T-Zell-Subtypen im Fettgewebe - Die Beziehung zwischen Fettgewebsinflammation und intestinaler Inflammation bei Patienten mit Morbus Crohn und Adipositas per magna

Einleitung: Die Inzidenz von Adipositas und Morbus Crohn (MC) steigt weltweit an. Beide Erkrankungen zeigen eine Inflammation des viszeralen Fettgewebes (VF). Im hypertrophierten VF adipöser Patienten besteht eine chronische niedriggradige Entzündung, die mit der Entwicklung einer Insulinresistenz assoziiert ist. Zudem ist bei Adipositas die intestinale Barrierefunktion gestört und bei gleichzeitig bestehendem MC besteht ein schwererer Krankheitsverlauf. Beim MC wurde das pathognomonische creeping fat (CF) um entzündete Darmanteile herum durch Burrill B. Crohn identifiziert. Die Funktion dieses Gewebes aus hyperplastischen Adipozyten und infiltrierenden Immunzellen bleibt ungeklärt. Protektive sowie entzündungsaggravierende Funktionen werden diskutiert. In dieser Arbeit wurden T-Zell-Subpopulationen im Fettgewebe von Patienten mit Adipositas per magna und chronisch entzündlichen Darmerkrankungen (CED) untersucht. Dabei wurde auch ein Fokus auf die Expression des Darm-spezifischen homing-Integrins α4β7 gelegt. Methoden: Fettgewebs- und (bei CED) Mukosaproben wurde in krankheitsassoziierten Operationen von 47 Patienten entnommen. Die Gewebeproben wurden teils fixiert und immunhistochemisch untersucht oder daraus Immunzellen isoliert, gefärbt und durchflusszytometrisch analysiert. In Fettgewebsüberständen wurde mittels cytometric bead array Zytokine quantifiziert. Ergebnisse: Im VF exprimierte ein signifikant höherer Anteil der T-Zellen α4β7 als im subkutanen Fettgewebe bei vorherrschendem pro-inflammatorischen T-Zell-Milieu. Das ileale und colonische CF bei MC-Patienten unterschied sich makrokopisch sowie histologisch, wobei verkleinerte Adipozyten und eine hohe absolute T-Zell-Infiltration nur im ilealen CF zu finden waren. Das CF des Colons ähnelte stärker dem mesocolischen Fett von Colitis ulcerosa (CU)-Patienten. Es zeigten sich distinkte T-Zell-Populationen im Fettgewebe, wobei das ileale CF durch einen höheren Anteil von regulatorischen T-Zellen (Treg) und central-memory T-Zellen hervorstach. Zudem unterschieden sich die T-Zell-Subgruppen zwischen ilealem CF und Mukosa. Eine Korrelation zwischen im Fett ansässigen T-Helferzellen und Treg mit der Krankheitsaktivität war invers bei Patienten mit ilealem und colonischen MC. Das VF adipöser Patienten beherbergte einen höheren Anteil pro-inflammatorischer T-Zellen als das ileale CF bei MC, zudem exprimierten mehr T-Zellen α4β7 als im CF. Schlussfolgerungen: Bei Patienten mit Adipositas finden sich im VF α4β7+ T-Zellen in einem überwiegend pro-inflammatorischen Mileu. Diese Zellen könnten durch intestinales homing an der Barrierestörung bei Adipositas und der Krankheitsaggravierung bei gleichzeitigem MC beteiligt sein. Das ileale CF von MC-Patienten zeigte eine ausgeglichenere T-Zell-Infiltration im Vergleich zum VF adipöser Patienten. Interessanterweise unterschieden sich ileales und colonisches CF makroskopisch, histologisch sowie hinsichtlich der T-Zell-Zusammensetzung und deren Korrelation mit der Krankheitsaktivität. Diese Erkenntnisse unterstützen genetische Daten, welche die Crohn-Colitis als eigene Entität zwischen ilealem MC und CU ansiedeln und sprechen dafür, dass verschiedene Ausprägungsformen des CF im Rahmen des MC existieren.Introduction: Obesity and Crohn´s disease (CD) respresent diseases increasing worldwide. Both involve an inflammation of visceral adipose tissue (VAT). In hypertrophic, obese VAT, a low-grade inflammation has been associated with insulin resistance. Observations in obese patients imply an impairment of intestinal barrier function and an altered disease course in obese CD patients. In CD patients a pathognomonic fat wrapping occurs around inflamed regions of the intestine, the so-called creeping fat (CF). However, the exact function of this tissue, consisting of a hyperplasia of small-sized adipocytes and infiltrating leukocytes, remains unsolved. Both a protective and a contributing role towards intestinal inflammation are discussed. This study characterized T-cell subpopulations in VAT from obese patients as well as adipose tissue and adjacent mucosa in inflammatory bowel disease (IBD) patients. The study also focuses on expression of the gut-homing integrin α4β7. Methods: VAT (and in IBD patients mucosal) samples were obtained from 47 patients undergoing disease-related surgery. Fractions were fixed and stained for immunohistochemistry. Immune cells were isolated, stained and analyzed by flow cytometry. Cytometric bead array (CBA) was performed to quantify cytokine release in adipose tissue supernatants. Results: In obese VAT a high proportion of T-cells expressed integrin α4β7, significantly more than in subcutaneous adipose tissue. VAT was dominated by a pro-inflammatory T-cell milieu. Macroscopic differences were found between colonic and ileal CF. Histology showed significantly smaller adipocytes and a high absolute T-cell infiltration only in ileal CF. Colonic CF resembled fat from UC patients. T-cell populations differed between IBD groups, ileal CF standing out by a higher amount of regulatory T-cells (Treg) and central memory T-cells. Furthermore, T-cells in ileal CF and mucosa consisted of distinct subgroups. Correlation of adipose tissue T-helper cell and Treg share with disease activity was inverse in ileal and colonic CF. When comparing obese VAT with ileal CF, VAT showed a higher amount of pro-inflammatory T-cells, also significantly more T-cells were α4β7-positive. Conclusion: In obese VAT α4β7+ T-cells are present in a pro-inflammatory milieu. These cells could be involved in intestinal barrier impairment and exacerbation of CD in obese individuals. Ileal CF from CD patients presents a more balanced T-cell infiltration compared to VAT in obese individuals. Interestingly, ileal and colonic CF differ regarding macroscopic and histological aspects, composition of T-cells and correlation with disease activity. This supports genetic data placing colonic CD between ileal CD and UC

ELM—the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instance

Structural and functional characterisation of peptide-ligand G protein coupled receptors : examples from the chemoattractant cluster

G protein coupled receptors (GPCRs) constitute the largest family of cell-surface receptors in mammals and are key players in signal transduction. By responding to a plethora of extracellular stimuli ranging from photons to amines to fatty acids to peptides and proteins, these receptors trigger intracellular signalling cascades and regulate a variety of cellular responses. Approximately 800 genes in humans encode GPCRs which are classified according to sequence conservation into rhodopsin-like, glutamate, adhesion, frizzled/taste2 and secretin receptors. GPCRs share a seven transmembrane domain fold undergoing a conformational change upon ligand binding which is translated to the intracellular surface of the receptor thereby allowing a heterotrimeric G protein to couple. Heterotrimeric G proteins consist of a Ga, Gb and Gg subunit and dissociate into their Ga and Gbg entities upon activation by a GPCR. Subsequently, distinct signalling cascades are triggered by each G protein protomer. Membrane proteins and GPCRs in particular, are highly important targets in drug design and development as currently approximately 60% of all marketed drugs target membrane proteins. Although these classes of proteins are of high therapeutic interest, our understanding of their mechanism of action and structure remains limited. The first structure of a human GPCR was determined in 2007 and required the development of protein engineering and innovative crystallisation techniques. Since then, approximately 130 GPCR structures of less than 40 individual receptors have been determined providing insights into the structural arrangement of the transmembrane helices, ligand binding pockets and G protein interactions. Combined with spectroscopic methods, these studies allowed a more detailed understanding of the molecular aspects of GPCR activation and signalling. Despite the tremendous advances in GPCR structural biology, certain aspects of GPCR function still remain poorly understood. Due to their size and inherent flexibility, the interaction of protein and peptide ligands with their receptors remains a challenging aspect in the structural characterisation of GPCRs. Moreover, structural information on subtype selectivity of peptide ligands continues to be scarce. To contribute functional and structural information on the molecular mechanisms of peptide interactions with GPCRs, this thesis focused on characterising receptors from the chemoattractant cluster using radioligand binding assays as well as NMR spectroscopy. The chemoattractant cluster mainly groups the kinin, angiotensin, anaphylatoxin chemotactic complement and apelin receptors according to conserved residues in their ligand binding cavities. All receptors in this cluster bind to peptide ligands deriving from high molecular weight protein precursors upon proteolytic processing. Comparable to the conserved binding pocket of the chemoattractant receptors, the peptide ligands display a certain sequence conservation although they differ strongly in size. The largest ligands used in this thesis are the anaphylatoxins complement 3a and 5a, comprising 77 or 74 residues, respectively. Due to their size and complex fold involving three intramolecular disulphide bonds, solid phase synthesis is impossible, which prompted us to develop a modified cell-free expression system to produce these ligands in tritiated form for subsequent functional characterisation of the complement receptors. To demonstrate the versatility of the developed system, it was applied to another disulphidebond containing peptide ligand, the 21 amino acid endothelin-1. We describe a reliable and multifaceted tool to generate custom labelled peptide ligands for the structural and functional characterisation of GPCRs. The system allows the production of custom radioligands, peptides labelled for NMR studies or with fluorescent amino acids. Apart from the modulation of GPCR activity by orthosteric ligands, GPCR signalling has long been described to be regulated by allosteric ligands including peptides, small molecules and ions. In this thesis, the influence of sodium ions on the activity state of the chemoattractant cluster receptors and in particular on the apelin, bradykinin 2 and angiotensin II type 1 receptors was examined. In recent high resolution crystal structures an allosteric sodium ion pocket beneath the orthosteric ligand binding cavity was identified and residues contributing to the coordination of sodium ions are conserved throughout the chemoattractant cluster receptors. This allosteric sodium ion coordinated within the transmembrane domain bundle has been described to negatively influence the affinity of agonists but not of antagonists. It was found that sodium ions have distinct influences on the affinity state as well as the available number of binding sites of the chemoattractant receptors. In case of the apelin and bradykinin 2 receptors, sodium ions drastically reduced the number of available binding sites whereas the affinity of peptide ligands to the bradykinin 2 receptors remained constant and the ligand binding affinities to the apelin receptor were completely abolished. In contrast, the angiotensin II type 1 receptor affinity state towards the endogenous peptide ligand angiotensin II is highly dependent on the presence of sodium ions, whereas binding of the synthetic peptide antagonist Sar1-Ile8-angiotensin II remained unaffected by the sodium ion concentration. As differential effects irrespective of the efficacy class but dependent on the amino acid composition of the applied ligands are observed, it can be concluded that electrostatic interactions between charged residues of the peptide ligands and amino acids on the extracellular surface of the receptors are influenced by sodium ions thereby adding another layer of complexity on GPCR signalling. To elucidate the structure-function relationship of ligand selectivity between the kinin receptors, the structure of desArg10-kallidin (DAK) bound to the bradykinin 1 receptor was determined using solid state NMR (SSNMR) in the course of this thesis. The kinin peptides DAK and bradykinin bind with high affinity and high selectivity to either the bradykinin 1 or bradykinin 2 receptor, respectively. The binding pockets of the receptors are highly conserved and the two peptide ligands only differ in one amino acid at their N- and C-termini whereas the remaining eight amino acids are fully conserved. DAK adopts a U-shaped structure when bound to the bradykinin 1 receptor which resembles a horse shoe-like conformation. Using 2D TEDOR spectroscopy it could furthermore be demonstrated that positively charged residues at the N-terminal part of the peptide engage in ionic interactions with negatively charged amino acids on the extracellular surface of the bradykinin 1 receptor. In contrast, bradykinin displays a distinct b-turn at the C-terminus and an S-shaped conformation of the N-terminal segment when bound to the bradykinin 2 receptor. By using SSNMR to study the binding mode of DAK on the bradykinin 1 receptor we could determine that subtype selectivity between the kinin receptors is conferred by distinct conformational restraints within the peptide ligands and by the formation of specific ionic interaction between charged residues on the peptide and receptor, respectively. In brief, this thesis contributes structural and functional data on the binding mechanisms and binding mode of different peptide-ligand GPCRs helping to understand subtype selectivity and allosteric modulation of the chemoattractant cluster receptors. In addition, a versatile cell-free expression system was developed that allows the custom synthesis of isotopically labelled peptides containing disulphide bonds for the functional characterisation of GPCRs.G Protein gekoppelte Rezeptoren (GPCR) repräsentieren die größte Klasse von Zelloberflächenrezeptoren in Säugetieren. Mit ungefähr 800 Genen, die im menschlichen Genom GPCR verschlüsseln, stellt diese Proteinklasse die wichtigste Familie an Signalüberträgern in der Zytoplasmamembran dar. GPCR werden durch eine Mannigfaltigkeit von extrazellulären Stimuli aktiviert, die Photonen, Amine, Fettsäuren, Peptide oder Proteine beinhalten können. Die Rezeptoren sind aus sieben Transmembransegmenten aufgebaut, welche durch Aktivierung des Rezeptors infolge der Bindung eines Liganden an die orthosterische Bindungstasche eine konformationelle Änderung erfahren, die die Kopplung eines intrazellulären heterotrimeren G Proteins erlaubt. Heterotrimere G Proteine bestehen aus einer Ga, Gb und Gg Untereinheit, die durch Aktivierung in ihre Ga und Gbg Bestandteile zerfallen. Diese wiederum aktivieren gezielt Signaltransduktionswege innerhalb der Zelle und regulieren so zelluläre Prozesse. Im Menschen werden GPCR aufgrund von Sequenzhomologien in fünf Unterfamilien klassifiziert: Glutamat-, Rhodopsin-ähnliche-, Adhäsions-, Frizzled/Taste2 und Secretin-Rezeptoren. Die am besten beschriebene GPCR Unterfamilie stellen die Rhodopsin-ähnlichen Rezeptoren dar, die auch Gegenstand dieser Arbeit sind. Membranproteine sind im Allgemeinen von hohem therapeutischem Interesse, da circa 60% aller zugelassenen Medikamente an dieser Proteinklasse wirken. Trotz ihrer Bedeutung für die Medikamentenentwicklung gestalten sich strukturelle Studien an Membranproteinen und im Speziellen an G Protein gekoppelten Rezeptoren aufgrund ihrer niedrigen natürlichen Expressionsrate, ihrer Instabilität und Flexibilität schwierig. Die erste Struktur eines humanen GPCR wurde im Jahr 2007 mittels Röntgenkristallographie bestimmt, wobei der Rezeptor mit Hinsicht auf eine Erhöhung der Expressionsrate, der Stabilität und der Formierung von Kristallkontakten stark modifiziert werden musste. Fortschritte in der Gestaltung der Expressionskonstrukte, der Wahl von Antiköpern zuro-Kristallisation sowie Entwicklungen technologischer Art führten seitdem zu ungefähr 130 GPCR Strukturen von nur weniger als 40 individuellen Rezeptoren. Diese haben fundamental dazu beigetragen die Architektur der Transmembransegmente und der Ligandbindetaschen sowie strukturelle Aspekte der G Protein Kopplung zu verstehen. Einer der am besten charakterisierten Rhodopsin-ähnlichen Rezeptoren ist der b2-adrenerge Rezeptor, für den es gelang Kristallstrukturen in verschiedenen Aktivitätszuständen zu bestimmen. Hierzu wurden Liganden verschiedener Effizienzklassen an den Rezeptor gebunden und dessen Struktur bestimmt. Hiermit konnte die hohe konformationelle Flexibilität der Rhodopsin-ähnlichen Rezeptoren demonstriert werden, da insbesondere die Transmembranhelices V und VI signifikant unterschiedliche Konformationen abhängig des Aktivierungszustandes des Rezeptors aufweisen. Um die Dynamik der GPCR Aktivierung besser verstehen zu können, wurden unterschiedliche spektroskopische Methoden angewendet, die es erlauben die konformationellen Änderungen der einzelnen Helices zu verfolgen. Trotz des immensen Fortschritts bei der strukturbiologischen Charakterisierung von G Protein gekoppelten Rezeptoren und verschiedener spektroskopischer und biochemischer Studien, bleiben einige Schlüsselkonzepte wie die Selektivität der Ligandenbindung an Rezeptorsubtypen, die Interaktion von Peptidliganden mit ihren Rezeptoren und der Einfluss allosterischer Modulatoren dennoch weitgehend unverstanden. In diesem Kontext setzte es sich diese Dissertation zum Ziel ausgewählte Peptidligand- GPCR aus der Chemoattraktanten Gruppe mittels Radioligand-Bindungsstudien, Kleinwinkelröntgenstreuung und Festphasen-Kernspinresonanzspektroskopie funktional sowie strukturell zu charakterisieren...

ELM--the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instances.Author has checked copyrightDG 09/11/12Names (!) JG 2012-11-2