DM facilitates formation of high affinity complexes of peptide–major histocompatibility complex (MHC) by release of class II MHC–associated invariant chain peptide (CLIP). This has been proposed to occur through discrimination of complex stability. By probing kinetic and conformational intermediates of the wild-type and mutant human histocompatibility leukocyte antigen (HLA)-DR1–peptide complexes, and examining their reactivities with DM, we propose that DM interacts with the flexible hydrophobic pocket 1 of DR1 and converts the molecule into a conformation that is highly peptide receptive. A more rigid conformation, generated upon filling of pocket 1, is less susceptible to DM effects. Thus, DM edits peptide–MHC by recognition of the flexibility rather than stability of the complex.

The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the β-chain (DR1βH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1βH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1βH81N complexes. Reintroduction of an appropriate hydrogen bond (DR1βH81N βV85H) restored DM-mediated peptide dissociation. Thus, DR1βH81N might represent a `post-DM effect' conformation. We suggest that DM may mediate peptide dissociation by a `hit-and-run' mechanism that results in conformational changes in MHC class II molecules and disruption of hydrogen bonds between βHis81 and bound peptide.Shortly after being synthesized in the antigen-presenting cell, major histocompatibility complex (MHC) class II αβ heterodimers form nonameric assemblies with invariant chain (Ii) in the endoplasmic reticulum and are then transported through the Golgi complex to the endocytic pathway 1, 2. During transport through the endocytic pathway, most Ii is removed from MHC class II molecules by low pH and acid proteases3, leaving a proteolytic fragment of Ii called `CLIP' bound to MHC class II molecule4. CLIP acts as a `placeholder' for the MHC class II groove, inhibiting conformational changes that render the groove closed5 -13 , and it must be removed to allow binding of exogenous peptides to nascent MHC class II complexes. Human HLA-DM (called `DM' here), or H2-M in mice, is a nonclassical HLA molecule that is critical in the displacement of CLIP 14-17. In addition to displacing CLIP, DM transiently interacts with empty MHC class II molecules to generate a peptide-receptive conformation and is active in the selection of specific peptide-MHC class II complexes during antigen processing18 -26. The two concurrent hypotheses for the recognition of certain peptide-MHC class II by DM relate to the intrinsic affinity between MHC class II © 2006 Nature Publishing Group Correspondence should be addressed to S.S-N (ssadegh@jhmi.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2011 January 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and the peptide 22,27,28 or to subtle structural variations among different peptide-MHC complexes 25,[29][30][31][32] , whereby structurally flexible complexes are susceptible to DM-induced dissociation, and `rigid' complexes are resistant to DM 25 . Although those studies may have brought greater understanding of the crite...

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE 2 ). It is in the family of G-protein-coupled receptors and is known to activate Ca 2ϩ signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE 2 stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1␣ (HIF-1␣), which can be completely blocked by pertussis toxin, indicating coupling to G i/o . This up-regulation of HIF-1␣ occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/ mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1␣, which involves decreased protein degradation, the up-regulation of HIF-1␣ by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1␣ observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1␣ is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE 2 biosynthesis could up-regulate the expression of HIF-1␣ and promote tumorigenesis. E-type prostanoid receptors (EP) are the receptors that mediate the actions of prostaglandin E 2 (PGE 2 ) and are members of the superfamily of G-protein coupled receptors.There are four primary subtypes of EP receptors, named EP1, EP2, EP3, and EP4. The EP1, EP2, and EP3 receptors were initially classified on the basis of their pharmacology and upon differences in their functional effects on various types of smooth muscle, as well as their activation of second-messenger signaling pathways (Coleman et al., 1994). Thus, PGE 2 stimulation of EP1 receptors produced contractile responses that could be selectively blocked with 8-chloro-dibenz [b,f][1,4]oxazepine-10(11H)-carboxy-(2-acetyl)hydrazide (SC-19220) and were involved in the mobilization of intracellular Ca 2ϩ . PGE 2 stimulation of EP2

Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-Daspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.Inhalational anesthetics have been in widespread use for more than 150 years and have been essential in the development of modern surgical procedures, but their molecular mechanisms have remained poorly understood. Early hypotheses based on nonspecific interactions of lipid-soluble anesthetics with the lipid bilayer of neuronal membranes have largely given way to the recent suggestion that anesthetics interact with multiple membrane-associated proteins involved in synaptic transmission (1-5). Inhibitory GABA A and glycine receptors and excitatory N-methyl-D-aspartate (NMDA), 1 nicotinic acetylcholine, and serotonin receptors have been demonstrated as possible physiological targets that underlie general anesthesia (6). Inhalational anesthetics have been shown to both enhance inhibitory receptor-mediated synaptic neurotransmission and depress excitatory receptor-mediated synaptic neurotransmission.Recent studies have revealed a much more complicated picture of excitatory receptor-mediated synaptic transmission than previously anticipated. For efficient synaptic transmission, the downstream effectors are targeted to the receptors by scaffolding proteins via a complex network of protein-protein interactions (7). The PDZ domain is one of the most common protein-protein recognition modules that have been found in diverse scaffolding and signaling proteins (8, 9). The name PDZ derives from the first three proteins (PSD-95/SAP90, Dlg, and ZO-1 (10)) in which these domains were identified. The PDZ domain recognizes specific C-terminal motifs found in target proteins, most often in the cytoplasmic tails of transmembrane receptors and channels (10, 11). The PDZ domain can also recognize structure-related internal motifs to form homo-and heteromeric PDZ-PDZ interactions (12-15). Therefore, PDZ domain-mediated protein-protein interactions provide a framework for the assembly of multiprotein signaling complexes at synapses and neuromuscular junctions. These interactions coordinate and guide the flow of regulatory information and regulate receptor and ion channel activities (16 -19).One of the best understood PDZ domain proteins at synapses is PSD-95, a modula...

Prostaglandin E 2 (PGE 2 ) is produced at high levels in the injured central nervous system, where it is generally considered a cytotoxic mediator of inflammation. The cellular actions of PGE 2 are mediated by G-protein signaling activated by prostanoid receptors termed EP 1 , EP 2 , EP 3 and EP 4 . Recent studies have implicated the EP 2 prostanoid receptor in apparently conflicting roles promoting neuronal death in some model systems and the survival of neurons in others. Here we show that treatment of immortalized human microglia and CCF-STTG1 astrocytes with either PGE 2 or the EP 2 selective agonist butaprost stimulates the release of brain-derived neurotrophic factor (BDNF). Both cell lines express mRNA for the EP 2 receptor, whereas transcripts for the other subtypes are not detected. Pharmacological studies using PGE 2 and modulators of cyclic AMP signaling implicate this pathway in PGE 2 -stimulated BDNF release. These results indicate that EP 2 prostanoid receptor activation induces BDNF secretion through stimulation of cyclic AMP dependent signaling. Our findings provide a mechanism by which endogenous PGE 2 might contribute to either neurotoxicity or neuroprotection in the injured brain via the induction of BDNF release from microglial cells and astrocytes. KeywordsProstaglandin E 2 ; microglia; astrocytes; BDNF; G-protein coupled receptor; brain derived neurotrophic factor; cyclic AMP Prostaglandin E 2 (PGE 2 ) is a prominent lipid autocrine/paracrine signaling mediator produced by the sequential metabolism of arachidonic acid by cyclooxygenase (COX) and PGE 2 synthase. PGE 2 is a major agent in local intercellular signaling associated with inflammation, pain, fever and immune responses (reviewed by Matsuoka & Narumiya, 2007;Khanapure et al., 2007;Harris et al., 2002). The cellular actions of PGE 2 are mediated by the activation of a group of G-protein coupled receptors that includes subtypes termed the EP 1 , EP 2 , EP 3 and EP 4 prostanoid receptors. These receptor subtypes are distinguished from one another by their selectivity for different ligands and by their unique coupling to intracellular signaling #Corresponding author (E-mail: regan@pharmacy.arizona.edu). * These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Funk et al., 1993) while stimulation of the EP 2 receptor promotes the production of intracellular cyclic AMP (Regan et al., 1994a). Activation of the EP 3 receptor, which is expressed as multiple isoforms through alternative mRNA splicing, inhibits cyclic AMP signaling (Regan et al., 1994b) ...

Tissue engineering is a possible method for long-term repair of cartilage lesions, but current tissue-engineered cartilage constructs have inferior mechanical properties compared to native cartilage. This problem may be due to the lack of an oriented structure in the constructs at the microscale that is present in the native tissue. In this study, we utilize contact guidance to develop constructs with microscale architecture for improved chondrogenesis and function. Stable channels of varying microscale dimensions were formed in collagen-based and polydimethylsiloxane membranes via a combination of microfabrication and soft-lithography. Human mesenchymal stem cells (MSCs) were selectively seeded in these channels. The chondrogenic potential of MSCs seeded in these channels was investigated by culturing them for 3 weeks under differentiating conditions, and then evaluating the subsequent synthesized tissue for mechanical function and by type II collagen immunohistochemistry. We demonstrate selective seeding of viable MSCs within the channels. MSC aligned and produced mature collagen fibrils along the length of the channel in smaller linear channels of widths 25-100 mm compared to larger linear channels of widths 500-1000 mm. Further, substrates with microchannels that led to cell alignment also led to superior mechanical properties compared to constructs with randomly seeded cells or selectively seeded cells in larger channels. The ultimate stress and modulus of elasticity of constructs with cells seeded in smaller channels increased by as much as fourfolds. We conclude that microscale guidance is useful to produce oriented cartilage structures with improved mechanical properties. These findings can be used to fabricate large clinically useful MSC-cartilage constructs with superior mechanical properties.

The peptide editor HLA-DM (DM) catalyzes the exchange of peptides bound to MHC class II molecules within antigen presenting cells by generating a “peptide-receptive” MHC class II conformation (MHCreceptive) to which peptides readily bind and rapidly unbind. While recent work has uncovered the determinants of DM recognition and effector functions, the nature of MHCreceptive and its interaction with DM remains unclear. Here, we show that DM induces but does not stabilize MHCreceptive in the absence of peptides. We demonstrate that DM is out-competed by certain superantigens, and increasing solvent viscosity inhibits DM-induced peptide association. We suggest that DM mediates peptide exchange by interacting transiently and repeatedly with MHC class II molecules, continually generating MHCreceptive. The simultaneous presence of peptide and DM in the milieu is thus crucial for the efficient generation of specific peptide-MHC class II complexes over time.

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