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EFFECTS OF CANNABINOID RECEPTOR INTERACTING PROTEIN (CRIP1a) ON CANNABINOID (CB1) RECEPTOR FUNCTION. By Tricia Hardt Smith, B.S., M.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2009. Major Director: Dana E. Selley, Ph.D., Department of Pharmacology and Toxicology This dissertation examines modulation of cannabinoid CB1 receptor function by Cannabinoid Receptor Interacting Protein (CRIP1a), a novel protein that binds the C-terminus of CB1 receptors. In Human embryonic kidney cells expressing human CB1 receptors (hCB1-HEK) and hCB1-HEK cells stably co-expressing CRIP1a (hCB1-HEK-CRIP1a), quantitative immunoblotting revealed a CRIP1a/CB1 molar ratio of 5.4 and 0.37, respectively, with no difference in CB1 receptor expression. To test the hypothesis that CRIP1a modulates CB1 receptor signaling, G-protein and effector activity were examined with and without full, partial and inverse agonists. [35S]GTPgS binding, which measures G-protein-coupled receptor (GPCR)-mediated G-protein activation, showed that CRIP1a inhibited constitutive CB1 receptor activity, as indicated by the decreased effect of the inverse agonist SR141716A. CRIP1a also decreased CB1 receptor-mediated G-protein activation by high efficacy agonists, whereas moderate and low efficacy agonists were unaffected. In experiments varying Na+ concentration, CRIP1a decreased spontaneous G-protein activation at low Na+ concentrations, where spontaneous GPCR activity is highest. This effect was eliminated by pertussis toxin pre-treatment, indicating that CRIP1a only inhibits GPCR-mediated activity. To determine whether CRIP1a modulates receptor adaptation, hCB1-HEK (±CRIP1a) cells were pretreated with WIN or THC. Both ligands desensitized CB1 receptor-mediated G-protein activation, but desensitization was unaffected by CRIP1a. In contrast, CRIP1a attenuated downregulation of CB1 receptor binding sites by WIN, but not THC. Downstream, CRIP1a attenuated constitutive CB1 receptor-mediated inhibition of cAMP, as indicated by elimination of SR141716A-stimulated cAMP, without affecting agonist-induced cAMP inhibition. Constitutive inhibition was not due to endocannabinoids because LC-ESI-MS-MS did not detect endocannabinoids in hCB1-HEK (±CRIP1a) cells. To determine whether effects of CRIP1a were conserved among cell types, Chinese Hamster Ovary cells expressing CB1 receptors were stably co-transfected with CRIP1a, and had a CRIP1a/CB1 receptor molar ratio of 15 and 1900 with and without CRIP1a over-expression, respectively. In this model, CRIP1a inhibited constitutive CB1 receptor-mediated G-protein activity, but activation by agonists was enhanced, suggesting CRIP1a effects were dependent on stoichiometry of CRIP1a/CB1 receptor or cell type. Overall, these results indicate that CRIP1a decreases constitutive CB1 receptor activity, modulates agonist efficacy, and inhibits CB1 receptor downregulation, in a ligand- and cellular environment-dependent manner.

<p>The Muoio lab studies the interplay between lipid whole body energy balance,</p><p>mitochondrial function and insulin action in skeletal muscle. Data from our lab suggests that lipid-induced insulin resistance in skeletal muscle may stem from excessive incomplete oxidation of fatty acids, which occurs when high rates of &beta;-­oxidation exceed TCA cycle flux (Koves et al., 2005; Koves et al., 2008). Most notably, we have shown that mice with a genetically engineered decrease in mitochondrial uptake and oxidation of fatty acids are protected against diet-­induced insulin resistance (Koves et al., 2008). This</p><p>suggests that an excessive and/or inappropriate metabolic burden on muscle</p><p>mitochondria provokes insulin resistance. Our working model predicts that: 1) high rates of incomplete &beta;-oxidation reflect a state of &rdquo;mitochondrial stress,&rdquo; and 2) that energy-overloaded mitochondria generate a yet unidentified signal that mediates insulin</p><p>resistance. One possibility is that this putative mitochondrial-derived signal stems from redox imbalance and disruptions in redox sensitive signaling cascades. Therefore, we are interested in identifying molecules that link redox balance, mitochondrial function and insulin action in skeletal muscle. The work described herein identifies thioredoxin-interacting protein (TXNIP) as an attractive candidate that regulates both glucose homeostasis and mitochondrial fuel selection.</p><p>TXNIP is a redox sensitive, &alpha;-arrestin protein that has been implicated as a negative regulator of glucose control. Mounting evidence suggested that TXNIP might play a key role in regulating mitochondrial function; however, the molecular nature of this relationship was poorly defined. Previous studies in TXNIP knockout mice reported that deficiency of this protein compromises oxidative metabolism, increases glycolytic activity and promotes production of reactive oxygen species (ROS), while also affording protection against insulin resistance. Therefore, we hypothesized that TXNIP might serve as a nutrient sensor that couples cellular redox status to the adjustments in mitochondrial function. We tested this hypothesis by exploiting loss of function models to evaluate the effects of TXNIP deficiency on mitochondrial metabolism and respiratory function.</p><p>In chapter 3, we comprehensively evaluated oxidative metabolism, substrate</p><p>selection, respiratory kinetics and redox balance in mice with total body and skeletal muscle-­specific TXNIP deficiency. Targeted metabolomics, comprehensive bioenergetics analysis, whole-body respirometry and conventional biochemistry showed that TXNIP deficiency results in reduced exercise tolerance with marked impairments in skeletal muscle oxidative metabolism. The deficits in substrate oxidation were not secondary to decreased mitochondrial mass or increased H₂O₂ emitting potential from the electron transport chain. Instead, the activities of several mitochondrial dehydrogenases involved in branched-chain amino acid and ketone catabolism, the tricarboxylic acid (TCA) cycle and fatty acid &beta;-oxidation were significantly diminished in TXNIP null muscles. These deficits in mitochondrial enzyme activities were accompanied by decreased protein abundance without changes in mRNA expression. Taken together, these results suggest that in skeletal muscle TXNIP plays an essential role in maintaining protein synthesis and/or stability of a subset of mitochondrial dehydrogenase enzymes that permit muscle use of alternate fuels under conditions of glucose deprivation.</p><p>Based on these conclusions, we questioned whether additional regulatory</p><p>mechanisms could contribute to the reduced oxidative metabolism in the absence ofTXNIP. Several metabolic enzymes of the TCA cycle have been shown to be redox-sensitive protein targets regulated by the thioredoxin (TRX1/TRX2) and glutathione (GSH) redox-mediated circuits. TXNIP has been shown to respond to oxidative stress by shuttling to the mitochondria where it binds to TRX2 and/or other proteins, thus affecting downstream signaling pathways, such as the apoptotic cascade. Therefore, we speculated whether there was a role for redox imbalance in mediating the mitochondrial phenotype of the TXNIP knockout (TKO) mice. In chapter 4, we present preliminary evidence that increased glucose uptake promotes non-mitochondrial ROS production, causing a shift in redox balance, decreased GSH/GSSG, and S-glutathionylation of &alpha;-­ketoglutarate dehydrogenase (&alpha-KGD). This post-translational modification protects the protein from permanent oxidative damage, but at the cost of reversible loss of activity and subsequent disruption of TCA cycle flux that contributes, in part, to the diminished oxidative metabolism observed in the TXNIP deficient mice.</p><p>In aggregate, this work sheds new light onto the physiological role of TXNIP in</p><p>skeletal muscle as it pertains to substrate metabolism and fuel switching in response to nutrient availability. This work has important implications for metabolic diseases such as obesity and type 2 diabetes, which are characterized by marked disruptions in fuel selection.</p> / Dissertation

Alix (ALG-2 Interacting Protein X) est une protéine cytoplasmique impliquée dans divers processus cellulaires allant de l'apoptose à la cytocinèse en passant par le bourgeonnement des virus, la réparation membranaire et la régulation de la voie endosomale. Toutes ces fonctions sont étroitement associées à l'interaction d'Alix avec ses partenaires impliqués dans la déformation des membranes telles que les endophilines A, Tsg-101 et CHMP4B du complexe ESCRT (Endosomal Sorting Complex Required for Transport). Le but de ce projet est de caractériser le phénotype de la souris Alix ko récemment développée au laboratoire, dans l'espoir de mieux comprendre le rôle physiologique d'Alix. Ces souris, viables et fertiles, sont caractérisées par une microcéphalie apparaissant au cours de l'embryogenèse. Ce phénotype est accompagné d'une apoptose massive touchant les progéniteurs neuronaux durant la neurogenèse et d'une altération du développement de l'arborisation dendritique après la naissance. Les souris adultes présentent également des défauts de plasticité synaptique accompagnés d'une altération du recyclage des vésicules synaptiques. L'ensemble de ces processus repose sur la capacité d'Alix à contrôler le remodelage de la membrane plasmique. Au niveau moléculaire, nos travaux sur les neurones en cultures et sur les fibroblastes montrent une régulation de l'endocytose indépendante de la clathrine (CIE) par Alix et les endophilines A qui pourrait être à l'origine du phénotype neuronal de la souris. Cependant, l'association d'Alix avec CHMP4B du complexe ESCRT pourrait également être nécessaire au développement du système nerveux puisque l'interaction Alix-CHMP4B est nécessaire pour le contrôle de la CIE et de la mort neuronale.L'ensemble de ces résultats mets en évidence qu'à travers des mécanismes et des partenaires bien caractérisés, Alix est requise pour de nouvelles fonctions nécessaires au développement et au fonctionnement du système nerveux. / Alix (ALG-2 Interacting Protein X) is a cytoplasmic protein implicated in multiple processes including apoptosis, endosome function, membrane repair, viral budding and cytokinesis. Most of these involve modifications of plasma or endosomal membrane organization. Consistent with this, Alix is known to interact with diverse proteins modulating membrane deformation, such as endophilins or Tsg-101 and CHMP4B of the Endosomal Sorting Complex Required for Transport (ESCRT). By studying the phenotype of Alix ko mice that we recently developed, we aim to better understand the precise role of Alix in vivo. These mice are viable and fertile but develop microcephaly even at embryonic stages. This microcephaly is associated with an increase of apoptosis in neural progenitor cells in embryos and a defect in neurite outgrowth at post-natal stages. Later on, these mice also develop defects in synaptic plasticity related to an alteration of synaptic vesicle recycling. All of these processes are tightly dependent on membrane shaping and remodelling. These features of the phenotype can be related to a new function of Alix with endophilin A in the control of clathrin-independent endocytosis (CIE), as described in Alix ko fibroblasts and cultured neurons. However, these defects might also be related to the well-known Alix partner CHMP4B, since we now know that this interaction is needed for controlling CIE and neuronal cell death.All together, these results shed light on novel functions of Alix in the developing and adult central nervous system, all relying on well-known molecular mechanisms and partners.

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24983号 / 生博第512号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 鈴木 淳, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Ptf1a/p48 is a pancreas specific bHLH transcription factor that is required at early stages of embryonic development for pancreas formation and, during adulthood, for the proper exocrine pancreatic function. P48 also exerts an antiproliferative effect, which may exert a tumor suppressor activity. In this study, based on a yeast two-hybrid approach, we have identified new p48 partners that modulate the activity of p48. Among the newly identified putative interactors we found p/CAF, which is a coactivator that potentiates its transcriptional activity, and ICAT, an inhibitor of the &#946;-catenin/TCF signaling pathway. ICAT binds to p48 and is coexpressed with it in the pancreas during development and postnatally. Using different cellular models, ICAT overexpression in acinar tumor cells resulted in changes of the pancreatic specific gene expression pattern. Furthermore, high levels of ICAT inhibited the interaction between p48 and p/CAF. While this hetero-oligomeric complex is required for the acinar gene expression, ICAT itself is shown to be present in a reconstituted PTF1 complex in vivo. Importantly, altered ICAT expression is demonstrated in several histological types of pancreatic tumors, possibly contributing to their differentiation phenotype and neoplastic properties. / Ptf1a/p48 es un factor de transcripción bHLH específico del páncreas necesario durante los estadios tempranos del desarrollo embrionario para la formación del mismo, y para el correcto funcionamiento del páncreas exocrino en el adulto. P48 desempeña también una función antiproliferativa, la cual puede resultar en una actividad de supresión tumoral. En el presente estudio, basado en una estrategia de cribado de doble-híbrido en levadura, han sido identificadas nuevas proteínas que interaccionan y que modulan la actividad específica de p48. Entre las posibles proteínas que interaccionan y han sido identificadas de novo se encuentra p/CAF, un co-activador que potencia la actividad transcripcional de p48, y ICAT, un inhibidor de la vía de señalización de la &#946;-catenina. Se ha demostrado que ICAT se une a p48 y ambos son co-expresados en el páncreas durante el desarrollo y en el adulto. Utilizando diferentes modelos celulares, la sobreexpresión de ICAT en células tumorales acinares resultó en un cambio en el patrón de expresión de genes específicos del páncreas. Al mismo tiempo, se observó que niveles elevados de ICAT inhiben la interacción entre p48 y su co-activador p/CAF. Mientras que este complejo hetero-oligomérico es necesario para la expresión de los genes acinares, se demostró que ICAT está presente en un complejo PTF1 reconstituido in vivo. Finalmente, se observaron alteraciones en la expresión de ICAT en varios tipos histológicos de tumores pancreáticos, que posiblemente contribuyen a su fenotipo de diferenciación y propiedades neoplásicas.