The search for novel mRNAs targeted by the RNA-destabilizing protein tristetraprolin for degradation

Genexpression ist auf den Ebenen der Transkription, mRNA Stabilität und Translation reguliert. Die Stabilität der mRNA wird in den meisten Fällen von regulatorischen Elementen, welche sich in der 3´ untranslatierten Region der mRNA befinden, reguliert. Die am besten charakterisierten Elemente sind die sogenannten Adenin-Uridin-reichen Elemente (ARE). AREs werden von RNA-bindenden Proteinen erkannt, welche stabilisierende oder destabilisierende Effekte bewirken können. Eines dieser Proteine ist Tristetraprolin (TTP), welches dafür bekannt ist Entzündungsreaktionen durch die Beschleunigung von Cytokin mRNA Abbau zu unterdrücken. In dieser Arbeit beschreiben wir drei neue mRNAs wessen Abbau von TTP reguliert wird. Durch Untersuchung von primären Makrophagen und Reporter Systemen zeigen wir, dass TTP die mRNAs von IL1α, IL6 und Cxcl2 binden und destabilisieren kann. Zusätzlich zeigen wir, dass die Expression eines Luziferase-Reporters in Anwesenheit von TTP deutlich reduziert wird wenn die Luziferase mRNA an die 3´ UTR von IL1α, IL6, IL12β und Cxcl2 gekoppelt ist. Einige der identifizierten Ziele von TTP, wie VEGF, COX-2 und IL3, sind essentiell an der Entstehung und dem Fortschreiten von Tumoren beteiligt. In dieser Arbeit beschreiben wir die Rolle von TTP in der Entwicklung von v-abl bedingtem B-Zell Lymphom. Zellen, welche von TTP-/- Mäusen isoliert wurden, zeigen eine erhöhte Tendenz stabile Kolonien zu bilden nachdem das v-abl Onkogen eingebracht wurde. In Versuchen an lebenden Mäusen, welchen transformierte Zellen von Wildtyp und TTP-/- Mäusen injiziert wurden, bilden TTP -/- Zellen signifikant größere Tumore aus, was die Rolle von TTP als Tumor-Unterdrücker weiter untermauert.Gene expression is regulated at the level of transcription, mRNA stability and translation. The stability of mRNA is in most cases regulated by cis-acting regulatory elements located in the 3´ untranslated regions. The best characterized elements are the so called adenine-uridine-rich sequences (AREs). These AREs are recognized by RNA-binding proteins that may have stabilizing or destabilizing effects. One of these proteins is tristetraprolin (TTP) wich is known to suppress inflammation by accelerating the degradation of cytokine mRNAs. By promoting mRNA decay, TTP significantly contributes to cytokine homeostasis. In this diploma thesis three new TTP targets were characterized. This study shows that TTP can bind and destabilize the mRNA of IL1α, IL6 and Cxcl2 through assays performed with both primary macrophages and through rabbit β-globin reporter assays. Additionally, the presence of TTP significantly reduces the expression of a luciferase gene fused to the 3´ UTR of IL1α, IL6, Cxcl2 and IL12β in reporter assay experiments. Some of TTPs targets, such as VEGF, COX-2 and IL3, have been shown to play a fundamental role in the formation and progression of various cancers, including colon cancer and mast-cell carcinoma. In this thesis, an involvement of TTP in v-abl induced B-cell lymphoma formation is described. Cells isolated from TTP-deficient mice show an increased ability to form transformed cell lines after transduction with the v-abl oncogene. Subcutaneous injection of v-abl-transformed wild-type and TTP-/- B-cells into recipient mice show an increase in tumor size, further solidifying the hypothesis that TTP acts as a tumor suppressor

Enzymatic synthesis of antibody-human serum albumin conjugate for targeted drug delivery using tyrosinase from Agaricus bisporus

Highly specific targeted drug delivery devices can be obtained with antibody-human serum albumin (mAb-HSA) conjugates. However, their conventional production involves several reaction steps including chemical modification and activation of both proteins followed by cross-linking often involving toxic chemicals. Here, we describe the enzymatic synthesis of mAb-HSA conjugates for targeted drug delivery devices using tyrosinase from Agaricus bisporus under mild reaction conditions (pH 6.8, 25 [degree]C). Reaction conditions were optimized by using fluorescence labeled HSA to facilitate SDS-PAGE analysis with fluorescence scanning. Enzymatic cross-linking in the presence of natural low molecular weight phenolic compounds (e.g. caffeic acid) resulted in reaction products in the molecular weight range of [similar]216 kDa, corresponding to mAb-HSA conjugates. The composition of the conjugates was confirmed with tryptic digestion followed by LC-MS/MS analysis of the resulting peptide fragments. Successful binding of mAb-HSA conjugates (in contrast to free HSA) to MHC II molecules, located on antigen-presenting cells, was demonstrated by both ELISA and flow cytometry analysis.This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL and FWF, DK: Metabolic and Cardiovascular Disease: W1226-B18. We thank Tamara Reiter, Graz University of Technology for technical support with SEC; Exbio from the Czech Republic for providing the mAbs and Britta Obrist, Medical University of Graz and the Austrian Centre of Industrial Biotechnology, for technical assistance with LC-MS/MS analysis

Characterization of the protein microenviroment of the folate receptor beta

Marker für diese funktionell unterschiedlichen Makrophagen mithilfe eines bispezifischen Antikörpers zu verbessern. In weiterer Folge, haben wir die Informationen über die Proteinumgebung zur funktionellen Charakterisierung von FR genutzt. Als Resultat dieser Strategie haben wir eine funktionelle Interaktion zwischen FR und dem Integrin Heterodimer CD11b/CD18 entdeckt, welche in einer Konformationsänderung von CD11b/CD18 resultiert. Diese Interaktion verringert die Fähigkeit von primären menschlichen Makrophagen sowie von FR-transduzierten THP-1 Zellen auf Kollagen zu adhärieren und durch kollagenreiche Gewebe zu migrieren. Daher haben wir FR als neuen Regulator der Migration von Makrophagen identifiziert. hinsichtlich der daran beteiligten Zellen genau zu beschreiben und erlaubt damit Rückschlüsse, auf den zeitlichen und qualitativen Ablauf der Abwehrreaktion im Rahmen eines ARDS.Macrophages are a heterogeneous population of immune cells that fulfill essential functions in the defense against pathogens as well as the maintenance of tissue integrity and remodeling of tissues. However, errant macrophages pose a serious threat to the organism as a whole as they can either fuel inflammation in chronic inflammatory disorders or block effective immune surveillance in the tumor microenvironment. This heterogeneity complicates effective therapy, as indiscriminative targeting of macrophages leads to severe side effects in tissue homeostasis or fighting infections. Folate receptor beta (FR), a GPI anchored protein that is selectively expressed in activated macrophages, was proposed as a promising marker of harmful macrophage populations. To further investigate the applicability of FR, we characterized the protein microenvironment of FR to identify interacting proteins for use in bispecfic targeting of specific macrophage subpopulations and utilized this proteomic information to decipher the function of FR. As a result of this strategy, we detected FR-proximal proteins, which can be utilized to discriminate functionally diverse macrophage populations. More importantly, we detected a functional interaction between FR and the integrin heterodimer CD11b/CD18. This interaction changes the conformation of CD11b/CD18 and regulates adhesion to collagen in FR-transduced THP-1 cells and human primary macrophages. Further, we found that FR-expressing cells display a reduced ability to migrate through collagen-rich matrixes and thereby identified FR as a novel regulator of macrophage homing.submitted by Mag. rer. nat. Christian MachacekZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeB

Neuronal control of Drosophila walking direction

Most land animals normally walk forward but switch to backward walking upon sensing an obstacle or danger in the path ahead. A change in walking direction is likely to be triggered by descending “command” neurons from the brain that act upon local motor circuits to alter the timing of leg muscle activation. Here we identify descending neurons for backward walking in Drosophila—the MDN neurons. MDN activity is required for flies to walk backward when they encounter an impassable barrier and is sufficient to trigger backward walking under conditions in which flies would otherwise walk forward. We also identify ascending neurons, MAN, that promote persistent backward walking, possibly by inhibiting forward walking. These findings provide an initial glimpse into the circuits and logic that control walking direction in Drosophila

Effectiveness of Biologics in Clinical Practice in German patients: Week 12 Primary Outcomes from the International Observational Psoriasis Study of Health Outcomes (PSoHo)

Schwarz B, Effendy I, Kors C, et al. Effectiveness of Biologics in Clinical Practice in German patients: Week 12 Primary Outcomes from the International Observational Psoriasis Study of Health Outcomes (PSoHo). In: Special Issue:Abstractband anlässlich der 52. Tagung der Deutschen Dermatologischen Gesellschaft, Vereinigung Deutschsprachiger Dermatologen e. V. 26. bis 29. April 2023, Berlin. Journal der Deutschen Dermatologischen Gesellschaft. Vol 21. Hoboken: Wiley; 2023: 64

HSA nanocapsules functionalized with monoclonal antibodies for targeted drug delivery

The chronic autoimmune disorder rheumatoid arthritis (RA) affects millions of adults and children every year. Chronically activated macrophages secreting enzymes and inflammatory cytokines play a key role in RA. Distinctive marker molecules on the macrophage surface could be used to design a targeted drug delivery device for the treatment of RA without affecting healthy cells and tissues. Here, different methods for covalent attachment of antibodies (mAb) recognizing MHC class II molecules found on macrophages onto human serum albumin (HSA) nanocapsules were compared. HSA nanocapsules were prepared with a hydrodynamic diameter of 500.7 ± 9.4 nm and a narrow size distribution as indicated by a polydispersity index (PDI) of 0.255 ± 0.024. This was achieved by using a sonochemical process avoiding toxic cross linking agents and emulsifiers. Covalent binding of mAb on the surface of HSA nanocapsules was realized using polyethyleneglycol (PEG)3000 as spacer molecule. The presence of mAb was confirmed by confocal laser scanning microscopy (CLSM) and enzyme-linked immunosorbent assay (ELISA). Specific binding of mAb-HSA nanocapsules to MHC class II molecules on antigen-presenting cells was demonstrated by flow cytometry analysis.This work has received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. We thank Exbio from Czech Republic for providing antibodies

Journal of Leukocyte Biology / The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6phosphate/insulinlike growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is upregulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogeninduced efferocytosis. The level of uptake of plasminogencoated apoptotic cells inversely correlates with the TNF production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an uptonow undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.(VLID)341171