Nitric Oxide:Synthesis, Signaling & Pharmacology

Danyelle M. Townsend Associate Professor

Director, Analytical Redox Biochemistry Nebraska, June 13, 2019

Nitric OxideEndothelium derived relatation factor 1977

1998 Nobel Prize (Robert F. Furchgott, Louis J. Ignarro and Ferid Murad*)

Diatomic free radical with a half life of a few secondsNot to be confused w/ N2O or NO2

Lipid soluble easy passage between cell membranes, can travel the length of ~ 5 cells

Direct and indirect effectsIndirect effects become more pronounced with sustained NO●

production in inflammatory conditions

N O

Functional Roles of NO in the human body

Nervous system – signaling molecule involved in nerve action potentials

Circulatory system – Vasodilator released in response to Acetyl choline & bradykinin

Muscular system – originally described as endothelium derived relaxation factor

Immune system (iNOS)- inhibits viral replication & bacterial growth

Digestive system- adaptive smooth muscle relaxation in response to stomach filling

Dependent and Independent NO synthesis

NITRIC OXIDE SYNTHESIS

www.researchgate.net/publication/23471814_Nitric_oxide_and_cardiovascular_effects_New_insights_in_the_role_of_nitric_oxide_for_the_management_of_osteoarthritis

Nitric Oxide Synthase (NOS)Constitutive Inducible (cytokines)

Neuronal (nNOS / NOS1)Central / peripheral neurons

Endothelial (eNOS/NOS3)Vascular endothelial cell

Inducible (iNOS/ NOS2) Nucleated cells

Ca2+dependent Ca2+Independent

Short bursts Sustained production

Low levels of NO● Higher levels of NO●

Nitric Oxide Synthase

Effects of NO

Direct-NO coordinates with Metal complexes (Heme)

-Can activate and inactivate many proteins

•Lipid radicals

Indirect-Is due to formation of N203

And ONOO-

•S-Nitrosation on Cys residue

•DNA strand breaks

•Nitration on Tyr residue

Direct nitric oxide signaling

NO●

binds to iron of heme-containing proteins

guanylyl cyclase (GC)

results in... nitrosyl-heme formation and enzyme stimulation

Increased cyclic guanosine monophosphate (cGMP)

cGMP interacts with binding sites on target proteins (kinases, phospho-diesterases,

cyclic nucleotide-gated ion channels

downstream effects

cytochrome P-450 enzymes cytochrome c oxidase

Altered drug metabolism Altered cellular energetics

cGMP independent

results in... results in...

NO Direct Effects:Guanylate Cyclase (GC) is the primary receptor for NO

• There are membrane-bound and soluble forms of GC.

• GC is activated by ligand binding.

• GTP is converted to cGMP (2nd

messenger).

• cGMP activates PKG and downstream signaling involved in smooth muscle relaxation, cell division, angiogenesis.

• Phosphodiesterases (PDEs) convert cGMP to GMP, which shuts off signaling.

– Therapeutic Target --

• NO● reacts to cause the formation of intermediate byproducts capable of:

• 1) Post-translationally modifying proteins– Tyrosine Nitration (ONOO-)– Cysteine Nitrosylation (NO●)

• 2) Damaging lipids and DNA – OH●, hydroxyl radical – O2

-, superoxide- Nitrosation of secondary and tertiary amines to form nitrosamines (chemical carcinogens) via N2O3

Indirect effects of Nitric Oxide

Journal of Cell Death 6(1):27-35 · March 2013

Protein Nitration is Irreversible

Front. Plant Sci., 15 November 2016

www.creative-proteomics.com

Post-translational modifications: Highly Regulated Complexity of Life

# cysteines in genome

Biological complexity

200,000 cysteines in human proteome

https://www.researchgate.net/publication/232226611_Oxidative_Modification_of_Proteins_An_Emerging_Mechanism_of_Cell_Signaling

GSH

NOGSNOSSG SNO

S-glutathionylation

GSH / TrxGSH/ 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

S-Nitrosylation of Proteins

Target-Selective Protein S-Nitrosylation by Sequence Motif Recognition Cell :Volume 159, Issue 3, Pages 623-634 (October 2014)

Free Radical Biology and MedicineVolume 110, September 2017, Pages 19-30

Denitrosylation: Directly catalyzed by TrxIndirectly via GSH / GSNOR

Chem Biol. 2015 July 23; 22(7): 965–975.

Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation

Molecular Cell : Volume 69, Issue 3, Pages 438-450.e5 (February 2018)

Copyright © 2017 The Author(s) Terms and Conditions

Indirect approaches to detect Cysteine PTM

Mol Biosyst. 2017 May 02; 13(5): 816–829.

Biotin Switch Assay: Gold Standard for Detection of P-SNO

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

34DLGTTYSC(glut)VGVFK46MH+ = 1694.74 DaM+2H+ = 847.87 Da

y2294.16

y4450.23

b5-H2O470.22

b4387.23

y5549.34

(y11-129)2+

669.46

y112+

733.98

y3393.26

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

774.56

(M+2H-129)2+

783.53

y6-129-H2O810.65

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

829.60

y7-129915.44

y6957.50

y71044.46

y8-1291078.50

y81207.56

y9-1291179.58

y91308.57

b91245.51

b11-1291272.50

y11-1291337.61

b111401.63

y111466.72

b121548.49

b9-1291116.51

BiP is s-glutathionylated on C41 and C420

BiP

PSSG

Merge

Cell Death and Differentiation (2011) 18, 1478–1486

Disulfide Formation

SH SHSulfydryl

S-SSH SH

PDIPDI

Disulfide

Disulfide Isomerizations

SH SH

PDI

Hindawi Publishing Corporation International Journal of Cell Biology Volume 2013, Article ID 797914, 15 pages http://dx.doi.org/10.1155/2013/797914Review Article

The Role of S-Nitrosylation andS-Glutathionylation of Protein Disulphide Isomerase in Protein Misfolding andNeurodegeneration

M.Halloran,1 S. Parakh,2 and J. D. Atkin2

nNOS,NO

Reduction

PDIRys

Isomerase

Chaperone

RNS,ROS

(A)

PDI

(B)

NMDAr

(C)

Mitochondria

(D)ER

GSH:GSSG

UPR(E)

NO

NO

NO

NO NO

NO

SNO-Rys

SNO-PDI

Ca2+

Ca2+

Ca2+

Ca2+

Figure 2: Cellsurface PDI, NO,andSNO-PDI. (A) Cellsurface PDI reducesNOfrom extracellular SNOproteins (SNO-P) andin theprocess2+undergoes thiol modification. (B) Hyperactivation of the NMDAr leads to an intracellular influx of Ca ions (NMDAr may also undergo

reversibleS-nitrosylationtoameliorate excessive activity).(C) Inhibition of mitochondriacontributes toan increaseinintracellularNOwhich2is potentially oxidized by O leading to an increase in NO, nNOS, ROS, and RNS. (D) Increases in RNS/ROS alters the ER redoxenvironment,

2+ 2+and NO S-nitrosylates Ca ryanodine (Ryn) receptor leading to a disruption in Ca homeostasis. (E) ER-resident proteins such as PDI arevulnerable to S-nitrosylation, deactivating its isomerase and chaperone activity, leading to accumulation of misfolded proteins, ER stress, and UPR induction. P-SSG-PDI CXXC CXXC Chaperone

Figure 3: S-glutathionylation of PDI. Nitrosative stress from an exogenous agent (PABA/NO) increases intracellular NO and leads to the production of SNO-PDI. However, this may result in a decrease in GSSG/GSH ratio and increases in the free cellular pool of GSH. GSH then

Nitrosative stress

NO NO

CXXC CXXCSNO-PDI

GSSG GSH

GSH GSH Isomerase

binds to the catalytic (a, a ) domains of PDI, resulting in S-glutathionylation (P-SSG) of its cysteine residues and attenuation of its protectiveisomerase and chaperone activity.

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

S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegenerationTakashi Uehara1,4, Tomohiro Nakamura1, Dongdong Yao1, Zhong-Qing Shi1, Zezong Gu1, Yuliang Ma2, Eliezer Masliah3, Yasuyuki Nomura4 and Stuart A.Lipton1,3

Cigarette smoke exposure of PDI results in cysteine and tyrosine oxidative modifications.

Harshavardhan Kenche et al. J. Biol.Chem. 2016;291:4763-4778

PDIFLFL is refractory to S-glutathionylation

WB: PDI

WB: P-SSG

In VitroPABA/NO

PDIWT

0 25 50 100

PDIFLFL

0 25 50100PDIWT PDIFLFL

- + - +

Rotenone

FLAG-PDIWT

- +

FLAG-PDIFLFL

- + PABA/NO+

In Neuronal CellsCtr

IP: FLAG

WB: PDI-PSSGWB: PDI

WB: P-SSG

WB: PDI

500 1000 Rotenone (µM)0 100 250

Ying and Townsend: (2012) Internation J. Cancer

Rotenone (uM)

Mea

nO

.D.

0

2

4

6

0

10

5

0

2

4

PDI

+ --+

PDIWT PDIFLFL

+ --+

PDIWT PDIFLFL

- + -PDIWT

+PDIFLFL

BIP

CHO

Pm

RN

Afo

ldin

crea

sem

RN

Afo

ldin

crea

sem

RN

Afo

ldin

crea

se

S-glutathionylation refractory PDI

1) Blunts the toxic effects of rotenone2) Diminishes activation of the UPR

in PC12 neuronal cells

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

Nitric Oxide Donors as Therapeutics

• Inhaled nitric oxide gas• Sodium nitroprusside

• Organic Nitrates– Metabolized by cytochrome P450s to release NO●

– e.g., Nitroglycerin, Isosorbide dinitrate

• Used to treat angina pectoris, myocardial infarction, congestive heart failure, pulmonary hypertension

Nitric oxide donors as drugs

PDE inhibitors• Early idea to use PDE inhibitors

to treat angina, pulmonary hypertension.

• Studies led to the discovery of erectogenesis as a side effect (corpus cavernosum is relatively enriched in PDE5)

• Quickly recognized as a potential revolutionary treatment for ED

(phosphodiesterase)

• Sildenafil (Viagra, ½ life = 4 hr; onset 30-60 min)

• Tadalafil (Cialis, ½ life = 17.5 hr; onset 60-120 min)

PDE inhibitors

• Molecular basis of side effects?• Tissue distribution of PDE enzyme subtypes• Specificity of drugs• “Unsafe drop in blood pressure”

– Pulmonary Vasodilation• “Blurred vision”

– PDE6 is important in the retina– Figure 33.24, visual signal transduction

PABA/NO: GSTπ activated prodrug

COOHNO2O2N

O NN N+

O–

N

COOHNO22ON

NGS

–O

NN

O–

2HN+

NH3+ 2NO

CH3

CH3

CH3

CH3

GST

PABA/NO

IP:a -PDI

IP:IgG

DTT

- - + + ++ - - + +

- + + - -- - - - +

250105

7550

WB:PSSG

WB:PDI

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

Glutathione S-transferase P is an NO carrier

Mechanism to protect from toxicity sequester toxic GSNO / NO*

Potential role as S-nitrosylase proteins

Fluorescence equilibrium titration of cysteine mutants of GSTP1-1 with GSNO. A, representation of dimeric human GSTP1-1 (Protein Data Bank code 6GSS).

David Balchin et al. J. Biol. Chem. 2013;288:14973-14984

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

RAMIRO TELLEZ-SANZ,1 ELEONORA CESAREO,2 MARZIA NUCCETELLI,3ANA M. AGUILERA,1 CARMEN BARO N,1 LORIEN J. PARKER,4 JULIAN J. ADAMS,4 CRAIG J. MORTON,4 MARIO LO BELLO,2 MICHAEL W. PARKER,4 AND

LUIS GARCI´A-FUENTES1

1Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, Faculty of Experimental Sciences, University of Almer ıa, 04120 Almer ıa, Spain2Department of Biology and 3Internal Medicine, University of Rome ‘‘Tor Vergata,’’ 00133 Rome, Italy4Biota Structural Biology Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065, Australia

(RECEIVED December 20, 2005; FINAL REVISION January 31, 2006; ACCEPTED January 31, 2006)

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione(GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound todimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolu- tion. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multipleinteractions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with theenzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at morephysiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation ofa sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which theflexible helix a2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positivecooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix a2 forming newinteractions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.

Protein Science (2006), 15:1093–1105. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society

Biochim Biophys Acta Gen Subj. 2017 May;1861(5 Pt A):995-999. doi: 10.1016/j.bbagen.2017.02.021. Epub 2017 Feb 17.

Regulation and control of nitric oxide (NO) in macrophages: Protecting the "professional killer cell" from its own cytotoxic arsenal via MRP1 and GSTP1

Free Radical Research, 2015; 49(12): 1438–1448

ORIGINAL ARTICLE

Glutathione S-transferase P1 suppresses iNOS protein stability in RAW264.7 macrophage-like cells after LPS stimulationXiang Cao1∗, Xiuqin Kong1∗, Yi Zhou1, Lei Lan1, Lan Luo2 & Zhimin Yin1

AbstractGlutathione S-transferase P1 (GSTP1) is a ubiquitous expressed protein which plays an important role in the detoxification and xenobiotics metabolism. Previous studiesshowed that GSTP1 was upregulated by the LPS stimulation in RAW264.7 macrophage-like cells and GSTP1 overexpression downregulated lipopolysaccharide (LPS)induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Here we show that GSTP1 physically associates with the oxygenase domainof iNOS by the G-site domain and decreases the protein level of iNOS dimer. Both overexpression and RNA interference (RNAi) experiments indicate that GSTP1downregulates iNOS protein level and increases S-nitrosylation and ubiquitination of iNOS. The Y7F mutant type of GSTP1 physically associates with iNOS, butshows no effect on iNOS protein content, iNOS S-nitrosylation, and changes in iNOS from dimer to monomer, suggesting the importance of enzyme activity of GSTP1in regulating iNOS S-nitrosylation and stability. GSTM1, another member of GSTs shows no significant effect on regulation of iNOS. In conclusion, our study revealsthe novel role of GSTP1 in regulation of iNOS by affecting S-nitrosylation, dimeriza- tion, and stability, which provides a new insight for analyzing the regulation ofiNOS and the anti-inflammatory effects of GSTP1.

GSTP forms protein: protein interactions with iNOS

GSTP iNOS-SNO

decreases protein stability

Does GSTP have Nitrosylase activity?

Summary:

• NO is an important signaling molecule with direct & indirect functionality

PhysiologyPathophysiology

• NO induces post-translational modifications:• Cys (nitrosylation / glutathionylation) reversible (Trx)• Tyr (Nitration) irreversible

Pharmacologic modulation of NO is important clinically

Front. Plant Sci., 16 August 2013 | https://doi.org/10.3389/fpls.2013.00314

http://ej.iop.org/images/0022-3727/45/26/263001/Full/jphysd400847f32_online.jpg

Reactive Nitrogen Species (RNS) = Nitrogen containing oxidantsnitric oxide (NO.) peroxynitrite (ONOO.) nitrogen dioxide (NO2)

GSH

NOGSNOSSG SNO

S-glutathionylation

GSH / TrxGSH/ 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