The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

Selective inhibition of cannabinoid CB1 receptor-evoked signalling by the interacting protein GAP43.

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.

Control of a hippocampal recurrent excitatory circuit by cannabinoid receptor-interacting protein Gap43.

The type-1 cannabinoid receptor (CB1R) is widely expressed in excitatory and inhibitory nerve terminals, and by suppressing neurotransmitter release, its activation modulates neural circuits and brain function. While the interaction of CB1R with various intracellular proteins is thought to alter receptor signaling, the identity and role of these proteins are poorly understood. Using a high-throughput proteomic analysis complemented with an array of in vitro and in vivo approaches in the mouse brain, we report that the C-terminal, intracellular domain of CB1R interacts specifically with growth-associated protein of 43 kDa (GAP43). The CB1R-GAP43 interaction occurs selectively at mossy cell axon boutons, which establish excitatory synapses with dentate granule cells in the hippocampus. This interaction impairs CB1R-mediated suppression of mossy cell to granule cell transmission, thereby inhibiting cannabinoid-mediated anti-convulsant activity in mice. Thus, GAP43 acts as a synapse type-specific regulatory partner of CB1R that hampers CB1R-mediated effects on hippocampal circuit function.

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis.

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP+/- mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP+/- double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP+/- mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP+/- mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB1) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB1 receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.

Identification of BiP as a CB1 Receptor-Interacting Protein That Fine-Tunes Cannabinoid Signaling in the Mouse Brain.

Cannabinoids, the bioactive constituents of cannabis, exert a wide array of effects on the brain by engaging Type 1 cannabinoid receptor (CB1R). Accruing evidence supports that cannabinoid action relies on context-dependent factors, such as the biological characteristics of the target cell, suggesting that cell population-intrinsic molecular cues modulate CB1R-dependent signaling. Here, by using a yeast two-hybrid-based high-throughput screening, we identified BiP as a potential CB1R-interacting protein. We next found that CB1R and BiP interact specifically in vitro, and mapped the interaction site within the CB1R C-terminal (intracellular) domain and the BiP C-terminal (substrate-binding) domain-α. BiP selectively shaped agonist-evoked CB1R signaling by blocking an "alternative" Gq/11 protein-dependent signaling module while leaving the "classical" Gi/o protein-dependent inhibition of the cAMP pathway unaffected. In situ proximity ligation assays conducted on brain samples from various genetic mouse models of conditional loss or gain of CB1R expression allowed to map CB1R-BiP complexes selectively on terminals of GABAergic neurons. Behavioral studies using cannabinoid-treated male BiP+/- mice supported that CB1R-BiP complexes modulate cannabinoid-evoked anxiety, one of the most frequent undesired effects of cannabis. Together, by identifying BiP as a CB1R-interacting protein that controls receptor function in a signaling pathway- and neuron population-selective manner, our findings may help to understand the striking context-dependent actions of cannabis in the brain.SIGNIFICANCE STATEMENT Cannabis use is increasing worldwide, so innovative studies aimed to understand its complex mechanism of neurobiological action are warranted. Here, we found that cannabinoid CB1 receptor (CB1R), the primary molecular target of the bioactive constituents of cannabis, interacts specifically with an intracellular protein called BiP. The interaction between CB1R and BiP occurs selectively on terminals of GABAergic (inhibitory) neurons, and induces a remarkable shift in the CB1R-associated signaling profile. Behavioral studies conducted in mice support that CB1R-BiP complexes act as fine-tuners of anxiety, one of the most frequent undesired effects of cannabis use. Our findings open a new conceptual framework to understand the striking context-dependent pharmacological actions of cannabis in the brain.

Multivalent cationic dendrofullerenes for gene transfer: synthesis and DNA complexation.

Non-viral nucleic acid vectors able to display high transfection efficiencies with low toxicity and overcoming the multiple biological barriers are needed to further develop the clinical applications of gene therapy. The synthesis of hexakis-adducts of [60]fullerene endowed with 12, 24 and 36 positive ammonium groups and a tridecafullerene appended with 120 positive charges has been performed. The delivery of a plasmid containing the green fluorescent protein (EGFP) gene into HEK293 (Human Embryonic Kidney) cells resulting in effective gene expression has demonstrated the efficacy of these compounds to form polyplexes with DNA. Particularly, giant tridecafullerene macromolecules have shown higher efficiency in the complexation and transfection of DNA. Thus, they can be considered as promising non-viral vectors for transfection purposes.

Molecular dissection of the membrane aggregation mechanisms induced by monomeric annexin A2.

Annexins are a multigene family of proteins involved in aggregation and fusion processes of biological membranes. One of its best-known members is annexin A2 (or p36), capable of binding to acidic phospholipids in a calcium-dependent manner, as occurs with other members of the same family. In its heterotetrameric form, especially with protein S100A10 (p11), annexin A2 has been involved as a determinant factor in innumerable biological processes like tumor development or anticoagulation. However, the subcellular coexistence of different pools of the protein, in which the monomeric form of annexin A2 is growing in functional relevance, is to date poorly described. In this work we present an exhaustive structural and functional characterization of monomeric human annexin A2 by using different recombinant mutants. The important role of the amphipathic N-terminal α-helix in membrane binding and aggregation has been analyzed. We have also studied the potential implication of lateral "antiparallel" protein dimers in membrane aggregation. In contrast to what was previously suggested, formation of these dimers negatively regulate aggregation. We have also confirmed the essential role of three lysine residues located in the convex surface of the molecule in calcium-free and calcium-dependent membrane binding and aggregation. Finally, we propose models for annexin A2-mediated vesicle aggregation mechanisms.

Subcellular Targeting of Nitric Oxide Synthases Mediated by Their N-Terminal Motifs.

From a catalytic point of view, the three mammalian nitric oxide synthases (NOSs) function in an almost identical way. The N-terminal oxygenase domain catalyzes the conversion of l-arginine to l-citrulline plus ·NO in two sequential oxidation steps. Once l-arginine binds to the active site positioned above the heme moiety, two consecutive monooxygenation reactions take place. In the first step, l-arginine is hydroxylated to make Nω-hydroxy-l-arginine in a process that requires 1 molecule of NADPH and 1 molecule of O2 per mol of l-arginine reacted. In the second step, Nω-hydroxy-l-arginine, never leaving the active site, is oxidized to ·NO plus l-citrulline and 1 molecule of O2 and 0.5 molecules of NADPH are consumed. Since nitric oxide is an important signaling molecule that participates in a number of biological processes, including neurotransmission, vasodilation, and immune response, synthesis and release of ·NO in vivo must be exquisitely regulated both in time and in space. Hence, NOSs have evolved introducing in their amino acid sequences subcellular targeting motifs, most of them located at their N-termini. Deletion studies performed on recombinant, purified NOSs have revealed that part of the N-terminus of all three NOS can be eliminated with the resulting mutant enzymes still being catalytically active. Likewise, NOS isoforms lacking part of their N-terminus when transfected in cells render mislocalized, active proteins. In this review we will comment on the current knowledge of these subcellular targeting signals present in nNOS, iNOS, and eNOS.

Molecular Basis for the Protein Recognition Specificity of the Dynein Light Chain DYNLT1/Tctex1: CHARACTERIZATION OF THE INTERACTION WITH ACTIVIN RECEPTOR IIB.

It has been suggested that DYNLT1, a dynein light chain known to bind to various cellular and viral proteins, can function both as a molecular clamp and as a microtubule-cargo adapter. Recent data have shown that the DYNLT1 homodimer binds to two dynein intermediate chains to subsequently link cargo proteins such as the guanine nucleotide exchange factor Lfc or the small GTPases RagA and Rab3D. Although over 20 DYNLT1-interacting proteins have been reported, the exact sequence requirements that enable their association to the canonical binding groove or to the secondary site within the DYNLT1 surface are unknown. We describe herein the sequence recognition properties of the hydrophobic groove of DYNLT1 known to accommodate dynein intermediate chain. Using a pepscan approach, we have substituted each amino acid within the interacting peptide for all 20 natural amino acids and identified novel binding sequences. Our data led us to propose activin receptor IIB as a novel DYNLT1 ligand and suggest that DYNLT1 functions as a molecular dimerization engine bringing together two receptor monomers in the cytoplasmic side of the membrane. In addition, we provide evidence regarding a dual binding mode adopted by certain interacting partners such as Lfc or the parathyroid hormone receptor. Finally, we have used NMR spectroscopy to obtain the solution structure of human DYNLT1 forming a complex with dynein intermediate chain of ∼74 kDa; it is the first mammalian structure available.

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase: CHARACTERIZATION OF THE INTERACTION WITH THE TIGHT JUNCTION PROTEIN CLAUDIN-3.

Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2 By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier.

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor.

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.

Binding of PDZ domains to the carboxy terminus of inducible nitric oxide synthase boosts electron transfer and NO synthesis.

iNOS lacks any phosphorylatable residue at its C-terminus despite displaying a 25-residue extension known to block electron transfer and activity. We report that C-terminal deletions of iNOS increased the cytochrome c reduction rate. Moreover, the interaction of the iNOS C-terminus with the PDZ domains of EBP50 or CAP70 resulted not only in augmented reductase activity and greater NO synthesis but also anticipated the formation of the air-stable semiquinone generated after NADPH addition. Hence, the C-terminus of iNOS regulates the activity of the enzyme, albeit, unlike nNOS and eNOS, displacement of the autoinhibitory element occurs upon binding to proteins with PDZ domains.