC_HUMAN Lysozyme C OS=Homo sapiens GN=LYZ PE=1 SV=1 16526 43 H3C_HUMAN Histone H3.3C OS=Homo sapiens GN=H3F3C PE=1 SV=3 15204 42

SERA_HUMAN D-3-phosphoglycerate dehydrogenase OS=Homo sapiens

GN=PHGDH PE=1 SV=4 56614 41

DDX3X_HUMAN ATP-dependent RNA helicase DDX3X OS=Homo sapiens GN=DDX3X

PE=1 SV=3 73198 40

RL27A_HUMAN 60S ribosomal protein L27a OS=Homo sapiens GN=RPL27A PE=1

SV=2 16551 39

SND1_HUMAN Staphylococcal nuclease domain-containing protein 1 OS=Homo

sapiens GN=SND1 PE=1 SV=1 101934 39

RL26L_HUMAN 60S ribosomal protein L26-like 1 OS=Homo sapiens GN=RPL26L1

PE=1 SV=1 17246 39

HNRPL_HUMAN Heterogeneous nuclear ribonucleoprotein L OS=Homo sapiens

GN=HNRNPL PE=1 SV=2 64092 37

NPM_HUMAN Nucleophosmin OS=Homo sapiens GN=NPM1 PE=1 SV=2 32555 36

CSDE1_HUMAN Cold shock domain-containing protein E1 OS=Homo sapiens

GN=CSDE1 PE=1 SV=2 88829 35

TRFL_HUMAN Lactotransferrin OS=Homo sapiens GN=LTF PE=1 SV=6 78132 34

ELMO1_HUMAN Engulfment and cell motility protein 1 OS=Homo sapiens GN=ELMO1

PE=1 SV=2 83776 33

RL30_HUMAN 60S ribosomal protein L30 OS=Homo sapiens GN=RPL30 PE=1 SV=2 12776 31

TCPH_HUMAN T-complex protein 1 subunit eta OS=Homo sapiens GN=CCT7 PE=1

SV=2 59329 30

SRSF1_HUMAN Serine/arginine-rich splicing factor 1 OS=Homo sapiens GN=SRSF1

PE=1 SV=2 27728 30

RL18A_HUMAN 60S ribosomal protein L18a OS=Homo sapiens GN=RPL18A PE=1

SV=2 20749 30

Table 2. Diamide-induced sulfiredoxin disulfide-forming targets

FLAG-SRXN1 over-expressing HEK cells were treated with diamide. The dimer of

sulfiredoxin was pulled down in reduced condition, and eluted with β-mercaptoehtanol

to release the possible disulfide-binding partners. Proteins that only found in

diamide-treated group are listed here, and the complete dataset is listed in Appendix

Table S2.

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