The Deubiquitinating Enzyme USP17 Blocks N-Ras Membrane Trafficking and Activation but Leaves K-Ras Unaffected

The proto-oncogenic Ras isoforms (H, N, and K) have a C-terminal CAAX motif and undergo the same post-translational processing steps, although they traffic to the plasma membrane through different routes. Previously, we have shown that overexpression of the deubiquitinating enzyme USP17 inhibits H-Ras localization to the plasma membrane. Now we report that whereas H-Ras and N-Ras were unable to localize to the plasma membrane in the presence of USP17, K-Ras4b localization was unaffected. EGF stimulation was unable to induce N-Ras membrane localization in USP17-expressing cells. In addition, N-Ras activity and downstream signaling through the MAPK MEK/ERK and PI3K/JNK pathways were blunted. However, we still detected abundant N-Ras localization at the ER and Golgi in USP17-expressing cells. Collectively, our data showed that the deubiquitinating enzyme USP17 blocks EGF-induced N-Ras membrane trafficking and activation, but left K-Ras unaffected.

Immune activation in Retinal Aging: A Gene Expression Study.

PURPOSE
To investigate changes in gene expression during aging of the retina in the mouse.

METHODS
Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkagemoderated t-statistics method. Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.01. Differential expression in 11 selected genes was further verified by real-time PCR. Functional pathways involved in retinal ageing were analyzed by an online software package (DAVID-2008) in differentially expressed gene lists. Age-related changes in differential expression in the identified retinal molecular pathways were further confirmed by immunohistochemical staining of retinal flat mounts and retinal cryosections.

RESULTS
With ageing of the retina, 298 genes were upregulated and 137 genes were downregulated. Functional annotation showed that genes linked to immune responses (Ir genes) and to tissue stress/injury responses (TS/I genes) were most likely to be modified by ageing. The Ir genes affected included those regulating leukocyte activation, chemotaxis, endocytosis, complement activation, phagocytosis, and myeloid cell differentiation, most of which were upregulated, with only a few downregulated. Increased microglial and complement activation in the aging retina was further confirmed by confocal microscopy of retinal tissues. The most strongly upregulated gene was the calcitonin receptor (Calcr; >40-fold in old versus young mice).

CONCLUSIONS
The results suggest that retinal ageing is accompanied by activation of gene sets, which are involved in local inflammatory responses. A modified form of low-grade chronic inflammation (para-inflammation) characterizes these aging changes and involves mainly the innate immune system. The marked upregulation of Calcr in ageing mice most likely reflects this chronic inflammatory/stress response, since calcitonin is a known systemic biomarker of inflammation/sepsis. © Association for Research in Vision and Ophthalmology.

The effect of increased dietary fruit and vegetable consumption on endothelial activation, inflammation and oxidative stress in hypertensive volunteers

Background and aims

Public health campaigns recommend increased fruit and vegetable (FV) consumption as an effective means of cardiovascular risk reduction. During an 8 week randomised control trial among hypertensive volunteers, we noted significant improvements in endothelium-dependent vasodilatation with increasing FV consumption. Circulating indices of inflammation, endothelial activation and insulin resistance are often employed as alternative surrogates for systemic arterial health. The responses of several such biomarkers to our previously described FV intervention are reported here.
Methods and results

Hypertensive volunteers were recruited from medical outpatient clinics. After a common 4 week run-in period during which FV consumption was limited to 1 portion per day, participants were randomised to 1, 3 or 6 portions daily for 8 weeks. Venous blood samples for biomarker analyses were collected during the pre and post-intervention vascular assessments. A total of 117 volunteers completed the 12 week study. Intervention-related changes in circulating levels of high sensitivity C-reactive protein (hsCRP), soluble intracellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) did not differ significantly between FV groups. Similarly, there were no significant between group differences of change in homeostasis model assessment (HOMA) scores.
Conclusions

Despite mediating a significant improvement in acetylcholine induced vasodilatation, increased FV consumption did not affect a calculated measure of insulin resistance or concentrations of the circulating biomarkers measured during this study. Functional indices of arterial health such as endothelium-dependent vasomotion are likely to provide more informative cardiovascular end-points during short-term dietary intervention trials.

Two Arginine-Glutamate Ionic Locks Near the Extracellular Surface of FFAR1 Gate Receptor Activation

Activation of a number of class A G protein-coupled receptors (GPCRs) is thought to involve two molecular switches, a rotamer toggle switch within the transmembrane domain and an ionic lock at the cytoplasmic surface of the receptor; however, the mechanism by which agonist binding changes these molecular interactions is not understood. Importantly, 80% of GPCRs including free fatty acid receptor 1 (FFAR1) lack the complement of amino acid residues implicated in either or both of these two switches; the mechanism of activation of these GPCRs is therefore less clear. By homology modeling, we identified two Glu residues (Glu-145 and Glu-172) in the second extracellular loop of FFAR1 that form putative interactions individually with two transmembrane Arg residues (Arg-183(5.39) and Arg-258(7.35)) to create two ionic locks. Molecular dynamics simulations showed that binding of agonists to FFAR1 leads to breakage of these Glu-Arg interactions. In mutagenesis experiments, breakage of these two putative interactions by substituting Ala for Glu-145 and Glu-172 caused constitutive receptor activation. Our results therefore reveal a molecular switch for receptor activation present on the extracellular surface of FFAR1 that is broken by agonist binding. Similar ionic locks between the transmembrane domains and the extracellular loops may constitute a mechanism common to other class A GPCRs also.

Atomistic Insights into Rhodopsin Activation from a Dynamic Model

Rhodopsin, the light sensitive receptor responsible for blue-green vision, serves as a prototypical G protein-coupled receptor (GPCR). Upon light absorption, it undergoes a series of conformational changes that lead to the active form, metarhodopsin II (META II), initiating a signaling cascade through binding to the G protein transducin (G(t)). Here, we first develop a structural model of META II by applying experimental distance restraints to the structure of lumi-rhodopsin (LUMI), an earlier intermediate. The restraints are imposed by using a combination of biased molecular dynamics simulations and perturbations to an elastic network model. We characterize the motions of the transmembrane helices in the LUMI-to-META II transition and the rearrangement of interhelical hydrogen bonds. We then simulate rhodopsin activation in a dynamic model to study the path leading from LUMI to our META II model for wild-type rhodopsin and a series of mutants. The simulations show a strong correlation between the transition dynamics and the pharmacological phenotypes of the mutants. These results help identify the molecular mechanisms of activation in both wild type and mutant rhodopsin. While static models can provide insights into the mechanisms of ligand recognition and predict ligand affinity, a dynamic model of activation could be applicable to study the pharmacology of other GPCRs and their ligands, offering a key to predictions of basal activity and ligand efficacy.

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecularbasis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct phannacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation. (c) 2007 Elsevier B.V. All rights reserved.

Identification of residues important for agonist recognition and activation in GPR40

GPR40 was formerly an orphan G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs). The receptor, now named FFA receptor 1, has been implicated in the pathophysiology of type 2 diabetes and is a drug target because of its role in FFA-mediated enhancement of glucose-stimulated insulin release. Guided by molecular modeling, we investigated the molecular determinants contributing to binding of linoleic acid, a C18 polyunsaturated FFA, and GW9508, a synthetic small molecule agonist. Twelve residues within the putative GPR40-binding pocket including hydrophilic/positively charged, aromatic, and hydrophobic residues were identified and were subjected to site-directed mutagenesis. Our results suggest that linoleic acid and GW9508 are anchored on their carboxylate groups by Arg183, Asn244, and Arg258. Moreover, His86, Tyr91, and His137 may contribute to aromatic and/or hydrophobic interactions with GW9508 that are not present, or relatively weak, with linoleic acid. The anchor residues, as well as the residues Tyr12, Tyr91, His137, and Leu186, appear to be important for receptor activation also. Interestingly, His137 and particularly His86 may interact with GW9508 in a manner dependent on its protonation status. The greater number of putative interactions between GPR40 and GW9508 compared with linoleic acid may explain the higher potency of GW9508.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Asymmetric rhodium carbene insertion into the Si-H bond: identification of new dirhodium(II) carboxylate catalysts using parallel synthesis techniques

Decomposition of methyl 2-diazophenylacetate in the presence of silanes and a chiral dirhodium(11) catalyst results in Si-H insertion of the intermediate carbenoid with varying degrees of enantioselectivity. New chiral dirhodium(11) carboxylate catalysts were identified using solution phase parallel synthesis techniques. (C) 2003 Elsevier Science Ltd. All rights reserved.