Proteolytische Freisetzung von NKG2D Liganden durch Tumorzellen

The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumour initiation by activating NK cells and costimulating CD8 T cells. MICA and other ligands of NKG2D are frequently expressed by tumour cells. Human tumour cells are thought to avert NKG2D-mediated-immunosurveillance by shedding MICA and other NKG2D ligands (NKG2DL). This thesis shows that the GPI-anchored NKG2DL ULBP2 is released from the cell surface of tumour cells by the action of metalloproteases similarly to the type I transmembrane proteins MICA and MICB. In addition, soluble ULBP2 was detected in the serum of patients with hematopoetic malignancies. Shedding of MICA and ULBP2 from tumours was induced by activation of protein kinase C (PKC) and was inhibited by the same compounds suggesting that ULBP2 and MIC molecules are released by the same or closely related proteases. Further, the molecular mechanisms of MICA shedding were defined to characterise the proteases involved. Amino acid deletions in the membrane-proximal stalk region of the MICA ectodomain greatly impaired MICA shedding, whereas amino acid substitutions had no significant effect. Further, MICA shedding was blocked by specific inhibitors of “a disintegrin and metalloprotease” (ADAM) proteases and was markedly reduced when ADAM10 and/or ADAM17 were down-regulated by RNA-interference. Altogether, these data demonstrate that ADAM10 and ADAM17 are critically involved in the proteolytic release of soluble MICA by tumours and thereby likely contribute to tumour immune evasion. Therefore, therapeutic blockade of ADAM10 and ADAM17 activities may represent a novel attractive approach to improve the efficacy of immunotherapeutic cancer treatment.NKG2D ist ein aktivierender NK-Zell Rezeptor und ein kostimulierender Rezeptor auf CD8 T Zellen. Die Expression der NKG2D Liganden (NKG2DL) wird durch Zellstress, virale Infektion und im Zuge maligner Transformation induziert und ermöglicht so dem Immunsystem die Erkennung und Elimination von veränderten und potentiell „gefährlichen“ Körperzellen (z. B. Tumoren). Die Freisetzung von löslichen NKG2D Liganden durch Metalloproteasen wird als ein wichtiger Mechanismus der Tumorzellen zur Vermeidung einer Immunantwort erachtet. In dieser Arbeit konnte gezeigt werden, dass auch der NKG2DL ULBP2, wie z. B. die MIC-Moleküle MICA und MICB, in löslicher Form von der Zelloberfläche von Tumorzellen freigesetzt wird. Lösliches ULBP2 konnte im Serum von Leukämie-Patienten nachgewiesen werden. Die Tumor-assoziierte Freisetzung sowohl von ULBP2 als auch von MICA ließ sich durch Aktivierung der Protein Kinase C verstärken und durch die gleichen Inhibitoren blockieren. Dies führte zu der Schlussfolgerung, dass sowohl die MIC Moleküle als auch ULBP2, als Vertreter der GPI-verankerten ULBPs, durch die gleichen oder nah verwandte Proteasen freigesetzt werden. Zur Identifizierung dieser Proteasen wurde beispielhaft der NKG2D Ligand MICA gewählt. Es konnte gezeigt werden, dass MICA in der Stielregion, die sich zwischen der Transmembranregion und der Ektodomäne befindet, gespalten wird. Wichtig für die Freisetzung ist nicht die Aminosäuresequenz, sondern die Länge dieser Stielregion. Des Weiteren konnte die Bildung von löslichem MICA durch Inhibitoren, die spezifisch gegen Mitglieder der ADAM (eine Disintegrin und Metalloprotease) Proteasen gerichtet sind, verringert werden. Die aufgrund dieser Experimente getätigte Annahme, dass ADAM10 und ADAM17 an der Freisetzung von löslichem MICA beteiligt sind, konnte in zwei unabhängigen Versuchsansätzen durch transiente Expressionssuppression dieser ADAMs in zwei Tumorzelllinien bestätigt werden. Zusammenfassend konnte gezeigt werden, dass die Metalloproteasen ADAM10 und ADAM17 an der Freisetzung von löslichem MICA von Tumorzellen beteiligt sind und somit einen interessanten Ansatzpunkt zur Verbesserung der Immuntherapien von Tumoren bieten

Spencer's cave :an Adirondack anomaly

This Report is a morphological and mineralogical survey of Spencer's Cave in Essex County, New York. Discovered in 1978 by Ranger Spencer Cram of the New York State Department of Environmental Conservation (DEC), this small cave is situated in a geologically complex area of anorthosites, syenites, gneisses, calc-silicates, and metasediments. Spencer's Cave is a water carved feature in a body of white, green, or blue marble containing small amounts of augite, magnetite, and many diverse members of the diopside-hedenbergite solid solution series. The rocks surrounding the marble body are garnetiferous and represent the effects of regional and contact metamorphism, assimilation, and metasomatism. Formation of the cave was probably controlled by differential dissolution along a zone of fracture in Grenville metasediments.No embarg

Zur Anthropologie der Liven : Inaugural-Dissertation zur Erlangung des Grades eines Doctors der Medicin / verfasst und mit Bewilligung Einer Hochverordneten Medicinischen Facultät der Kaiserlichen Universität zu Dorpat zur öffentlichen Verteidigung bestimmt von Ferdinand Waldhauer

http://www.ester.ee/record=b4115737*es

Metropolitan food supply: commitment mission Egypt

The present study is to investigate the commitment of potential participants to contribute to these opportunities and what their conditions are to the 3 main activities

Metropolitan food supply in Egypt : hydroponics production of leafy vegetables

This study incorporates the follow up activities of the two earlier missions of Wageningen UR/Food & Biobased Research (FBR) to Egypt, the exploration mission of 2013 (Broek and Boerrigter, 2014a) and the commitment mission of 2014 (Broek, Boerrigter and Waldhauer, 2014b), targeting the improvement of food security in Egypt in general and the reduction of post-harvest losses in particular

Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform

NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform

BBF RFC 105: The Intein standard - a universal way to modify proteins after translation

This Request for Comments (RFC) proposes a new standard that allows for easy and flexible cloning of intein constructs and thus makes this technology accessible to the synthetic biology community