Nuevos aspectos en la regulación de receptores G en el sistema nervioso: estudio comparativo del receptor opioide [mu] y cannabinoide CB1

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-06-201

Characterization of a baculovirus expression vector for producing an universal vaccine against human common cold virus

Motivation: Human rhinovirus (HRV) is the primary etiologic agent of the common cold, which triggers approximately 50% of asthma and chronic obstructive pulmonary diseases. Direct and indirect costs associated to the common cold are approximately $50B per year only in the USA. Therefore, a vaccine decreasing the incidence of common cold is needed and has got a big potential market. The main challenges for developing a vaccine are: the high variety of virus serotypes and the need of a reliable and economically sustainable manufacturing process. HRV has an icosahedral capsid consists of 60 copies of the four structural proteins VP1, VP2, VP3 and VP4 (1). Bionaturis' partner in Maryland (USA) has modified the sequence of the structural proteins in order to raise universal immunity. Bionaturis is optimizing a method of manufacturing of a recombinant vaccine based on viral-like particles (VLPs) of the universal version of HRV. Methods: For manufacturing the HRV-VLPs, Bionaturis is using its proprietary platform FLYLIFE, which consists in using lepidopteran larvae as biofactories taking advantage of the baculovirus expression technology. This type of manufacturing is more flexible and economically sustainable than traditional expression systems based on cell fermentors. The first part of the manufacturing process is to produce and characterize the working viral bank (WVB), i.e. a recombinant baculovirus able to accumulate the protein of interest when it infects lepidopteran larvae (2). Subsequently, the larvae are infected and harvested for purification of the HRV-VLPs. The product of interest is analyzed using techniques of basic proteomics like SDS-PAGE and immunodetection by Western blot.Results: The WVB was successfully produced by cotransfection of insect cells Sf21 of Spodoptera frugiperda ovary with Bionaturis' Master Viral DNA based on the genome of the baculovirus AcMNPV (Autographa californica) and a transfer vector containing the expression cassette for making the HRV-VLPs (3). The purity and identity of the WVB generated was characterized by using genomic and proteomic techniques, certifying adequacy of its use for manufacturing HRV-VLPs in Bionaturis' FlyLife platform. Conclusions: Flylife is a good candidate platform for manufacturing of a universal vaccine against human rhinovirus, combining aimed properties like lower capital investment, less space, and greater biological safety

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins

<p>Abstract</p> <p>Background</p> <p>In general, opioids that induce the recycling of μ-opioid receptors (MORs) promote little desensitization, although morphine is one exception to this rule. While morphine fails to provoke significant internalization of MORs in cultured cells, it does stimulate profound desensitization. In contrast, morphine does promote some internalization of MORs in neurons although this does not prevent this opioid from inducing strong antinociceptive tolerance.</p> <p>Results</p> <p>In neurons, morphine stimulates the long-lasting transfer of MOR-activated Gα subunits to proteins of the RGS-R7 and RGS-Rz subfamilies. We investigated the influence of this regulatory process on the capacity of morphine to promote desensitization and its association with MOR recycling in the mature nervous system. In parallel, we also studied the effects of [D-Ala², <it>N</it>-MePhe⁴, Gly-ol⁵] encephalin (DAMGO), a potent inducer of MOR internalization that promotes little tolerance. We observed that the initial exposure to icv morphine caused no significant internalization of MORs but rather, a fraction of the Gα subunits was stably transferred to RGS proteins in a time-dependent manner. As a result, the antinociception produced by a second dose of morphine administered 6 h after the first was weaker. However, this opioid now stimulated the phosphorylation, internalization and recycling of MORs, and further exposure to morphine promoted little tolerance to this moderate antinociception. In contrast, the initial dose of DAMGO stimulated intense phosphorylation and internalization of the MORs associated with a transient transfer of Gα subunits to the RGS proteins, recovering MOR control shortly after the effects of the opioid had ceased. Accordingly, the recycled MORs re-established their association with G proteins and the neurons were rapidly resensitized to DAMGO.</p> <p>Conclusion</p> <p>In the nervous system, morphine induces a strong desensitization before promoting the phosphorylation and recycling of MORs. The long-term sequestering of morphine-activated Gα subunits by certain RGS proteins reduces the responses to this opioid in neurons. This phenomenon probably increases free Gβγ dimers in the receptor environment and leads to GRK phosphorylation and internalization of the MORs. Although, the internalization of the MORs permits the transfer of opioid-activated Gα subunits to the RGSZ2 proteins, it interferes with the stabilization of this regulatory process and recycled MORs recover the control on these Gα subunits and opioid tolerance develops slowly.</p

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

Abstract Background Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated. Results Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids. Conclusion In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.</p

Calcium/calmodulin-dependent protein kinase II supports morphine antinociceptive tolerance by phosphorylation of glycosylated phosducin-like protein

The long isoform of the phosducin-like protein (PhLPl) is widely expressed in the brain and it is thought to influence G-protein signalling by regulating the activity of Gβγ dimers. We show that in the mature nervous system, PhLPl exists as both a 38 kDa non-glycosylated isoform and as glycosylated isoforms of about 45, 100 and 150 kDa. Additionally, neural PhLPl is subject to serine phosphorylation, which augments upon the activation of Mu-opioid receptors (MORs), as does its association with Gβγ subunits and 14-3-3 proteins. While the intracerebroventricular (icv) administration of morphine to mice rapidly reduced the association of MORs with G proteins, it increased the serine phosphorylation of these receptors. Moreover, activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) accumulated in the MOR environment and phosphorylated PhLPl was seen to co-precipitate with these opioid receptors. Opioid-induced phosphorylation of PhLPl was impaired by inhibiting the activity of CaMKII and, in these circumstances, the association of PhLPl with Gβγ dimers and 14-3-3 proteins was diminished. Furthermore, these events were coupled with the recovery of G protein regulation by the MORs, while there was a decrease in serine phosphorylation of these receptors and morphine antinociceptive tolerance diminished. It seems that CaMKII phosphorylation of PhLPl stabilizes the PhLPl·Gβγ complex by promoting its binding to 14-3-3 proteins. When this complex fails to bind to 14-3-3 proteins, the association of PhLPl with Gβγ is probably disrupted by GαGDP subunits and the MORs recover control on G proteins. © 2007 Elsevier Ltd. All rights reserved.Peer Reviewe

NMDAR-nNOS generated zinc recruits PKCγ to the HINT1-RGS17 complex bound to the C terminus of Mu-opioid receptors

In neurons, the C terminus of the Mu-opioid receptor (MOR) binds to the protein kinase C-interacting protein/histidine triad nucleotide binding protein 1 (PKCI/HINT1) which in turn binds the regulator of G-protein signalling RGSZ1/Z2 (RGSZ) protein. In this study, we found that intracerebroventricular (icv) administration of morphine recruits PKC isoforms, mostly PKCγ, to the MOR via the HINT1/RGSZ complex. There, diacylglycerol (DAG) activates this PKCγ to phosphorylate the MOR and thus, its signal strength was reduced. When PKCI/HINT1 expression is depressed, morphine produces stronger analgesic effects and neither the PKCγ-MOR complex nor serine phosphorylation of this receptor is detected. This MOR-PKC association involves the cysteine rich domains (CRDs) in the regulatory C1 region of PKC, as well as requiring free zinc ions, HINT1 and RGSZ proteins. Increasing the availability of this metal ion recruits inactive PKCγ to the MOR, while phorbol esters prevent this binding and even disrupt it. The nitric oxide donor (S)-Nitroso-N-acetylpenicillamine (SNAP) foments the association of PKCγ with the MORs, effect that was prevented by the heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), suggesting a role for endogenous zinc and neural nitric oxide synthase. The N-methyl-D-aspartate receptor (NMDAR) antagonist, MK801, also prevented PKCγ recruitment to MORs and serine phosphorylation of the receptors following icv morphine. These results indicate that the NMDAR/nNOS cascade, activated via MORs, provide the free zinc ions required for inactive PKCγ to bind to HINT1/RGSZ complex at the C terminus of the receptor. © 2008 Elsevier Inc. All rights reserved.Peer Reviewe

Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins-4

<p><b>Copyright information:</b></p><p>Taken from "Morphine induces endocytosis of neuronal μ-opioid receptors through the sustained transfer of Gα subunits to RGSZ2 proteins"</p><p>http://www.molecularpain.com/content/3/1/19</p><p>Molecular Pain 2007;3():19-19.</p><p>Published online 17 Jul 2007</p><p>PMCID:PMC1947952.</p><p></p>olerance was monitored at various intervals post-opioid administration by measuring the analgesia produced by the release of the opioid. Groups of 10 mice were sacrificed at different intervals and the dorsal horns of the cervical-dorsal spinal cords were removed. To analyze the phosphorylation and presence of MORs in the plasma membrane, the immunoprecipitation was performed under denaturing conditions. For every post-opioid interval analyzed, densitometric signals associated with 55–65, 70–75, and 90–100 kDa were pooled and normalized to those obtained probing the anti-MOR IgGs (heavy chain). The assay was repeated twice and the results were comparable. Further details as in Fig. 1