Sudha K. Shenoy - US grants

DESCRIPTION (provided by applicant): The beta 1 and beta 2 adrenergic receptors are members of the super-family of G protein-coupled receptors (GPCRs). This receptor super-family comprises some of the most important pharmacological targets for the treatment of cardiovascular (e.g. heart failure, hypertension) and pulmonary diseases (e.g. asthma). A major cause for the observed attenuation of adrenergic response in heart failure and asthma is receptor downregulation, which results from chronic agonist exposure. Recently, ubiquitination of cell-surface receptors has been implicated as an important mechanism for post-endocytic sorting to lysosomes. Ubiquitination is a post-translational modification of proteins orchestrated by a well-defined process involving a cascade of three enzymatic activities. Of these the final step, catalyzed by the enzyme, E3 ubiquitin ligase, determines the specificity of substrate ubiquitination. We have demonstrated that two representative GPCRs, the beta2 adrenergic receptor and the V2 vasopressin receptor and their adaptor protein beta-arrestin become ubiquitinated upon agonist stimulation. Ubiquitination of beta-arrestin is crucial for receptor internalization, whereas ubiquitination of the receptor is essential for the proper sorting and downregulation of the internalized and activated receptors. We hypothesize that ubiquitination and deubiquitination coordinate the trafficking and signaling of GPCRs and involve a specific set of endocytic adapter proteins including beta-arrestins. The specific aims are: 1) to define the molecular mechanisms of receptor ubiquitination leading to receptor downregulation, and 2) to determine the roles of deubiquitinating enzyme(s) in receptor trafficking. Unraveling the molecular mechanisms governing the regulation of GPCRs, especially beta ARs, by ubiquitination could have a great impact on the development of novel therapeutic strategies for cardiovascular and pulmonary diseases.

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) constitute the largest cell-surface receptor family and at least 35% of currently prescribed drugs act on these receptor molecules. GPCR signaling is critically involved in many aspects of cardiovascular function. The magnitude and extent of GPCR signaling is determined by several governing factors including the lifetime of the receptor molecule itself. During the first period of funding, we have found that ubiquitination of the cell-surface b2 adrenergic receptor (b2AR) determines its degradation in lysosomes, thus providing an 'off switch' for attenuating cellular responses. We have identified specific enzymatic activities involved in regulating the intracellular trafficking of agonist-activated b2ARs. Thus, the RING-domain containing E3 ubiquitin ligase Mdm2 ubiquitinates the receptor associated adaptor protein b-arrestin2 and is involved in early steps of receptor internalization while the HECT- domain containing E3 ligase Nedd4 ubiquitinates the b2AR leading to receptor degradation in the lysosomes. Recruitment of both ligases to the b2AR is agonist-dependent and occurs sequentially. We have also shown that two related deubiquitinases (DUBS), USP20 and USP33 reverse this ubiquitination and prevent receptor degradation while concomitantly promoting receptor recycling to the plasma membrane. The central hypothesis for the proposed work in this competing continuation application is: b-adrenergic signaling is intimately linked to trafficking pathways and involves dynamic regulation by distinct E3 ligases and deubiquitinases. By using aortic vascular smooth muscle cells and neonatal ventricular myocytes as cellular model systems, RNAi and knockout mice, we will define the impact of ubiquitination/deubiquitination dynamics on bAR responsiveness in the cardiovascular system. The specific aims are: 1) To determine the effects of lysosomal trafficking in regulating bAR signaling, 2) To elucidate the molecular mechanisms that define the recruitment and/or activation of deubiquitinases during bAR resensitization and 3) To elucidate the mechanistic role of Mdm2 in bAR signaling in the heart. The long-term goal of this project is to understand the molecular mechanisms that integrate G protein-coupled receptor trafficking and signaling, which could play a critical role in balancing physiological responsiveness.

DESCRIPTION (provided by applicant): Atherosclerosis remains a leading cause of death in the United States. Accumulating evidence suggests that aortic wall smooth muscle cell gene expression contributes substantially to atherogenesis. ß-arrestin2 (ßarr2) an endocytic and signaling adaptor for G protein-coupled receptors (GPCRs), growth factor receptors and ion- channels is also known to promote neointimal hyperplasia and atherosclerosis in mice. Reversible ßarr2 ubiquitination, as regulated by deubiquitinases (DUBs) is a critical post-translational modification that is required for ßarr2's adaptor functions in mediating cell-signaling. Our Preliminary Studies suggests that the DUB USP20 might affect ßarr2 ubiquitination as well as NF?B signaling induced by the atherogenic Toll-like receptor 4 (TLR4). To delineate the role(s) of ßarr2 ubiquitination/deubiquitination dynamics in vivo and to evaluate whether the ubiquitination status of ßarr2 could engender pro-inflammatory signaling in SMCs, we have generated transgenic mice expressing USP20 or its catalytically inactive isoform (DN-USP20) under control of the SMC-specific SM22? promoter. In this model, we expect that by de-ubiquitinating ßarr2, USP20 would reduce ßarr2 activity and thereby reduce the SMC pro-atherogenic proliferation and migration that engenders neointimal hyperplasia, whereas the DN-USP20 would have reciprocal effects. By utilizing these and additional novel reagents and in vivo methods involving diet and gene-dependent atherosclerosis and in vitro techniques employing primary vascular smooth muscle cells we will test the hypotheses that USP20 in SMCs mitigates atherosclerosis through mechanisms involving deubiquitination of ßarr2, and/or deubiquitination of TRAF6 or TRAF2 in a manner dependent upon ßarr2 scaffolding by accomplishing following specific aims: (1) To determine the atheroprotective role of SMC USP20, and whether USP20's mechanism of action requires de-ubiquitination of ßarr2 (2) To determine whether USP20 activity regulates ßarr2-dependent SMC proliferation, migration and signaling triggered by inflammatory stimuli and (3) To elucidate the mechanistic basis of USP20's effects on ßarr2-dependent signaling.

PROJECT SUMMARY We recently demonstrated that ubiquitin-specific protease-20 (USP20) is scaffolded by the adaptor protein known as ?-arrestin2 (?arr2), and that USP20 desensitizes ubiquitin-dependent signaling from Toll-like receptor-4 (TLR4) to NF?B activation by deubiquitinating TRAF6 and ?arr2. Using transgenic mice expressing dominant-negative USP20 in smooth muscle cells, we found that USP20 reduces neointimal hyperplasia after arterial injury and that USP20 activity in SMCs reduces atherosclerosis in Ldlr-/- mice. To establish anti-atherogenic effects of systemically expressed USP20, and to elucidate further molecular mechanisms by which USP20 protects against atherosclerosis, this project will test the hypothesis that USP20 attenuates atherosclerosis by deubiquitinating several substrate proteins that were previously unassociated with USP20 but that are important in signaling pathways that activate NF?B: ?arr1, TRAF6, TRAF2, and RIPK1. Furthermore, because USP20 employs ?arr2 as a scaffold to facilitate association with distinct proteins, and because ?arr1 reduces vascular inflammation, this project will test whether USP20?s anti-atherogenic activity involves ?arr1-mediated scaffolding. To these ends, this project will study systemic effects of USP20 on atherosclerosis by comparing Usp20-/- /Ldlr-/- versus Ldlr-/- mice, on a background of ?arr1+/+ or ?arr1-/+. To determine the effects of endothelial USP20 on atherosclerosis, this project will compare atherosclerosis among VECad-Cre- ERT2/Usp20flox/flox/Ldlr-/- vs. Usp20flox/flox/Ldlr-/- mice treated ± tamoxifen; furthermore, we will investigate cytokine secretion, and dynamic ubiquitination of signaling proteins in primary aortic endothelial cells that are WT, Usp20-/-, Usp20-/-/?arr1-/+, or ?arr1-/-. To determine what kinase in endothelial cells phosphorylates USP20 on Ser333 (and thereby abrogates USP20 deubiquitinase activity), this project will test IRAK1, PAK1, and ROCK1 with several loss-of function approaches, including a USP20 minigene, in primary endothelial cells. These studies collectively may identify USP20 phosphorylation as novel therapeutic target for atherosclerosis.

PROJECT SUMMARY Chronic pressure overload that leads to left ventricular hypertrophy (LVH) and adverse cardiac remodeling is one of the leading causes of heart failure and affects millions of Americans. In addition to pathological insults and biomechanical factors, neurohormonal pathways that involve coordinated signaling through ?1 and ?2 adrenergic receptors (ARs) can play a major role in myocardial adaptation to pressure overload. The factors that shift the myocardial response to pressure overload from adaptive to maladaptive LVH remain largely obscure. Our research has focused on the regulation of ?AR trafficking and signaling by ubiquitination/deubiquitination mechanisms. We recently discovered that differential regulation of ?1AR and ?2AR endocytic trafficking can be achieved by the deubiquitinase (DUB) called ubiquitin-specific protease-20 (USP20), which we found deubiquitinates both ?ARs. In response to the ?AR agonist (-)isoproterenol (Iso), USP20 is phosphorylated on Ser333 by protein kinase A (PKA); this phosphorylation inhibits USP20 DUB activity toward the ?2AR and regulates trafficking of the ?2AR to autophagosomes. USP20 phosphorylation occurs in vivo, as well: (1) USP20 phosphorylation is significantly elevated in failing human hearts when compared with non-failing hearts and (2) pressure overload achieved by transverse aortic constriction (TAC) triggers USP20 phosphorylation in cardiomyocytes of WT, but not ?1AR KO mice. Accordingly, during LVH, USP20 phosphorylation requires ?1AR activation in the heart and may play a role in pathologic remodeling in LVH and/or heart failure. We hypothesize that ?USP20 and its phosphorylation status fine-tune ?AR signal transduction, endocytic trafficking and autophagy, thus impacting cardiac remodeling in LVH.? We will test our hypotheses by addressing the following specific aims: (1) To determine the role of cardiomyocyte-USP20 in the development of cardiac dysfunction, (2) To determine the regulation of myocardial ?AR signaling by USP20 Ser333 phosphorylation and (3) To determine the coordinated roles of ?1AR and USP20 in regulating ubiquitination status of the autophagy protein Beclin1 and its effect in cardiomyocyte autophagy.