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Dive into the research topics where Russell Desnoyer is active.

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Featured researches published by Russell Desnoyer.


Cell | 2015

Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography

Haitao Zhang; Hamiyet Unal; Cornelius Gati; Gye Won Han; Wei Liu; Nadia A. Zatsepin; Daniel James; Dingjie Wang; Garrett Nelson; Uwe Weierstall; Michael R. Sawaya; Qingping Xu; Marc Messerschmidt; Garth J. Williams; Sébastien Boutet; Oleksandr Yefanov; Thomas A. White; Chong Wang; Andrii Ishchenko; Kalyan C. Tirupula; Russell Desnoyer; Jesse Coe; Chelsie E. Conrad; Petra Fromme; Raymond C. Stevens; Vsevolod Katritch; Sadashiva S. Karnik; Vadim Cherezov

Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.


Journal of Biological Chemistry | 2015

Structural Basis for Ligand Recognition and Functional Selectivity at Angiotensin Receptor

Haitao Zhang; Hamiyet Unal; Russell Desnoyer; Gye Won Han; Nilkanth Patel; Vsevolod Katritch; Sadashiva S. Karnik; Vadim Cherezov; Raymond C. Stevens

Background: Angiotensin receptor (AT1R) blockers are critical therapeutics used to treat cardiovascular disease. Results: We solved the AT1R-olmesartan structure and identified specific interactions for olmesartan derivatives with different functions. Conclusion: Our results identified residues critical for the binding of different ligands and allosteric modulation by sodium ion. Significance: Our results provide new insights into the structural basis for ligand recognition and functional selectivity at AT1R. Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N1113.35A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the β-arrestin-biased peptide TRV120027.


Cardiovascular Research | 2010

Role of nuclear unphosphorylated STAT3 in angiotensin II type 1 receptor-induced cardiac hypertrophy

Hong Yue; Wei Li; Russell Desnoyer; Sadashiva S. Karnik

AIMS Cardiac hypertrophy is a risk factor independent of blood pressure; however, the mechanisms that distinguish pathological remodelling due to local cues from pressure overload are unresolved. This study was aimed at discovering a novel gene expression mechanism in heart failure. METHODS AND RESULTS In angiotensin II type 1 receptor (AT1R) transgenic mice (TG), we found a significant increase of mRNA and total STAT3 (T-STAT3) protein, but not STAT3 phosphorylated at residues Y705 and S727. A net increase in nuclear accumulation of this unphosphorylated form of STAT3 (U-STAT3) correlated with the development of cardiac hypertrophy and dysfunction, which are associated with abnormal expression of osteopontin and regulator of G protein signalling 2 genes. Nuclear accumulation of U-STAT3 is induced by angiotensin II treatment in neonatal cardiac myocytes, fibroblasts, and AT1R-expressing human embryonic kidney 293 (HEK-AT1R) cells. Chromatin immunoprecipitation demonstrated that U-STAT3 binds to the target gene promoter, and siRNA-mediated knockdown of STAT3 expression significantly altered the expression of target genes in HEK-AT1R cells. T-STAT3 in TG mouse hearts and the phosphorylation-deficient Y705F mutant STAT3 in HEK-AT1R cells physically interacted with transcription co-activator p300. CONCLUSION Chronic activation of AT1R induces unregulated expression of the Stat3 gene, leading to nuclear accumulation of U-STAT3, which significantly correlated with progression of cardiac hypertrophy.


Journal of Biological Chemistry | 2013

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

Hamiyet Unal; Rajaganapathi Jagannathan; Anushree Bhatnagar; Kalyan C. Tirupula; Russell Desnoyer; Sadashiva S. Karnik

Background: The binding of ligands to the orthosteric pocket induces ligand-specific conformational changes all through domains of G protein-coupled receptors. Results: Loss of function and gain of function mutations in the transmembrane domain spontaneously induce changes similar to a ligand-specific conformation in the extracellular domain. Conclusion: Coupling between domains determines the overall signaling state of GPCRs. Significance: Targeting domain interfaces might be the future for GPCR-specific drug development. The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific “lid” conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar1,Gln2,Ile8] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.


PLOS ONE | 2014

Atypical signaling and functional desensitization response of MAS receptor to peptide ligands

Kalyan C. Tirupula; Russell Desnoyer; Robert C. Speth; Sadashiva S. Karnik

MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data suggest that peptide ligands induce atypical signaling and functional desensitization of MAS.


PLOS ONE | 2013

Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

Anushree Bhatnagar; Hamiyet Unal; Rajaganapathi Jagannathan; Suma Kaveti; Zhong Hui Duan; Sandro L. Yong; Amit Vasanji; Michael Kinter; Russell Desnoyer; Sadashiva S. Karnik

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ2 with integral components of chromatin and effects of Gβ2 depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ2 levels in the nucleus and Gβ2 interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ2 repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ2 interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ2-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.


PLOS ONE | 2010

AT1 Receptor Induced Alterations in Histone H2A Reveal Novel Insights into GPCR Control of Chromatin Remodeling

Rajaganapathi Jagannathan; Suma Kaveti; Russell Desnoyer; Belinda Willard; Michael Kinter; Sadashiva S. Karnik

Chronic activation of angiotensin II (AngII) type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR) induces gene regulatory stress which is responsible for phenotypic modulation of target cells. The AT1R-selective drugs reverse the gene regulatory stress in various cardiovascular diseases. However, the molecular mechanisms are not clear. We speculate that activation states of AT1R modify the composition of histone isoforms and post-translational modifications (PTM), thereby alter the structure-function dynamics of chromatin. We combined total histone isolation, FPLC separation, and mass spectrometry techniques to analyze histone H2A in HEK293 cells with and without AT1R activation. We have identified eight isoforms: H2AA, H2AG, H2AM, H2AO, H2AQ, Q96QV6, H2AC and H2AL. The isoforms, H2AA, H2AC and H2AQ were methylated and H2AC was phosphorylated. The relative abundance of specific H2A isoforms and PTMs were further analyzed in relationship to the activation states of AT1R by immunochemical studies. Within 2 hr, the isoforms, H2AA/O exchanged with H2AM. The monomethylated H2AC increased rapidly and the phosphorylated H2AC decreased, thus suggesting that enhanced H2AC methylation is coupled to Ser1p dephosphorylation. We show that H2A125Kme1 promotes interaction with the heterochromatin associated protein, HP1α. These specific changes in H2A are reversed by treatment with the AT1R specific inhibitor losartan. Our analysis provides a first step towards an awareness of histone code regulation by GPCRs.


Journal of Biological Chemistry | 2008

Site-specific Cleavage of G Protein-coupled Receptor-engaged β-Arrestin: INFLUENCE OF THE AT1 RECEPTOR CONFORMATION ON SCISSILE SITE SELECTION*

ChangWoo Lee; Sumantha Bhatt; Anita Shukla; Russell Desnoyer; Satya Prakash Yadav; Mijin Kim; Sei Heon Jang; Sadashiva S. Karnik

The discovery of β-arrestin-related ∼46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT1R)-dependent proteolytic cleavage of β-arrestin(s). Receptor-ligand induced proteolysis of β-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT1R-engaged isoforms of β-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT1R complex is associated with full-length and ∼46-kDa β-arrestin forms. Mass spectrometric analysis of the AT1R-associated short form suggested a scissile site located within the Arg363–Arg393 region in the bovine β-arrestin 1. Edman degradation analysis of a β-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe388 and a minor cleavage after Asn375. Rather unexpectedly, the inverse agonist EXP3174-bound AT1R generated different fragmentation of bovine β-arrestin 1, at Pro276. The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca2+-mediated signaling pathways. The proteolysis of β-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that β-arrestin cleavage upon AT1R stimulation is a part of the unraveling β-arrestin-mediated G protein-coupled receptor signaling diversity.The discovery of beta-arrestin-related approximately 46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT(1)R)-dependent proteolytic cleavage of beta-arrestin(s). Receptor-ligand induced proteolysis of beta-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT(1)R-engaged isoforms of beta-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT(1)R complex is associated with full-length and approximately 46-kDa beta-arrestin forms. Mass spectrometric analysis of the AT(1)R-associated short form suggested a scissile site located within the Arg(363)-Arg(393) region in the bovine beta-arrestin 1. Edman degradation analysis of a beta-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe(388) and a minor cleavage after Asn(375). Rather unexpectedly, the inverse agonist EXP3174-bound AT(1)R generated different fragmentation of bovine beta-arrestin 1, at Pro(276). The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca(2+)-mediated signaling pathways. The proteolysis of beta-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that beta-arrestin cleavage upon AT(1)R stimulation is a part of the unraveling beta-arrestin-mediated G protein-coupled receptor signaling diversity.


PLOS ONE | 2017

Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts

Arunachal Chatterjee; John Barnard; Christine S. Moravec; Russell Desnoyer; Kalyan C. Tirupula; Sadashiva S. Karnik; James W. Porter

Perspectives on whether the functions of MAS, a G protein-coupled receptor, are beneficial or deleterious in the heart remain controversial. MAS gene knockout reduces coronary vasodilatation leading to ischemic injury. G protein signaling activated by MAS has been implicated in progression of adaptive cardiac hypertrophy to heart failure and fibrosis. In the present study, we observed increased expression of MAS, connective tissue growth factor (CTGF) and collagen genes in failing (HF) human heart samples when compared to non-failing (NF). Expression levels of MAS are correlated with CTGF in HF and NF leading to our hypothesis that MAS controls CTGF production and the ensuing expression of collagen genes. In support of this hypothesis we show that the non-peptide MAS agonist AR234960 increases both mRNA and protein levels of CTGF via ERK1/2 signaling in HEK293-MAS cells and adult human cardiac fibroblasts. MAS-mediated CTGF expression can be specifically blocked by MAS inverse agonist AR244555 and also by MEK1 inhibition. Expression of CTGF gene was essential for MAS-mediated up-regulation of different collagen subtype genes in HEK293-MAS cells and human cardiac fibroblasts. Knockdown of CTGF by RNAi disrupted collagen gene regulation by the MAS-agonist. Our data indicate that CTGF mediates the profibrotic effects of MAS in cardiac fibroblasts. Blocking MAS-CTGF-collagen pathway should be considered for pharmacological intervention for HF.


Journal of Chemical Information and Modeling | 2017

Divergent Spatiotemporal Interaction of Angiotensin Receptor Blocking Drugs with Angiotensin Type 1 Receptor

Khuraijam Dhanachandra Singh; Hamiyet Unal; Russell Desnoyer; Sadashiva S. Karnik

Crystal structures of the human angiotensin II type 1 receptor (AT1R) complex with the antihypertensive agent ZD7155 (PDB id: 4YAY ) and the blood pressure medication Benicar (PDB id: 4ZUD ) showed that binding poses of both antagonists are similar. This finding implies that clinically used angiotensin receptor blocking (ARB) drugs may interact in a similar fashion. However, clinically observed differences in pharmacological and therapeutic efficacies of ARBs lead to the question of whether the dynamic interactions of AT1R with ARBs vary. To address this, we performed induced-fit docking (IFD) of eight clinically used ARBs to AT1R followed by 200 ns molecular dynamic (MD) simulation. The experimental Ki values for ARBs correlated remarkably well with calculated free energy with R2 = 0.95 and 0.70 for AT1R-ARB models generated respectively by IFD and MD simulation. The eight ARB-AT1R complexes share a common set of binding residues. In addition, MD simulation results validated by mutagenesis data discovered distinctive spatiotemporal interactions that display unique bonding between an individual ARB and AT1R. These findings provide a reasonably broader picture reconciling the structure-based observations with clinical studies reporting efficacy variations for ARBs. The unique differences unraveled for ARBs in this study will be useful for structure-based design of the next generation of more potent and selective ARBs.

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Michael Kinter

Oklahoma Medical Research Foundation

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