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Posts Tagged ‘S-Nitrosylation Signaling’


Lesson 3 Cell Signaling & Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Updated 7/15/2019

Lesson 3 Powerpoint (click link below):

cell signaling and motility 3 finalissima sjw

Four papers to choose from for your February 11 group presentation:

Structural studies of G protein Coupled receptor

Shapiro-2009-Annals_of_the_New_York_Academy_of_Sciences

G protein as target in neurodegerative disease

fish technique

 

 

Today’s lesson 3 explains how extracellular signals are transduced (transmitted) into the cell through receptors to produce an agonist-driven event (effect).  This lesson focused on signal transduction from agonist through G proteins (GTPases), and eventually to the effectors of the signal transduction process.  Agonists such as small molecules like neurotransmitters, hormones, nitric oxide were discussed however later lectures will discuss more in detail the large growth factor signalings which occur through receptor tyrosine kinases and the Ras family of G proteins as well as mechanosignaling through Rho and Rac family of G proteins.

Transducers: The Heterotrimeric G Proteins (GTPases)

An excellent review of heterotrimeric G Proteins found in the brain is given by

Heterotrimeric G Proteins by Eric J Nestler and Ronald S Duman.

 

 

from Seven-Transmembrane receptors – Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Examples-of-heterotrimeric-G-protein-effectors_tbl1_11180073 [accessed 4 Feb, 2019] and see references within

 

 

See below for the G Protein Cycle

 

 

 

 

 

 

 

 

<a href=”https://www.researchgate.net/figure/32-The-G-protein-cycle-In-the-absence-of-agonist-A-GPCRs-are-mainly-in-the-low_fig2_47933733″><img src=”https://www.researchgate.net/profile/Veli_Pekka_Jaakola/publication/47933733/figure/fig2/AS:669499451781133@1536632516635/32-The-G-protein-cycle-In-the-absence-of-agonist-A-GPCRs-are-mainly-in-the-low.ppm&#8221; alt=”.3.2: The G protein cycle. In the absence of agonist (A), GPCRs are mainly in the low affinity state (R). After agonist binding, the receptor is activated in the high affinity state (R*), and the agonist-GPCR-G protein complex is formed. GTP replaces GDP in Gα. After that the G protein dissociates into the Gα subunit and the Gβγ heterodimer, which then activate several effector proteins. The built-in GTPase activity of the Gα subunit cleaves the terminal phosphate group of GTP, and the GDP bound Gα subunit reassociates with Gβγ heterodimer. This results in the deactivation of both Gα and Gβγ. The G protein cycle returns to the basal state. RGS, regulator of G protein signalling.”/></a>

 

From Citation: Review: A. M. Preininger, H. E. Hamm, G protein signaling: Insights from new structures. Sci. STKE2004, re3 (2004)

 

For a tutorial on G Protein coupled receptors (GPCR) see

https://www.khanacademy.org/test-prep/mcat/organ-systems/biosignaling/v/g-protein-coupled-receptors

 

 

 

cyclic AMP (cAMP) signaling to the effector Protein Kinase A (PKA)

from https://courses.washington.edu/conj/gprotein/cyclicamp.htm

Cyclic AMP is an important second messenger. It forms, as shown, when the membrane enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).

 

The cyclic AMP then goes on the activate specific proteins. Some ion channels, for example, are gated by cyclic AMP. But an especially important protein activated by cyclic AMP is protein kinase A, which goes on the phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP activates protein kinase A.

Updated 7/15/2019

Additional New Studies on Regulation of the Beta 2 Adrenergic Receptor

We had discussed regulation of the G protein coupled beta 2 adrenergic receptor by the B-AR receptor kinase (BARK)/B arrestin system which uncouples and desensitizes the receptor from its G protein system.  In an article by Xiangyu Liu in Science in 2019, the authors describe another type of allosteric modulation (this time a POSITIVE allosteric modulation) in the intracellular loop 2.  See below:

Mechanism of β2AR regulation by an intracellular positive allosteric modulator

Xiangyu Liu1,*, Ali Masoudi2,*, Alem W. Kahsai2,*, Li-Yin Huang2, Biswaranjan Pani2Dean P. Staus2, Paul J. Shim2, Kunio Hirata3,4, Rishabh K. Simhal2, Allison M. Schwalb2, Paula K. Rambarat2, Seungkirl Ahn2, Robert J. Lefkowitz2,5,6,Brian Kobilka1

Positive reinforcement in a GPCR

Many drug discovery efforts focus on G protein–coupled receptors (GPCRs), a class of receptors that regulate many physiological processes. An exemplar is the β2-adrenergic receptor (β2AR), which is targeted by both blockers and agonists to treat cardiovascular and respiratory diseases. Most GPCR drugs target the primary (orthosteric) ligand binding site, but binding at allosteric sites can modulate activation. Because such allosteric sites are less conserved, they could possibly be targeted more specifically. Liu et al. report the crystal structure of β2AR bound to both an orthosteric agonist and a positive allosteric modulator that increases receptor activity. The structure suggests why the modulator compound is selective for β2AR over the closely related β1AR. Furthermore, the structure reveals that the modulator acts by enhancing orthosteric agonist binding and stabilizing the active conformation of the receptor.

Abstract

Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2– over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

 

Recent structures of GPCRs bound to allosteric modulators have revealed that receptor surfaces are decorated with diverse cavities and crevices that may serve as allosteric modulatory sites (1). This substantiates the notion that GPCRs are structurally plastic and can be modulated by a variety of allosteric ligands through distinct mechanisms (2-7). Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive states (1). To date, only a single structure of an active GPCR bound to a small-molecule positive allosteric modulator (PAM) has been reported, namely, the M2 muscarinic acetylcholine receptor with LY2119620 (8). Thus, mechanisms of PAMs and their potential binding sites remain largely unexplored.

F1.large

 

Fig 1. Structure of the active state T4L-B2AR in complex with the orthosteric agonist BI-167107, nanobody 689, and compound 6FA.  (A) The chemical structure of compound-6FA (Cmpd-6FA). (B) Isoproterenol (ISO) competition binding with 125I-cyanopindolol (CYP) to the β2AR reconstituted in nanodisks in the presence of vehicle (0.32% dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding–log IC50 (median inhibitory concentration) curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. (C) Analysis of Cmpd-6FA interaction with the BI-167107–bound β2AR by ITC. Representative thermogram (inset) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (KD = 1.2 ± 0.1 μM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal mol−1), and entropy (ΔS =13 ± 2.0 cal mol−1 deg−1). (D) Side view of T4L-β2AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. (E) Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2Fo– Fc electron density contoured at 1.0 σ (green mesh).From Science  28 Jun 2019:
Vol. 364, Issue 6447, pp. 1283-1287

 

F3.large

Fig 3. Fig. 3 Mechanism of allosteric activation of the β2AR by Cmpd-6FA.

(A) Superposition of the inactive β2AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β2AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β2AR are depicted, and the hydrogen bond formed between Asp1303.49and Tyr141ICL2 in the active state is indicated by a black dashed line. (B) Topography of Cmpd-6FA binding surface on the active β2AR (left, blue) and the corresponding surface of the inactive β2AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β2AR is represented by a purple asterisk. (C) Overlay of the β2AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β2AR–Gscomplex (PDB code: 3SN6). The inset shows the position of Phe139ICL2 relative to the α subunit of Gs. (D) Superposition of the active β2AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β2AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TM helices 3, 5, and 6 upon activation and their relative distances.

 

 

 

 

Allosteric sites may not face the same evolutionary pressure as do orthosteric sites, and thus are more divergent across subtypes within a receptor family (2426). Therefore, allosteric sites may provide a greater source of specificity for targeting GPCRs.

 

 

  1. D. M. Thal, A. Glukhova, P. M. Sexton, A. Christopoulos, Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018). doi:10.1038/s41586-018-0259-zpmid:29973731CrossRefPubMedGoogle Scholar

 

  1. D. Wacker, R. C. Stevens, B. L. Roth, How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414–427 (2017).

doi:10.1016/j.cell.2017.07.009pmid:28753422CrossRefPubMedGoogle Scholar

 

  1. D. P. Staus, R. T. Strachan, A. Manglik, B. Pani, A. W. Kahsai, T. H. Kim, L. M. Wingler, S. Ahn, A. Chatterjee, A. Masoudi, A. C. Kruse, E. Pardon, J. Steyaert, W. I. Weis, R. S. Prosser, B. K. Kobilka, T. Costa, R. J. Lefkowitz, Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535, 448–452 (2016). doi:10.1038/nature18636pmid:27409812CrossRefPubMedGoogle Scholar

 

  1. A. Manglik, T. H. Kim, M. Masureel, C. Altenbach, Z. Yang, D. Hilger, M. T. Lerch, T. S. Kobilka, F. S. Thian, W. L. Hubbell, R. S. Prosser, B. K. Kobilka, Structural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling. Cell 161, 1101–1111 (2015). doi:10.1016/j.cell.2015.04.043pmid:25981665CrossRefPubMedGoogle Scholar

 

5,   L. Ye, N. Van Eps, M. Zimmer, O. P. Ernst, R. S. Prosser, Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533, 265–268 (2016). doi:10.1038/nature17668pmid:27144352CrossRefPubMedGoogle Scholar

 

  1. N. Van Eps, L. N. Caro, T. Morizumi, A. K. Kusnetzow, M. Szczepek, K. P. Hofmann, T. H. Bayburt, S. G. Sligar, O. P. Ernst, W. L. Hubbell, Conformational equilibria of light-activated rhodopsin in nanodiscs. Proc. Natl. Acad. Sci. U.S.A. 114, E3268–E3275 (2017). doi:10.1073/pnas.1620405114pmid:28373559Abstract/FREE Full TextGoogle Scholar

 

  1. R. O. Dror, H. F. Green, C. Valant, D. W. Borhani, J. R. Valcourt, A. C. Pan, D. H. Arlow, M. Canals, J. R. Lane, R. Rahmani, J. B. Baell, P. M. Sexton, A. Christopoulos, D. E. Shaw, Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature 503, 295–299 (2013). doi:10.1038/nature12595pmid:24121438CrossRefPubMedWeb of ScienceGoogle Scholar

 

  1. A. C. Kruse, A. M. Ring, A. Manglik, J. Hu, K. Hu, K. Eitel, H. Hübner, E. Pardon, C. Valant, P. M. Sexton, A. Christopoulos, C. C. Felder, P. Gmeiner, J. Steyaert, W. I. Weis, K. C. Garcia, J. Wess, B. K. Kobilka, Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013). doi:10.1038/nature12735pmid:24256733

 

 

Additional information on Nitric Oxide as a Cellular Signal

Nitric oxide is actually a free radical and can react with other free radicals, resulting in a very short half life (only a few seconds) and so in the body is produced locally to its site of action (i.e. in endothelial cells surrounding the vascular smooth muscle, in nerve cells). In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980s he and others identified this substance as being NO.

Nitric oxide is produced from metabolism of endogenous substances like L-arginine, catalyzed by one of three isoforms of nitric oxide synthase (for link to a good article see here) or release from exogenous compounds like drugs used to treat angina pectoris like amyl nitrate or drugs used for hypertension such as sodium nitroprusside.

The following articles are a great reference to the chemistry, and physiological and pathological Roles of Nitric Oxide:

46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/

47. Nitric Oxide Function in Coagulation – Part II

Curator and Author: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/

48. Nitric Oxide, Platelets, Endothelium and Hemostasis

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/

49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/14/interaction-of-nitric-oxide-and-prostacyclin-in-vascular-endothelium/

50. Nitric Oxide and Immune Responses: Part 1

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/

51. Nitric Oxide and Immune Responses: Part 2

Curator and Author:  Aviral Vatsa PhD, MBBS

https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/

56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

57. New Insights on Nitric Oxide donors – Part IV

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

Curator and Author: Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-         a-concomitant-influence-on-mitochondrial-function/

Biochemistry of the Coagulation Cascade and Platelet Aggregation: Nitric Oxide: Platelets, Circulatory Disorders, and Coagulation Effects

Nitric Oxide Function in Coagulation – Part II

Nitric oxide is implicated in many pathologic processes as well.  Nitric oxide post translational modifications have been attributed to nitric oxide’s role in pathology however, although the general mechanism by which nitric oxide exerts its physiological effects is by stimulation of soluble guanylate cyclase to produce cGMP, these post translational modifications can act as a cellular signal as well.  For more information of NO pathologic effects and how NO induced post translational modifications can act as a cellular signal see the following:

Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?

58. Crucial role of Nitric Oxide in Cancer

Curator and Author: Ritu Saxena, Ph.D.

https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/

Note:  A more comprehensive ebook on Nitric Oxide and Disease Perspectives is found at

Cardiovascular Diseases, Volume One: Perspectives on Nitric Oxide in Disease Mechanisms

available on Kindle Store @ Amazon.com

http://www.amazon.com/dp/B00DINFFYC

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Reinforced disordered cell expression

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Diabetes, Alzheimer’s Share Molecular Pathways, Part of Same Vicious Cycle

http://www.genengnews.com/gen-news-highlights/diabetes-alzheimer-s-share-molecular-pathways-part-of-same-vicious-cycle/81252206/

http://www.genengnews.com/Media/images/GENHighlight/thumb_Jan8_2016_Fotolia_30836005_JigsawPuzzleBrainAndHead1904910113.jpg

A molecular-level link has been found that helps explain the poorly understood association between diabetes and Alzheimer’s disease. Both disorders can drive and be driven by the same pathological process, the disruption of a particular kind of post-translational modification called S-nitrosylation. Thus, the disorders can reinforce each other. [© freshidea/Fotolia]

 

Though they appear to be distinct, diabetes and Alzheimer’s disease have much in common at the molecular level. In fact, recent findings indicate that either disease can worsen the other by disrupting the same chemical process—S-nitrosylation, a form of post-translational modification that is necessary for the proper functioning of multiple enzymes.

S-nitrosylation, it turns out, can be disrupted by excess sugar or β-amyloid protein, either of which can wreak havoc by increasing the levels of nitric oxide and other free radical species. Once S-nitrosylation is disturbed and poorly functioning enzymes are produced, the downstream effects include abnormal increases in both insulin and β-amyloid protein.

Thus, diabetes and Alzheimer’s can drive, and be driven by, the same vicious cycle. Furthermore, either can contribute to the other’s progress. These results emerged from a study completed by researchers based at the Sanford Burnham Prebys Medical Discovery Institute and the Scintillon Institute. The research team was led by Stuart A. Lipton, M.D., Ph.D., a physician-scientist affiliated with both institutions.

“This work points to a new common pathway to attack both type 2 diabetes, along with its harbinger, metabolic syndrome, and Alzheimer’s disease,” stated Dr. Lipton.

The researchers published their work January 8 in the journal Nature Communications in an article entitled, “Elevated glucose and oligomeric β-amyloid disrupt synapses via a common pathway of aberrant protein S-nitrosylation.” This article describes how the scientists used a so-called “disease-in-a-dish” model to discover molecular pathways that are in common in both diabetes and Alzheimer’s.

Specifically, the scientists genetically reprogrammed the skin of human patients to make induced pluripotent stem cells, which were then used to derive nerve cells. They also used mouse models of each disease to analyze the combined effects of high blood sugar and β-amyloid protein in living animals.

“[We] report in human and rodent tissues that elevated glucose, as found in [metabolic syndrome and type 2 diabetes] and oligomeric β-amyloid (Aβ) peptide, thought to be a key mediator of [Alzheimer’s disease], coordinately increase neuronal Ca2+ and nitric oxide (NO) in an NMDA receptor-dependent manner,” wrote the authors of the Nature Communications article. “The increase in NO results in S-nitrosylation of insulin-degrading enzyme (IDE) and dynamin-related protein 1 (Drp1), thus inhibiting insulin and Aβ catabolism as well as hyperactivating mitochondrial fission machinery.”

The scientists also found that the changes in enzyme activity led to damage of synapses, the region where nerve cells communicate with one another in the brain. The combination of high sugar and β-amyloid protein caused the greatest loss of synapses. Since loss of synapses correlates with cognitive decline in Alzheimer’s, high sugar and β-amyloid coordinately contribute to memory loss.

“The NMDA receptor antagonist memantine attenuates these effects,” the authors continued. “Our studies show that redox-mediated posttranslational modification of brain proteins link Aβ and hyperglyaemia to cognitive dysfunction in [metabolic syndrome/type 2 diabetes] and [Alzheimer’s disease].”

“[Our work] means that we now know these diseases are related on a molecular basis, and hence, they can be treated with new drugs on a common basis,” stated Dr. Ambasudhan, a senior author of the study and an assistant professor at Scintillon.

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Analysis of S-nitrosylated Proteins: Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry

Author and Curator: Larry H Bernstein, MD, FACP 

 

Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.

Han P; Chen C
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Rapid Commun Mass Spectrom. 2008 Apr; 22(8):1137-45.   http://dx.doi.org/10.1002/rcm.3476.    PMID: 18335467

 

High-throughput proteomic analysis based on a biotin switch combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS)

  • enables simultaneous identification of S-nitrosylated sites and
  • their cognate proteins in complex biological mixtures, which is a great help
    • in elucidating the functions and mechanisms of this redox-based post-translational modification.

However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 adopted in these systems, which are hard to fully remove in the subsequent MS-based analyses,

  1. can suppress the peptide signals and
  2. influence the SNO-Cys site identification and
  3. the reproducibility of the experiments.

Here we developed a detergent-free biotin-switch method, which applied

  1. urea to replace detergents, and successfully
  2. combined it with LC/MS/MS in the analysis of S-nitrosylated proteins.

With this approach, 44 SNO-Cys sites were specified on 35 distinct proteins in S-nitrosoglutathione (GSNO)-treated HeLa cell extracts of proteins with good reproducibility.

The LC/MS performance was greatly improved as

  • analyzed with Pep3D and the amount of samples for analysis reduced from 40 mg used in the literature to 3-5 mg.

For S-nitrosylated targets detected both in the control sample and in the GSNO-treated sample,

  • extracted ion chromatography (XIC) was employed to
  • estimate the quantitative change of S-nitrosylation (S-nitrosation),

which facilitates the judgment on ‘accept or reject’ of the identified targets.

 

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G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

This recently published article delineates a role of G-protein-coupled receptor with S-nitrosylation in outcomes for acute coronary syndrome.

Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome to Cardiac Ischemic Injury

Z. Maggie Huang1, Erhe Gao1, Fabio Vasconcelos Fonseca2,3, Hiroki Hayashi2,3, Xiying Shang1, Nicholas E. Hoffman1, J. Kurt Chuprun1, Xufan Tian4, Doug G. Tilley1, Muniswamy Madesh1, David J. Lefer5, Jonathan S. Stamler2,3,6, and Walter J. Koch1*
1 Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA
2 Institute for Transformative Molecular Medicine, Case Western Reserve Univ SOM, Cleveland, OH
3 Department of Medicine, Case Western Reserve University, Cleveland, OH
4 Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA
5 Department  Surgery, Div of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA
6 University Hospitals Harrington Discovery Institute, Cleveland, OH

Sci. Signal., 29 Oct 2013; 6(299), p. ra95         http:dx.doi.org/10.1126/scisignal.2004225

Abstract

Heart failure caused by ischemic heart disease is a leading cause of death in the developed world. Treatment is currently centered on regimens involving

  • G protein–coupled receptors (GPCRs) or nitric oxide (NO).

These regimens are thought to target distinct molecular pathways. We showed that

  • these pathways are interdependent and converge on the effector GRK2 (GPCR kinase 2) to regulate myocyte survival and function.

Ischemic injury coupled to

  • GPCR activation, including GPCR desensitization and myocyte loss,
  • required GRK2 activation,

and we found that cardioprotection mediated by inhibition of GRK2 depended on

  • endothelial nitric oxide synthase (eNOS) and
  • was associated with S-nitrosylation of GRK2.

Conversely, the cardioprotective effects of NO bioactivity were absent in a knock-in mouse with a form of GRK2 that cannot be S-nitrosylated. Because GRK2 and eNOS inhibit each other,

the balance of the activities of these enzymes in the myocardium determined the outcome to ischemic injury. Our findings suggest new insights into

  • the mechanism of action of classic drugs used to treat heart failure and
  • new therapeutic approaches to ischemic heart disease.

* Corresponding author. E-mail: walter.koch@temple.edu
Citation: Z. M. Huang, E. Gao, F. V. Fonseca, H. Hayashi, X. Shang, N. E. Hoffman, J. K. Chuprun, X. Tian, D. G. Tilley, M. Madesh, D. J. Lefer, J. S. Stamler, W. J. Koch, Convergence of G Protein–Coupled Receptor and S-Nitrosylation Signaling Determines the Outcome t

 Editor’s Summary

Sci. Signal., 29 Oct 2013; 6(299), p. ra95 [DOI: 10.1126/scisignal.2004225]

NO More Heart Damage

Damage caused by the lack of oxygen and nutrients that occurs during myocardial ischemia can result in heart failure. A therapeutic strategy that helps to limit the effects of heart failure is to

  • increase signaling through G protein–coupled receptors (GPCRs)
  • by inhibiting GRK2 (GPCR kinase 2), a kinase that
    • desensitizes GPCRs.

Another therapeutic strategy provides S-nitrosothiols, such as nitric oxide, which can be

  • added to proteins in a posttranslational modification called S-nitrosylation.

Huang et al. found that the ability of S-nitrosothiols to enhance cardiomyocyte survival after ischemic injury required the S-nitrosylation of GRK2, a modification that inhibits this kinase. Mice bearing a form of GRK2 that could not be S-nitrosylated 

  • were more susceptible to cardiac damage after ischemia.

These results suggest that therapeutic strategies that promote the S-nitrosylation of GRK2 could be used to treat heart failure after myocardial ischemia.

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