Pierre-Yves Jean-Charles

Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

The non‐visual arrestins, β‐arrestin1, and β‐arrestin2 were originally identified as proteins that bind to seven‐transmembrane receptors (7TMRs, also called G protein‐coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non‐receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non‐receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β‐arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β‐arrestins in protein ubiquitination is evolutionarily conserved. β‐arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β‐arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β‐arrestin plays a critical role in stabilizing β‐arrestin‐7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin‐dependent regulation of 7TMRs as well as β‐arrestins and the potential role of reversible ubiquitination as a “biological switch” in signal transduction. J. Cell. Physiol. 231: 2071–2080, 2016.

Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress

Background: The mechanisms for recruiting and activating deubiquitinase(s) during GPCR trafficking are unknown. Results: PKA phosphorylation of USP20 Ser-333 inhibits β2AR interaction as well as deubiquitination and promotes receptor degradation via autolysosomes during physiological stress. Conclusion: USP20 activity and substrate-specific interaction involves a phosphorylation code. Significance: We identify a novel role for PKA in USP20 regulation and ubiquitin-dependent sorting of GPCRs. Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.

Insights into restrictive cardiomyopathy from clinical and animal studies

Cardiomyopathies are diseases that primarily affect the myocardium, leading to serious cardiac dysfunction and heart failure. Out of the three major categories of cardiomyopathies (hypertrophic, dilated and restrictive), restrictive cardiomyopathy (RCM) is less common and also the least studied. However, the prognosis for RCM is poor as some patients dying in their childhood. The molecular mechanisms behind the disease development and progression are not very clear and the treatment of RCM is very difficult and often ineffective. In this article, we reviewed the recent progress in RCM research from the clinical studies and the translational studies done on diseased transgenic animal models. This will help for a better understanding of the mechanisms underlying the etiology and development of RCM and for the design of better treatments for the disease.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein β-arrestin2. Although β-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic β-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of β-arrestin2 activity could be dictated by β-arrestin2s ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were β-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20 in vivo and in vitro. Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that β-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with β-arrestin2 was augmented when β-arrestin2 ubiquitination was prevented and reduced when β-arrestin2 ubiquitination was rendered constitutive. Constitutive β-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of β-arrestin2 are determined by β-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of β-arrestin2.

Gpcr signaling via β-arrestin-dependent mechanisms.

Abstract: &bgr;-arrestin1 (or arrestin2) and &bgr;-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein–coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that &bgr;-arrestins do not just “block” the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by &bgr;-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted &bgr;-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that &bgr;-arrestin–dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by &bgr;-arrestins and highlight the physiologic effects of &bgr;-arrestin–dependent signaling.

Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors

The seven-transmembrane containing G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. Transmembrane signaling by GPCRs is fundamental to many aspects of physiology including vision, olfaction, cardiovascular, and reproductive functions as well as pain, behavior and psychomotor responses. The duration and magnitude of signal transduction is tightly controlled by a series of coordinated trafficking events that regulate the cell-surface expression of GPCRs at the plasma membrane. Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs. Of the various molecular mechanisms that impart selectivity, sensitivity and strength of transmembrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR. Ubiquitination of proteins was originally discovered in the context of lysosome-independent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes. In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling. To date about 40 GPCRs are known to be ubiquitinated. For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes. This chapter will focus on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

Calcium desensitizer catechin reverses diastolic dysfunction in mice with restrictive cardiomyopathy

Diastolic dysfunction refers to an impaired relaxation and an abnormality in ventricular blood filling during diastole while systolic function is preserved. Cardiac myofibril hypersensitivity to Ca(2+) is a major factor that causes impaired relaxation of myocardial cells. The present study investigates the effect of the green tea extract catechins on myofibril calcium desensitization and restoration of diastolic function in a restrictive cardiomyopathy (RCM) mouse model with cardiac troponin mutations. Wild type (WT) and RCM mice were treated daily with catechin (epigallocatechin-3-gallate, EGCg, 50 mg/kg body weight) for 3 months. Echocardiography and cell based assays were performed to measure cardiac structure and flow-related variables including chamber dimensions, fraction shortening, trans-mitral flow patterns in the experimental mice. In addition, myocyte contractility and calcium dynamics were measured in WT and RCM cardiomyocytes treated in vitro with 5 μM EGCg. Our data indicated that RCM mice treated with EGCg showed an improved diastolic function while systolic function remained unchanged. At the cellular level, sarcomere relaxation and calcium decay were accelerated in RCM myocardial cells treated with EGCg. These results suggest that catechin is effective in reversing the impaired relaxation in RCM myocardial cells and rescuing the RCM mice with diastolic dysfunction.

Manifold roles of β-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9

β-Arrestin proteins fine-tune different GPCR-stimulated pathways that converge on ERK1/2 activation. The balancing act of β-arrestins G protein–coupled receptors (GPCRs) are thought to activate the kinases ERK1/2 through G protein– and β-arrestin–dependent pathways. The relative contribution of each is difficult to assess because β-arrestins prevent G protein coupling by GPCRs (see the Focus by Gurevich and Gurevich). Studies based on CRISPR/Cas9-generated cell lines suggested that β-arrestins are dispensable for ERK1/2 activation. Luttrell et al. compared the effects of siRNA-mediated and CRISPR/Cas9-mediated knockdown of β-arrestins on ERK1/2 activation by several GPCRs in independent clones. Their data showed that signaling rewiring in the CRISPR clones rendered GPCR-dependent ERK1/2 activation more G protein–dependent, which was reversed by reconstitution with β-arrestins. Together, these findings suggest that β-arrestins balance signaling through the different pathways from GPCRs to ERK1/2 and suggest that experiments with deletion of β-arrestins or G proteins should be interpreted with caution. G protein–coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β2 adrenergic receptors (β2ARs) and β1ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein–deleted cells to GPCR behavior in native systems.

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of β-adrenergic receptor signaling

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and β-arrestin2, thereby regulating β-adrenergic receptor (βAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects βAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac β1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable βAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued β1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of βAR signaling.

Dose-Dependent Arrhythmia and Cardiac Dysfunction in Restrictive Cardiomyopathy Mice Due to Troponin Mutations

Restrictive cardiomyopathy (RCM) is associated with a cardiac troponin I (cTnI) C-terminal mutation (R192H) in human patients. The transgenic mice expressing this mutation have confirmed a phenotype of a diastolic dysfunction and sudden cardiac death (SCD) (Du et al, 2006, 2008). In the present study, we generated transgenic mice (cTnI193His) expressing different levels of mutant cTnI R193H (mouse cTnI sequence) to investigate the dose-dependent cardiac dysfunction and to reveal the cause of the death in RCM mice. Our results indicated that the mice (cTnI193His/KO expressing only the mutant cTnI R193H at a wild type cTnI-null background had a dramatic early death at one-month old after birth. Telemetric ECG recording from these mice showed a significant bradycardia starting on day 22 or 23 after birth and a significant ischemia and arrhythmia 1-2 days before death. The diastolic function was deteriorated in these mice determined by echocardiography compared to wild type and the transgenic cTnI193His mice expressing 25% cTnI R193H and 75% wild type cTnI. Cell-based experiments indicated that myocardial contractility decreased significantly corresponding to the content of the mutant cTnI levels in cardiac myocytes and the alteration of Ca2+ dynamics in the mutant cTnI cardiac myocytes also showed a dose-dependent manner. Our study has demonstrated that cTnI R193H mutation-caused cardiac dysfunction is dose dependent. Bradycardia is likely an adaptive mechanism of RCM mice to compensate for the prolonged relaxation. The main cause of the death in RCM mice is associated with fatal arrhythmia and ventricular ischemia due to the restricted ventricles and enlarged atria. The transgenic mouse model provides us with a good tool to study the mechanisms and the cause of the death of RCM, which will be useful for the prevention and treatment of the disease.