Sequence features of HLA-DRB1 locus define putative basis for gene conversion and point mutations

<p>Abstract</p> <p>Background</p> <p>HLA/MHC class II molecules show high degree of polymorphism in the human population. The individual polymorphic motifs have been suggested to be propagated and mixed by transfer of genetic material (recombination, gene conversion) between alleles, but no clear molecular basis for this has been identified as yet. A large number of MHC class II allele sequences is publicly available and could be used to analyze the sequence features behind the recombination, revealing possible basis for such recombination processes both in HLA class II genes and other genes, which recombination acts upon.</p> <p>Results</p> <p>In this study we analyzed the vast dataset of human allelic variants (49 full coding sequences, 374 full exon 2 sequences) of the most polymorphic MHC class II locus, <it>HLA-DRB1</it>, and identified many previously unknown sequence features possibly contributing to the recombination. The CpG-dinucleotide content of exon 2 (containing the antigen-binding sites and subsequently a high degree of polymorphism) was much elevated as compared to the other exons despite similar overall G+C content. Furthermore, the CpG pattern was highly conserved. We also identified more complex, highly conserved sequence motifs in exon 2. Some of these can be identified as putative recombination motifs previously found in other genes, but most are previously unidentified.</p> <p>Conclusion</p> <p>The identified sequence features could putatively act in recombination allowing either less (CpG dinucleotides) or more specific DNA cleavage (complex sequences) or homologous recombination (complex sequences).</p

Cellular Signaling Mechanisms of Hypocretin/Orexin

Publisher Copyright: © 2021 The Author(s).Orexin receptors (OXRs) are promiscuous G-protein-coupled receptors that signal via several G-proteins and, putatively, via other proteins. On which basis the signal pathways are selected and orchestrated is largely unknown. We also have an insufficient understanding of the kind of signaling that is important for specific types of cellular responses. OXRs are able to form complexes with several other G-protein-coupled receptors in vitro, and one possibility is that the complexing partners regulate the use of certain signal transducers. In the central nervous system neurons, the main acute downstream responses of OXR activation are the inhibition of K+ channels and the activation of the Na+/Ca2+ exchanger and non-selective cation channels of unknown identity. The exact nature of the intracellular signal chain between the OXRs and these downstream targets is yet to be elucidated, but the Gq-phospholipase C (PLC) protein kinase C pathway - which is a significant signaling pathway for OXRs in recombinant cells - may be one of the players in neurons. The Gq-PLC pathway may also, under certain circumstances, take the route to diacylglycerol lipase, which leads to the production of the potent endocannabinoid (eCB), 2-arachidonoyl glycerol, and thereby connects orexins with eCB signaling. In addition, OXRs have been studied in the context of neurodegeneration and cancer cell death. Overall, OXR signaling is complex, and it can change depending on the cell type and environment.Peer reviewe

OX1 and OX2 orexin/hypocretin receptor pharmacogenetics

Orexin/hypocretin peptide mutations are rare in humans. Even though human narcolepsy is associated with orexin deficiency, this is only extremely rarely due to mutations in the gene coding prepro-orexin, the precursor for both orexin peptides. In contrast, coding and non-coding variants of the OX1 and OX2 orexin receptors have been identified in many human populations; sometimes, these have been associated with disease phenotype, although most confer a relatively low risk. In most cases, these studies have been based on a candidate gene hypothesis that predicts the involvement of orexins in the relevant pathophysiological processes. In the current review, the known human OX1/HCRTR1 and OX2/HCRTR2 genetic variants/polymorphisms as well as studies concerning their involvement in disorders such as narcolepsy, excessive daytime sleepiness, cluster headache, polydipsia-hyponatremia in schizophrenia, and affective disorders are discussed. In most cases, the functional cellular or pharmacological correlates of orexin variants have not been investigated-with the exception of the possible impact of an amino acid 10 Pro/Ser variant of OX2 on orexin potency-leaving conclusions on the nature of the receptor variant effects speculative. Nevertheless, we present perspectives that could shape the basis for further studies. The pharmacology and other properties of the orexin receptor variants are discussed in the context of GPCR signaling. Since orexinergic therapeutics are emerging, the impact of receptor variants on the affinity or potency of ligands deserves consideration. This perspective (pharmacogenetics) is also discussed in the review.Peer reviewe

Endocannabinoid Signaling in Embryonic Neuronal Motility and Cell-Cell Contact - Role of mGluR5 and TRPC3 Channels

Cell-cell communication plays a central role in the guidance of migrating neuronal precursor cells during the development of the cerebral cortex. Endocannabinoids (eCBs) have previously been shown to be one of the central factors regulating neuronal migration. In this study the effects of eCBs on different parameters, expected to affect embryonic cortical neuronal motility have been analyzed in neurosphere-derived neuroblasts using time-lapse microscopy. Increased endogenous production of the endocannabinoid 2-arachidonyl glycerol (2-AG) causes bursts of neuroblast motility. The neuroblasts move longer distances and show a low frequency of turning, and the number of neuron-neuron contacts are reduced. Similar changes occur interfering with the function of the metabotropic glutamate receptor 5 (mGluR5) or its transducer canonical transient receptor potential channel 3 (TRPC3) or the neuregulin receptor ErbB4. Blocking of 2-AG production reverses these effects. The data suggest that eCB-regulated neuronal motility is controlled by mGluR5/TRPC3 activity possibly via NRG/ErbB4 signaling. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Orexin receptor agonist Yan 7874 is a weak agonist of orexin/hypocretin receptors and shows orexin receptor-independent cytotoxicity

Two promising lead structures of small molecular orexin receptor agonist have been reported, but without detailed analyses of the pharmacological properties. One of them, 1(3,4-dichloropheny1)-242-imino-3-(4-methylbenzy1)-2,3-dihydro-1H-benzo[climidazol-1-yl] ethan-1-ol (Yan 7874), is commercially available, and we set out to analyze its properties. As test system we utilized human OX1 and OX2 orexin receptor -expressing Chinese hamster ovary (CHO) K1 cells as well as control CHO-K1 and neuro-2a neuroblastoma cells. Gq-coupling was assessed by measurement of intracellular Ca2+ and phospholipase C activity, and the coupling to G(i) and G(s) by adenylyl cyclase inhibition and stimulation, respectively. At concentrations above 1 pM, strong Ca' and low phospholipase C responses to Yan 7874 were observed in both OX1- and OX2-expressing cells. However, a major fraction of the response was not mediated by orexin receptors, as determined utilizing the nonselective orexin receptor antagonist N-biphenyl-2-y1-1-{[(1-methyl-1H-benzimidazol-2-y1) sulfanyl]acetyl}-L-prolinamide (TCS 1102) as well as control CHO-K1 cells. Yan 7874 did not produce any specific adenylyl cyclase response. Some experiments suggested an effect on cell viability by Yan 7874, and we thus analyzed this. Within a few hours of exposure, Yan 7874 markedly changed cell morphology (shrunken, rich in vacuoles), reduced growth, promoted cell detachment, and induced necrotic cell death. The effect was equal in cells expressing orexin receptors or not. Thus, Yan 7874 is a weak partial agonist of orexin receptors. It also displays strong off -target effects in the same concentration range, culminating in necrotic cell demise. This makes Yan 7874 unsuitable as orexin receptor agonist.Peer reviewe

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

AbstractWe wanted to analyze the basis for the distinction between OX1 and OX2 orexin receptors by the known agonists, orexin-A, orexin-B and Ala11, d-Leu15-orexin-B, of which the latter two show some selectivity for OX2. For this, chimaeric OX1/OX2 and OX2/OX1 orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca2+ elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor’s ligand profile

Azulene-based compounds for targeting orexin receptors

A library of 70 000 synthetically accessible azulene-based compounds was virtually screened at the OX2 receptor. Based on the results, a series of azulene derivatives was synthesized and the binding to and activation of both orexin receptor subtypes were assessed. Two most promising binders were determined to have inhibition constants in the 3-9 mu M range and two other compounds showed weak OX2 receptor agonism. Furthermore, three compounds exhibited a concentration-dependent potentiation of the response to orexin-A at the OX1 but not the OX2 receptors. Altogether this data opens new approaches for further development of antagonists, agonists, and potentiators of orexin response based on the azulene scaffold. (C) 2018 Elsevier Masson SAS. All rights reserved.Peer reviewe

Orexin-A measurement in narcolepsy : A stability study and a comparison of LC-MS/MS and immunoassays

Background: Orexin-A and-B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B. Methods: We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and-B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months. Results: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and-B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was <35 pg/mL (range <35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range <35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectable Conclusions: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS.Peer reviewe

Stapled truncated orexin peptides as orexin receptor agonists

The peptides orexin-A and -B, the endogenous agonists of the orexin receptors, have similar 19-amino-acid C-termini which retain full maximum response as truncated peptides with only marginally reduced potency, while further N-terminal truncations successively reduce the activity. The peptides have been suggested to bind in an α‐helical conformation, and truncation beyond a certain critical length is likely to disrupt the overall helical structure. In this study, we set out to stabilize the α‐helical conformation of orexin‐A15–33 via peptide stapling at four different sites. At a suggested hinge region, we varied the length of the cross-linker as well as replaced the staple with two α-aminoisobutyric acid residues. Modifications close to the peptide C‐terminus, which is crucial for activity, were not allowed. However, central and N‐terminal modifications yielded bioactive peptides, albeit with decreased potencies. This provides evidence that the orexin receptors can accommodate and be activated by α-helical peptides. The decrease in potency is likely linked to a stabilization of suboptimal peptide conformation or blocking of peptide backbone–receptor interactions at the hinge region by the helical stabilization or the modified amino acids.Peer reviewe

No evidence of autoimmunity to human OX1 or OX2 orexin receptors in Pandemrix-vaccinated narcoleptic children

Narcolepsy type 1, likely an immune-mediated disease, is characterized by excessive daytime sleepiness and cataplexy. The disease is strongly associated with human leukocyte antigen (HLA) DQB1∗06:02. A significant increase in the incidence of childhood and adolescent narcolepsy was observed after a vaccination campaign with AS03-adjuvanted Pandemrix influenza vaccine in Nordic and several other countries in 2010 and 2011. Previously, it has been suggested that a surface-exposed region of influenza A nucleoprotein, a structural component of the Pandemrix vaccine, shares amino acid residues with the first extracellular domain of the human OX2 orexin/hypocretin receptor eliciting the development of autoantibodies. Here, we analyzed, whether H1N1pdm09 infection or Pandemrix vaccination contributed to the development of autoantibodies to the orexin precursor protein or the OX1 or OX2 receptors. The analysis was based on the presence or absence of autoantibody responses against analyzed proteins. Entire OX1 and OX2 receptors or just their extracellular N-termini were transiently expressed in HuH7 cells to determine specific antibody responses in human sera. Based on our immunofluorescence analysis, none of the 56 Pandemrix-vaccinated narcoleptic patients, 28 patients who suffered from a laboratory-confirmed H1N1pdm09 infection or 19 Pandemrix-vaccinated controls showed specific autoantibody responses to prepro-orexin, orexin receptors or the isolated extracellular N-termini of orexin receptors. We also did not find any evidence for cell-mediated immunity against the N-terminal epitopes of OX2. Our findings do not support the hypothesis that the surface-exposed region of the influenza nucleoprotein A would elicit the development of an immune response against orexin receptors. © 2020 The AuthorsPeer reviewe