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The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex.
Design of novel IL-4 antagonists employing site-specific chemical and biosynthetic glycosylation
(2021)
The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases.
For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R.
This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist.
For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability.
The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab.
Regulatory T cells (Tregs) are the masters of immune regulation controlling inflammation and tolerance, tissue repair and homeostasis. Multiple immunological diseases result from altered Treg frequencies and Treg dysfunction. We hypothesized that augmenting Treg function and numbers would prevent inflammatory disease whereas inhibiting or depleting Tregs would improve cancer immunotherapy.
In the first part of this thesis, we explored whether in vivo activation and expansion of Tregs would impair acute graft-versus-host disease (aGvHD). In this inflammatory disease, Tregs are highly pathophysiological relevant and their adoptive transfer proved beneficial on disease outcome in preclinical models and clinical studies. IL-2 has been recognized as a key cytokine for Treg function. Yet, attempts in translating Treg expansion via IL-2 have remained challenging, due to IL-2s extremely broad action on other cell types including effector T cells, NK cells, eosinophils and vascular leakage syndrome, and importantly, due to poor pharmacokinetics in vivo. We addressed the latter issue using an IL-2-IgG-fusion protein (irrIgG-IL-2) with improved serum retention and demonstrated profound Treg expansion in vivo in FoxP3-luciferase reporter mice. Further, we augmented Treg numbers and function via the selective-TNF based agonists of TNFR2 (STAR2). Subsequently, we tested a next-generation TNFR2 agonist, termed NewSTAR, which proved even more effective. TNFR2 stimulation augmented Treg numbers and function and was as good as or even superior to the IL-2 strategy. Finally, in a mouse model of aGvHD we proved the clinical relevance of Treg expansion and activation with irrIgG-IL-2, STAR2 and NewSTAR. Notably, the TNFR2 stimulating constructs were outstanding as we observed not the IL-2 prototypic effects on other cell populations and no severe side effects.
In the second part of this thesis, we explored Tregs in pancreatic ductal adenocarcinoma (PDAC) and developed targeting strategies. Among several tumor entities in which Tregs impact survival, preclinical and clinical data demonstrated their negative role on PDAC. In our studies we employed the orthotopic syngeneic Panc02 model in immunocompetent mice. Based on flow cytometric analysis of the tumor microenvironment we propose TIGIT and TNFRSF members as novel therapeutic targets. Surprisingly, we found that blocking TNFR2 did not interfere with intratumoral Treg accumulation. However, we decreased the highly abundant intratumoral Tregs when we disrupted the tumor extracellular matrix. In PDAC, Treg manipulation alone did not lead to tumor regression and we propose that an additional immune boost may be necessary for efficient tumor immune surveillance and cancer clearance. This contrasts with aGvHD, in which Treg manipulation alone was sufficient to improve disease outcome.
Conclusively, we demonstrated the enormous medical benefit of Treg manipulation. Our promising data obtained with our newly developed powerful tools highlight the potential to translate our findings into clinical practice to therapeutically target human Tregs in patients. With novel TNFR2 agonists (STAR2, NewSTAR) we augmented Treg numbers and function as (or even more) effectively than with IL-2, without causing adverse side effects. Importantly, exogenous in vivo Treg expansion protected mice from aGvHD. For the therapy of PDAC, we identified novel targets on Tregs, notably TIGIT and members of the TNFRSF. We demonstrated that altering the extracellular tumor matrix can efficiently disrupt the Treg abundance in tumors. These novel targeting strategies appear as attractive new treatment options and they may benefit patients suffering from inflammatory disease and cancer in the future.
ABC-Transporter sind ein wichtiger Aspekt bei der Entwicklung von Resistenzen gegen Chemotherapeutika. Ziel dieser Studie war es, die Resistenzentwicklung in HNSCC-Zelllinien im Zusammenhang mit verschiedenen Cisplatinkozentrationen und Inkubationszeiten zu analysieren, sowie die Expression von ABC-Transportern via semi-quantitativer RT-PCR in diesen Zellen zu untersuchen. Die Zellen zeigten dabei keine relevante Resistenzentwick-lung im Sinne eines Anstiegs der IC50. Bei drei der Zelllinien konnte jedoch eine hohe intrin-sische Cisplatinresistenz beobachtet werden. Diese resistenten Zelllinien wiesen nach Inku-bation mit Cisplatin deutlich höhere Expressionswerte für TAP1, TAP2, ABCG2 sowie die ABCC-Transporterfamilie auf. Dabei zeigte sich, dass die Expression der ABCC-Familie mit zunehmender Inkubationsdauer abnahm. TAP1 in PCI-9 und PCI-68 war auch noch nach vierwöchiger Inkubation stark überexprimiert.
Die initiale IC50 dieser Zelllinien lag dabei deutlich über der Plasmakonzentration von Pati-enten mit Hochdosis-Chemotherapie. Die Expression der Transporter aus der ABCC-Familie ließ die Vermutung zu, dass diese Transporter initial zur Resistenz gegen Cisplatin beitrugen, allerdings mit zunehmender Inkubationsdauer an Bedeutung verloren. Diese Annahme wurde dadurch gestützt, dass die HNSCC-Zelllinien nach einem inkubationsfreien Intervall von vier Wochen im Anschluss an die Inkubation mit Cisplatin deutliche Überexpressionen der ABCC-Transporterfamilie zeigten. Auch für Transporter (ABCG2 und TAP-Transporter), die keine Effluxfunktion für Cisplatin besitzen, konnte ein Zusammenhang der Expression mit der Resistenz der HNSCC-Zellen beobachtet werden. Der Beitrag dieser Transporter zur Resistenz von Tumorzellen könnte über deren Funktionen im Metabolismus von Tumorzel-len, deren Fähigkeiten Tumorstammzellen zu bilden und dem Efflux endogener Zellstress verursachender Substrate erklärt werden. Allerdings werden diese Transporter erst seit kur-zem mit Resistenzen gegen Cisplatin in Verbindung gebracht. Aufbauend auf diese Studie wäre eine Verifizierung der Kausalität des Resistenzmechanismus durch knock-down und Inhibition der von uns untersuchten Transporter sinnvoll.
Induction of ectopic bone formation by site directed immobilized BMP2 variants \(in\) \(vivo\)
(2020)
In contrast to common bone fractures, critical size bone defects are unable to self-regenerate and therefore external sources for bone replacement are needed. Currently, the gold standard to treat critical size bone fractures, resulting from diseases, trauma or surgical interventions, is the use of autologous bone transplantation that is associated with several drawbacks such as postoperative pain, increased loss of blood during surgery and extended operative time.
The field of bone tissue engineering focuses on the combination of biomaterials and growth factors to circumvent these adverse events and thereby to improve critical size bone defects treatment.
To this aim, a promising approach is represented by using a collagen sponge soaked with one of the most powerful osteoinductive proteins, the bone morphogenetic protein 2 (BMP2). After the approval by the Food and Drug Administration (FDA), BMP2 was used to successfully treat several severe bone defects. However, the use of BMP2 delivery systems is associated with severe side effects such as inflammation, swelling, ectopic bone formation outside of the site of implantation and breathing problems if implanted in the area of the cervical spine. The occurrence of severe side effects is related to the supraphysiological amounts of the applied protein at the implantation site. The BMP2 is typically adsorbed into the scaffold and diffuses rapidly after implantation. Therefore, intensive research has been conducted to improve the protein’s retention ability, since a prolonged entrapment of the BMP2 at the implantation site would induce superior bone formation in vivo due to a minimized protein release. By controlling the release from newly designed materials or changing the protein immobilization methods, it seems possible to improve the osteoinductive properties of the resulting BMP2-functionalized scaffolds.
The combination of biocompatible and biodegradable scaffolds functionalized with a covalently immobilized protein such as BMP2 would constitute a new alternative in bone tissue engineering by eliminating the aforementioned severe side effects. One of the most common immobilization techniques is represented by the so-called EDC/NHS chemistry. This coupling technique allows covalent biding of the growth factor but in a non-site direct manner, thus producing an implant with uncontrollable and unpredictable osteogenic activities. Therefore, the generation of BMP2 variants harboring functional groups that allow a site-directed immobilization to the scaffold, would enable the production of implants with reproducible osteogenic activity.
The new BMP2 variants harbor an artificial amino acid at a specific position of the mature polypeptide sequence. The presence of the unnatural amino acid allows to use particular covalent immobilization techniques in a highly specific and site directed manner. The two selected BMP2 variants, BMP2 E83Plk and BMP2 E83Azide, were expressed in E. coli, renatured and purified by cation exchange chromatography. The final products were intensively analyzed in terms of purity and biological activity in vitro. The two BMP2 variants enabled the application of different coupling techniques and verify the possible options for site directed immobilization to the scaffold.
Intensive analyses on the possible side effects caused by the coupling reactions and on the quantification of the coupled protein were performed. Both click chemistry reactions showed high reaction efficacies when the BMP2 variants were coupled to functionalized fluorophores. Quantification by ELISA and scintillation counting of radioactively labeled protein revealed different outcomes. Moreover, the amounts of protein detected for the BMP2 variants coupled to microspheres were similar to that of the wild type protein. Therefore, it was not possible to conclude whether the BMP2 variants were covalently coupled or just adsorbed.
BMP2 variants being immobilized to various microspheres induced osteogenic differentiation of C2C12 cells in vitro, but only in those cells that were located in close proximity to the functionalized beads. This selectivity strongly indicates that the protein is for a great portion covalently coupled and not just adsorbed. Moreover, the difference between the covalently coupled BMP2 variants and the adsorbed BMP2 WT was confirmed in vivo. Injection of the BMP2-functionalized microspheres in a rat model induced subcutaneous bone formation.
The main aim of the animal experiment was to prove whether covalently coupled BMP2 induces bone formation at significant lower doses if compared to the amount being required if the protein is simply adsorbed. To this aim, several BMP2 concentrations were tested in this animal experiment. The BMP2 variants, being covalently immobilized, were hypothesized to be retained and therefore bio-available at the site of implantation for a prolonged time. However, in the animal experiments, lower doses of either coupled or adsorbed protein were unable to induce any bone formation within the 12 weeks.
In contrast, the highest doses induced bone formation that was first detected at week 4. During the 12 weeks of the experiment, an increase in bone density and a steady state bone volume was observed. These results were obtained only for the covalently coupled BMP2 E83Azide but not for BMP2 E83Plk that did not induce bone formation in any condition. The negative outcome after application of BMP2 E83Plk suggested that the coupling reaction might have provoked changes in the protein structure that extremely influenced its osteogenic capabilities in vivo.
However, the histological examination of the different ossicles induced either by BMP2 WT or BMP2 E83Azide, revealed clear morphological differences. BMP2 WT induced a bone shell-like structure, while the covalently coupled protein induced uniform bone formation also throughout the inner part. The differences between the two newly formed bones can be clearly associated with the different protein delivery mechanisms. Thus, the developed functionalized microspheres constitute a new interesting strategy that needs further investigations in order to be able to be used as replacement of the currently used BMP2 WT loaded medical devices.
The integrity of its DNA is fundamental for every living cell. However, DNA is constantly threatened by exogenous and endogenous damaging agents that can cause a variety of different DNA lesions. The severe consequences of an accumulation of DNA lesions are reflected in cancerogenesis and aging. Several DNA repair mechanisms ensure the repair of DNA lesions and thus maintain DNA integrity. One of these DNA repair mechanisms is nucleotide excision repair (NER), which is famous for its ability to address a large variety of structurally unrelated DNA lesions. A key component of eukaryotic NER is the transcription factor II H (TFIIH) complex, which is not only essential for DNA repair but also for transcription. The TFIIH complex is composed of ten subunits. How these subunits work together during NER to unwind the DNA around the lesion is, however, not yet fully understood. High-resolution structural data and biochemical insights into the function of every subunit are thus indispensable to understand the functional networks within TFIIH. The importance of an intact TFIIH complex is reflected in the severe consequences of patient mutations in the TFIIH subunits XPB, XPD or p8 leading to the hallmark diseases xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Defects in the NER pathway are further associated with several types of cancer including skin cancer.
The herein described work focused on five TFIIH subunits derived from the thermophilic fungus Chaetomium thermophilum, the p34/p44 pair and the ternary XPB/p52/p8 complex. The interaction between p34 and p44 was characterized based on a high-resolution structure of the p34_vWA/p44_RING minimal complex. Biochemical studies of the p34/p44 interaction led to the disclosure of an additional interaction between the p34 and p44 subunits, which had not been characterized so far. The p34/p44 interaction was shown to be central to TFIIH, which justifies the presence of several redundant interfaces to safeguard the interaction between the two proteins and might explain why so far, no patient mutations in these subunits have been identified. The p52 subunit of TFIIH was known to be crucial to stimulate the ATPase activity of XPB, which is required during NER. This work presents the first entire atomic resolution structural characterization of p52, which was derived of several crystal structures of p52 variants and a p52/p8 variant thereby demonstrating the interaction between p52 and p8. The precise structural model of p52 offered the possibility to investigate interactions with other TFIIH subunits in more detail. The middle domain 2 of p52 and the N-terminal domain of XPB were shown to mediate the main interaction between the two subunits. An analysis of the p52 crystal structures within recently published cryo-electron microscopy structures of TFIIH provides a model of how p52 and p8 stimulate the ATPase activity of XPB, which is essential for NER and transcription. The structural and biochemical findings of this work provide an additional building block towards the uncovering of the architecture and function of this essential transcription factor.
The molecular chaperone Hsp90 facilitates the folding and activation of a wide array of structurally and functionally diverse client proteins. Hsp90 presents a central node of protein homeostasis and is frequently involved in the development of many human diseases. Although Hsp90 is a promising target for disease treatment, the mechanism by which Hsp90 facilitates client recognition and maturation is poorly understood.
The shape of the homodimeric protein resembles a molecular clamp that opens and closes in response to binding and hydrolysis of ATP. Structural studies reveal a network of distinct local conformational rearrangements that coordinate the slow transition into the hydrolysis-active, closed state configuration (time order of minutes). However, the kinetics of local conformational changes remain elusive because spectroscopic tools that can detect them have been missing so far.
Fluorescence quenching of extrinsic fluorophores by the natural amino acid Tryptophan is based on a photoinduced electron transfer (PET) reaction, which requires sub-nanometer contact between fluorophore and Tryptophan. This quenching mechanism has been developed into a 1-nm spectroscopic tool for the detection of rapid protein folding dynamics. Within the scope of this doctoral thesis, PET-reporter systems were designed to investigate the kinetics of local conformational motions that are part of the mechanistic core of the Hsp90 chaperone cycle. ATP-triggered kinetics of closure of the ATP-lid as well as swapping of the N-terminal ß-strand across subunits and association of the N-terminal and middle-domain were estimated in solution. Bulk experiments revealed that local motions occur on similar timescales and are in good agreement with the ATP-hydrolysis rate. Functional mutations demonstrated that local motions act cooperatively. Furthermore, the lid was shown to close via a two-step process consisting of a rapid lid-reconfiguration in direct response to ATP-binding, followed by slow closure of the lid. The co-chaperone Aha1 seems to act early in the chaperone cycle by remodelling of the lid and by stabilization of apo Hsp90 in a NM-domain pre-associated conformation.
A two-colour single-molecule PET microscopy method was developed to observe local motions at remote positions simultaneously and in real-time. Thus, directionality within the network of local conformational changes could be revealed. In a first attempt, the feasibility of detecting PET-complexes on the single-molecule surface was tested on Hsp90 constructs that report on only one motion (one-colour single-molecule PET microscopy). PET-quenched complexes could be distinguished from photobleached fluorophores through oxidation by molecular oxygen, resulting in fluorescence recovery. In two-colour experiments, a dimmed state was identified for PET-quenched complexes, but not for all of the used PET-reporter systems. Results suggest that local motions occur simultaneously within the time-resolution of the experiment (0.3 sec). Furthermore, bi-exponential kinetics of transition into the closed clamp configuration indicate a more complex mechanism of clamp-closure than of clamp-opening, which could be well described by a mono-exponential function.
Several important cellular processes, including transcription, nucleotide excision repair and cell cycle control are mediated by the multifaceted interplay of subunits within the general transcription factor II H (TFIIH).
A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these pathways. This becomes especially important in the context of severe diseases like xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that arise from single point mutations in some of the TFIIH subunits.
In an attempt to structurally characterize the TFIIH complex, we harnessed the qualities of the eukaryotic thermophile Chaetomium thermophilum, a remarkable fungus, which has only recently been recognized as a novel model organism. Homologues of TFIIH from C. thermophilum were expressed in E. coli, purified to homogeneity and subsequently utilized for crystallization trials and biochemical studies.
The results of the present work include the first crystal structure of the p34 subunit of TFIIH, comprising the N-terminal domain of the protein. The structure revealed a von Willebrand Factor A (vWA) like fold, which is generally known to be involved in a multitude of protein-protein interactions. Structural comparison allowed to delineate similarities as well as differences to already known vWA domains, providing insight into the role of p34 within TFIIH. These results indicate that p34 assumes the role of a structural scaffold for other TFIIH subunits via its vWA domain, while likely serving additional functions, which are mediated through its
C-terminal zinc binding domain and are so far unknown.
Within TFIIH p34 interacts strongly with the p44 subunit, a positive regulator of the XPD helicase, which is required for regulation of RNA Polymerase II mediated transcription and essential for eukaryotic nucleotide excision repair. Based on the p34 vWA structure putative protein-protein interfaces were analyzed and binding sites for the p34 p44 interaction suggested. Continuous crystallization efforts then led to the first structure of a p34 p44 minimal complex, comprising the N-terminal vWA domain of p34 and the C-terminal C4C4 RING domain of p44. The structure of the p34 p44 minimal complex verified the previous hypothesis regarding the involved binding sites. In addition, careful analysis of the complex interface allowed to identify critical residues, which were subsequently mutated and analyzed with respect to their significance in mediating the p34 p44 interaction, by analytical size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The structure of the p34 p44 complex also revealed a binding mode of the p44 C4C4 RING domain, which differed from that of other known RING domains in several aspects, supporting the hypothesis that p44 contains a novel variation of this domain.
Bone morphogenetic proteins (BMPs) are involved in various aspects of cell-cell communication in complex life forms. They act as morphogens, help differentiate different cell types from different progenitor cells in development, and are involved in many instances of intercellular communication, from forming a body axis to healing bone fractures, from sugar metabolism to angiogenesis. If the same protein or protein family carries out many functions, there is a demand to regulate and fine-tune their biological activities, and BMPs are highly regulated to generate cell- and context-dependent outcomes.
Not all such instances can be explained yet. Growth/differentiation factor (GDF)5 (or BMP14) synergizes with BMP2 on chondrogenic ATDC5 cells, but antagonizes BMP2 on myoblastic C2C12 cells. Known regulators of BMP2/GDF5 signal transduction failed to explain this context-dependent difference, so a microarray was performed to identify new, cell-specific regulatory components. One identified candidate, the fibroblast growth factor receptor (FGFR)2, was analyzed as a potential new co-receptor to BMP ligands such as GDF5: It was shown that FGFR2 directly binds BMP2, GDF5, and other BMP ligands in vitro, and FGFR2 was able to positively influence BMP2/GDF5-mediated signaling outcome in cell-based assays. This effect was independent of FGFR2s kinase activity, and independent of the downstream mediators SMAD1/5/8, p42/p44, Akt, and p38. The elevated colocalization of BMP receptor type IA and FGFR2 in the presence of BMP2 or GDF5 suggests a signaling complex containing both receptors, akin to other known co-receptors of BMP ligands such as repulsive guidance molecules.
This unexpected direct interaction between FGF receptor and BMP ligands potentially opens a new category of BMP signal transduction regulation, as FGFR2 is the second receptor tyrosine kinase to be identified as BMP co-receptor, and more may follow. The integration of cell surface interactions between members of the FGF and BMP family especially may widen the knowledge of such cellular communication mechanisms which involve both growth factor families, including morphogen gradients and osteogenesis, and may in consequence help to improve treatment options in osteochodnral diseases.
The cytokine interleukin-5 (IL-5) is part of the TH2-mediated immune response. As a key regulator of eosinophilic granulocytes (eosinophils), IL-5 controls multiple aspects of eosinophil life. Eosinophils play a pathogenic role in the onset and progression of atopic diseases as well as hypereosinophilic syndrome (HES). Here, cytotoxic proteins and pro-inflammatory mediators stored in intracellular vesicles termed granula are released upon activation thereby causing local inflammation to fight the pathogen. However, if such inflammation persists, tissue damage and organ failure can occur. Due to the close relationship between eosinophils and IL-5 this cytokine has become a major pharmaceutical target for the treatment of atopic diseases or HES. As observed with other cytokines, IL-5 signals by assembling a heterodimeric receptor complex at the cell surface in a stepwise mechanism. In the first step IL-5 binds to its receptor IL-5Rα (CD125). This membrane-located complex then recruits the so-called common beta chain βc (CD131) into a ternary ligand receptor complex, which leads to activation of intracellular signaling cascades. Based on this mechanism various strategies targeting either IL-5 or IL-5Rα have been developed allowing to specifically abrogate IL-5 signaling. In addition to the classical approach of employing neutralizing antibodies against IL 5/IL-5Rα or antagonistic IL-5 variants, two groups comprising small 18 to 30mer peptides have been discovered, that bind to and block IL-5Rα from binding its activating ligand IL-5. Structure-function studies have provided detailed insights into the architecture and interaction of IL-5IL-5Rα and βc. However, structural information for the ternary IL-5 complex as well as IL-5 inhibiting peptides is still lacking.
In this thesis three areas were investigated. Firstly, to obtain insights into the second receptor activation step, i.e. formation of the ternary ligand-receptor complex IL-5•IL-5Rα•βc, a high-yield production for the extracellular domain of βc was established to facilitate structure determination of the ternary ligand receptor assembly by either X-ray crystallography or cryo-electron microscopy.
In a second project structure analysis of the ectodomain of IL-5Rα in its unbound conformation was attempted. Data on IL-5Rα in its ligand-free state would provide important information as to whether the wrench-like shaped ectodomain of IL-5Rα adopts a fixed preformed conformation or whether it is flexible to adapt to its ligand binding partner upon interaction. While crystallization of free IL-5Rα failed, as the crystals obtained did not diffract X rays to high resolution, functional analysis strongly points towards a selection fit binding mechanism for IL-5Rα instead of a rigid and fixed IL-5Rα structure. Hence IL-5 possibly binds to a partially open architecture, which then closes to the known wrench-like architecture. The latter is then stabilized by interactions within the D1-D2 interface resulting in the tight binding of IL-5.
In a third project X-ray structure analysis of a complex of the IL-5 inhibitory peptide AF17121 bound to the ectodomain of IL-5Rα was performed. This novel structure shows how the small cyclic 18mer peptide tightly binds into the wrench-like cleft formed by domains D1 and D2 of IL-5Rα. Due to the partial overlap of its binding site at IL-5Rα with the epitope for IL-5 binding, the peptide blocks IL-5 from access to key residues for binding explaining how the small peptide can effectively compete with the rather large ligand IL-5. While AF17121 and IL-5 seemingly bind to the same site at IL-5Rα, functional studies however showed that recognition and binding of both ligands differ. With the structure for the peptide-receptor complex at hand, peptide design and engineering could be performed to generate AF17121 analogies with enhanced receptor affinity. Several promising positions in the peptide AF17121 could be identified, which could improve inhibition capacity and might serve as a starting point for AF17121-based peptidomimetics that can yield either superior peptide based IL-5 antagonists or small-molecule-based pharmacophores for future therapies of atopic diseases or the hypereosinophilic syndrome.