nitric oxide: synthesis, signaling &...
TRANSCRIPT
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Nitric Oxide:Synthesis, Signaling & Pharmacology
Danyelle M. Townsend Associate Professor
Director, Analytical Redox Biochemistry Nebraska, June 13, 2019
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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
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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
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Dependent and Independent NO synthesis
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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
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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●
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Nitric Oxide Synthase
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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
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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...
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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 --
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• 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
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Journal of Cell Death 6(1):27-35 · March 2013
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Protein Nitration is Irreversible
Front. Plant Sci., 15 November 2016
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www.creative-proteomics.com
Post-translational modifications: Highly Regulated Complexity of Life
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# 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
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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
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S-Nitrosylation of Proteins
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Target-Selective Protein S-Nitrosylation by Sequence Motif Recognition Cell :Volume 159, Issue 3, Pages 623-634 (October 2014)
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Free Radical Biology and MedicineVolume 110, September 2017, Pages 19-30
Denitrosylation: Directly catalyzed by TrxIndirectly via GSH / GSNOR
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Chem Biol. 2015 July 23; 22(7): 965–975.
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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
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Indirect approaches to detect Cysteine PTM
Mol Biosyst. 2017 May 02; 13(5): 816–829.
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Biotin Switch Assay: Gold Standard for Detection of P-SNO
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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
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Cell Death and Differentiation (2011) 18, 1478–1486
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Disulfide Formation
SH SHSulfydryl
S-SSH SH
PDIPDI
Disulfide
Disulfide Isomerizations
SH SH
PDI
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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.
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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
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Cigarette smoke exposure of PDI results in cysteine and tyrosine oxidative modifications.
Harshavardhan Kenche et al. J. Biol.Chem. 2016;291:4763-4778
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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
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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
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Grek, C and Townsend, D.M. (2013)
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Nitric Oxide Donors as Therapeutics
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• 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
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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)
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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
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PABA/NO: GSTπ activated prodrug
COOHNO2O2N
O NN N+
O–
N
COOHNO22ON
NGS
–O
NN
O–
2HN+
NH3+ 2NO
CH3
CH3
CH3
CH3
GST
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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
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Glutathione S-transferase P is an NO carrier
Mechanism to protect from toxicity sequester toxic GSNO / NO*
Potential role as S-nitrosylase proteins
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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.
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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
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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
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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?
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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
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Front. Plant Sci., 16 August 2013 | https://doi.org/10.3389/fpls.2013.00314
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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)
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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