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Entstehung und Funktion von Peptid Endocannabinoiden

English title Origin and Functional Roles of Peptide Endocannabinoids
Applicant Gertsch Jürg
Number 141174
Funding scheme Project funding (Div. I-III)
Research institution Institut für Biochemie und Molekulare Medizin Universität Bern
Institution of higher education University of Berne - BE
Main discipline Pharmacology, Pharmacy
Start/End 01.05.2012 - 30.04.2015
Approved amount 391'672.00
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All Disciplines (4)

Discipline
Pharmacology, Pharmacy
Biochemistry
Organic Chemistry
Cellular Biology, Cytology

Keywords (7)

Endocannabinoid System; Monocytes; Macrophages; Hemoglobin; Novel Peptides; Receptor Pharmacology; Cannabinoid Receptors

Lay Summary (English)

Lead
Lay summary
The endocannabinoid system (ECS) is part of a fundamental regulatory lipid network that controls homeostasis in health and disease. The ECS comprises arachidonic acid-derived endogenous activators (agonists) and the cannabinoid receptors CB1 and CB2. While CB1 receptors are the most widely expressed G protein-coupled receptors in the brain where they modulate neurotransmission (e.g., pain perception and locomotion), in the periphery such as in liver they regulate lipogenesis and contribute to body weight. CB2 receptors are believed to be part of a protective system as they can resolve chronic inflammation in numerous tissues. CB2 receptors are interesting as drug target because they are mainly expressed in the periphery where they can attenuate inflammatory pain signals and also modulate bone mass. Both receptors have been suggested to play a role in cancer. In this project we will study a novel group of endogenous molecules that apparently specifically bind to CB receptors. These molecules are peptides derived from alpha hemoglobin (which is not only expressed in red blood cells) and we named them Pepcans. Preliminary data show that Pepcans are tissue-specifically expressed and found in the brain and in blood. Since nothing is known about the expression and function of Pepcans we will first generate the necessary biochemical tools (monoclonal antibodies, methods of quantification, etc.) to study the potential role of Pepcans in the ECS. The project addresses questions about the interaction of Pepcans with CB receptors (where do they bind) and what do they do in the body (how do they modulate CB receptors and what does that mean in a physiological context)? Ultimately, we try to elucidate whether peptide ligands play a major role in the ECS. Our group is collaborating with different other research groups worldwide.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.
Gachet Maria Salome, Rhyn Peter, Bosch Oliver, Quednow Boris, Gertsch Jürg (2014), A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma., in J Chromatogr B Analyt Technol Biomed Life Sci., 976-977, 6-18.
Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.
Bauer Mark, Gertsch Jürg (2012), Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors., in Journal of Biological Chemistry, 36944-36967.
Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla
Stefanie C. Hofera1 William T. Ralveniusb1 M. Salomé Gacheta Jean-Marc Fritschyb Hanns Ulrich Ze, Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla, in Neuropharmacology.

Collaboration

Group / person Country
Types of collaboration
Prof. Dr. Ullrich Zeilhofer Switzerland (Europe)
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Dr. Gerd Pluschke Switzerland (Europe)
- Publication
- Exchange of personnel
Prof. Dr. Beat Lutz Germany (Europe)
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
8th international metabolomics course Talk given at a conference LC-MS/MS for the quantification of arachidonic acid and related lipids 17.04.2015 Leiden, Netherlands Gachet Otanez Maria Salomé;
Pharmacology Meeting Dottorato in Scienza del Farmaco e delle Sostanze Bioattive Talk given at a conference Drug Discovery in the Endocannabinoid System high hopes for new therapies 27.04.2013 Pisa, Italy Gertsch Jürg;
Methods in Experimental and Clinical Neuroscience Talk given at a conference Endocannabinoid membrane transport - controversies and new insights 18.01.2013 Bern, Switzerland Gertsch Jürg;
ICRS Congress Freiburg Poster IDENTIFICATION OF A FAMILY OF PEPTIDE ENDOCANNABINOIDS (PEPCANS): EVIDENCE FOR ALLOSTERIC MODULATION OF CB1 RECEPTORS 22.06.2012 Freiburg, Germany Gachet Otanez Maria Salomé; Bauer Mark Tobias; Gertsch Jürg;


Self-organised

Title Date Place
1st Swiss Endocannabinoid Pharmacology Meeting 17.10.2014 Bern, Switzerland

Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved
Novartis Seminar CB2 Receptor Modulation : Novartis Institutes of Biomedical Research 13.06.2013 Basel, Switzerland Gertsch Jürg;


Communication with the public

Communication Title Media Place Year

Associated projects

Number Title Start Funding scheme
120672 Function and Regulation of Cannabinoid Receptors in Primary Monocytes and Osteoclasts 01.05.2008 Project funding (Div. I-III)
120672 Function and Regulation of Cannabinoid Receptors in Primary Monocytes and Osteoclasts 01.05.2008 Project funding (Div. I-III)
163359 Entstehung und Funktion von Peptid Endocannabinoiden 01.01.2016 Project funding (Div. I-III)
163359 Entstehung und Funktion von Peptid Endocannabinoiden 01.01.2016 Project funding (Div. I-III)

Abstract

Cannabinoid receptors are G protein-coupled receptors (GPCRs) that are expressed in the central nervous system (CB1 receptor) and in peripheral tissues (CB1 and CB2 receptors). They act via Gai/o pathways, depending on the cell type where they are expressed (neurons, phagocytes, hepatocyte, etc.). In vivo, CB receptors are activated by arachidonic acid-derived endocannabinoids, such as N-arachidonoylethanolamine (anandamide or AEA) and 2-arachidonoylglycerol (2-AG) and other putative minor endocannabinoids like noladin ether and N-arachidonoyl dopamine. To date, these lipid endocannabinoids are regarded as the only genuine endogenous ligands for CB receptors. In the presented project we will challenge this view and explore whether a novel family of peptide endocannabinoids (here called Pepcans), originating from alpha hemoglobin, play a role in CB receptor signaling.The present project is dedicated to the study of the generation and functional roles of novel peptide endocannabinoids (here termed Pepcans) from alpha hemoglobin that we have unambigously characterized in our laboratory in a preliminary explorative project. Although the existence of such peptides has already proposed by two research groups (vide infra), biochemical tools (i.e. monoclonal Abs) have been lacking to study (identification and quantification) these peptides in a cellular context. Continuing our ongoing research on macrophage biology and CB receptor pharmacology (specificity and promiscuity of natural and synthetic CB receptor ligands) we became interested in the peptide hemopressin (PVNFKLLSH, Hpa), which was proposed to be an endogenous antagonist (inverse agonist) of the CB1 receptor. In a preliminary project we were able to generate the first suitable monoclonal antibodies against the C-terminal part of Hpa. Our data show that Hpa is not detected in biological tissues, but a whole family of N-terminal extended variants (the Pepcans). We currently have two high-affinity monoclonal antibodies against the C-terminal part of RVDPVNFKLLSH recognizing two epitopes. Using immunoaffinity MS/MS experiments and Western blots these antibodies enabled us to detect, identify and quantify RVDPVNFKLLSH and related N-terminal extended peptides in mouse brain, human blood plasma and cerebrospinal fluid by competitive ELISA. We could show that these peptides differentially interact with CB1 and CB2 receptors at novel binding sites.To understand whether these peptides are present in different tissues under physiological and/or pathophysiological conditions we will establish the necessary analytical extraction protocols for optimal Pepcan quantification. Making use of a suitable fluorescently labeled peptide (Pepcan-F4) and conventional radioligand binding assays we have already obtained evidence that these peptides bind to a binding site distinct from the endocannabinoid binding site in CB1 and CB2 receptors. We are now motivated to generate the first antagonists for the novel Pepcan binding sites. In collaboration with other groups we further intend to screen sample tissues of healthy and disease states in order to elucidate whether the peptides are significantly overexpressed (regulated) under certain conditions and whether this correlates with lipid endocannabinoid levels that we can now also measure in our laboratory with GC/MS.A major aim of the project is to study the interaction of Pepcans with CB receptors in more detail. In particular, we want to address the question whether they bind to allosteric receptor sites (in a functional sense) from the classical endocannabinoids and modulate endocannabinoid action. Using heterologous CB1 and CB2 receptor expression systems (CHO-K1, HEK293, cancer cell lines, but also primary cells (PBMCs), we will characterize the signaling of these peptides. To date, almost nothing is known about CB receptor signal transduction by RVDPVNFKLLSH (Pepcan-12) in cells other than neurons, such as immune cells like monocytes/macrophages, and nothing is know about the effects of N-terminal extended peptides identified in our lab. We will measure cAMP, intracellular calcium and kinases to characterize the peptides as full or partial agonists or positive or negative allosteric modulators or silent. Based on our preliminary data, the cellular effects of Pepcans will be assessed on different immune cells (Th1/Th2 cytokine modulation). We will study whether this peptides are like lipid endocannabinoids (additive, synergistic) or different, and how they can modulate inflammatory stimuli and macrophage differentiation (osteoclastogenesis, foam cells generation, see preliminary data). To address the specificity of the CB receptor binding interactions we will profile the peptides on other receptors available in our laboratory, such as TRPV1, GPR55, GABAA and PPAR-gamma. Moreover, we are interested in the site of production of Pepcans in different cells, in particular in neurons and macrophages. We will address the hypothesis that alpha hemoglobin may be taken up by CD163 into macrophages where it could be enzymatically processed to realese the peptide cannabinoids. Alternatively, the hemoglobin is proteolytically processed outside the cells via soluble proteases. A second model is that alpha hemoglobin is expressed in cells (HBA1 gene expression) and the Pepcans are generated in situ. With our well characterized monoclonal antibodies we will study the processing (generation and transport) of Pepcans in neuronal and immune cells. We want to understand how (if) this peptides are transported out of the cell where it is generated and whether it is taken up by cells that express CB receptors and ultimately how it is degraded and by which proteases/peptidases. The tools established in our laboratory should be highly suitable to address these emerging questions. Since Pepcans are likely to be actively transported by an already known polypeptide transporter, in a later stage we want to elucidate the transporter using assays with cell lines of different origin. In addition to fluorescently labeled peptides will radiolabel the major peptide for CB receptor and transport studies.
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