Immunglobulin-basierte Strategien zur Verlängerung der Halbwertszeit rekombinanter Proteine

With numerous small recombinant proteins being developed for treatment of various diseases, half-life extension strategies are becoming increasingly vital for the creation of long lasting and efficient biotherapeutics. Because of their small size, these proteins are rapidly cleared from plasma circulation and lose a lot of their potential as therapeutic. Using immunoglobulin-binding domains (IgBD), in particular domain C3 from Streptococcus protein G (SpGC3), can substantially increase the half-life by forming transient complexes with endogenous IgG molecules. This complex formation needs to occur at neutral pH to prevent rapid renal clearance, but also under acidic conditions, facilitating the salvage from lysosomal degradation via the neonatal Fc receptor (FcRn). In order to enable FcRn recycling, the major binding site of SpGC3 to the IgG-Fc part was eliminated, since it is overlapping with the FcRn binding site, generating the CH1 specific domain SpGC3Fab. Affinity maturation towards the Fab fragment resulted in domains SpGC3FabRR and SpGC3FabRR,E15V showing significantly increased terminal half-lives as scDb-IgBD fusion proteins. Since the IgBDs originate from bacterial proteins, they might provoke immunogenic reactions and the production of anti-drug antibodies. For deimmunization, possible T-cell epitopes were identified by sequence and structure-based analysis and eliminated by combination of single amino acid substitutions. This resulted in deimmunized IgBDs that were able to strongly increase the terminal half-life as scDb fusion proteins. The application of SpGC3FabRR to recombinant human erythropoietin (huEPO) resulted in a roughly 5-fold increased terminal half-life compared to the unmodified huEPO that also translated into increased hemoglobin concentration after one single intravenous injection. Furthermore, a comparative study of various Fc fusion proteins revealed that a multitude of factors is jointly responsible for the long half-life of full-length IgG molecules. Although possessing an identical huIgG1 Fc part and affinity to the moFcRn, neither an scFv-Fc nor an scDb-Fc or a single-chain version of the IgG molecule (scFv-scCLCH1-Fc) could reach the pharmacokinetic properties of the IgG molecule, not fully explainable by factors like size, isoelectric point and stability. All in all, the utilization of immunoglobulins and FcRn-mediated salvage via IgBDs and Fc fusion proteins is a powerful tool for the generation of recombinant fusion proteins with favorable pharmacokinetic properties.Angesichts der Entwicklung einer Vielzahl kleiner rekombinanter Proteine, die für die Behandlung verschiedenster Krankheiten eingesetzt werden, nimmt das Interesse an Strategien zur Halbwertszeitverlängerung zu, um lang zirkulierende und effiziente Biotherapeutika zu entwerfen. Aufgrund ihrer geringen Größe werden diese Proteine schnell aus dem Blutkreislauf eliminiert und verlieren einen Großteil ihrer therapeutischen Wirksamkeit. Die Verwendung von Immunglobulin-bindenden Domänen (IgBD), insbesondere Domäne C3 von Protein G aus Streptococcus (SpGC3), führt zu einer deutlichen Erhöhung der Halbwertszeit rekombinanter Proteine, indem sie transiente Komplexe mit körpereigenen IgG Molekülen bildet. Diese Komplexbildung muss sowohl bei neutralem als auch saurem pH im Endosom auftreten und kann eine schnelle Ausscheidung in der Niere sowie den lysosomalen Abbau durch Recycling über den neonatalen Fc Rezeptor (FcRn) verhindern. Da die Bindestelle von SpGC3 und FcRn am IgG-Fc Teil überlappen, wurde diese bei der IgBD beseitigt und die CH1 spezifische Domäne SpGC3Fab generiert, um besseres FcRn Recycling zu ermöglichen. Die Domänen SpGC3FabRR und SpGC3FabRR,E15V, die durch CH1-gerichtete Affinitätsreifungen entstanden, zeigten signifikant erhöhte terminale Halbwertszeiten als scDb-IgBD Fusionsproteine. Da die IgBDs aus bakteriellen Proteinen hervorgingen, ist das Einsetzen einer Immunantwort und die damit verbundene Produktion von Antikörpern gegen das Medikament möglich. Für eine Deimmunisierung wurden daher mögliche T-Zell Epitope mittels sequenz- und strukturbasierter Untersuchung identifiziert und durch eine Kombination einzelner Aminosäureaustausche beseitigt. Die so entstandenen deimmunisierten IgBDs konnten die terminale Halbwertszeit als scDb Fusionsproteine deutlich steigern. Die Fusion von SpGC3FabRR mit rekombinantem humanem Erythropoietin (huEPO) erhöhte dessen Halbwertszeit ungefähr 5-fach, verglichen mit nicht modifiziertem huEPO. Diese verbesserte Pharmakokinetik spiegelte sich auch in einer erhöhten Hämoglobin Konzentration nach einmaliger intravenöser Gabe wieder. Außerdem zeigte eine vergleichende Studie verschiedener Fc Fusionsproteine, dass eine Vielzahl von Faktoren für die lange Halbwertszeit von ganzen IgG Molekülen mitverantwortlich ist. Obwohl alle Fusionsproteine einen identischen huIgG1 Fc Teil mit gleicher Affinität zum FcRn besaßen, konnten weder ein scFv-Fc, noch ein scDb-Fc oder eine einzelkettige Version des IgG Moleküls (scFv-scCLCH1-Fc) die pharmakokinetischen Eigenschaften des IgG Moleküls erreichen. Faktoren wie Größe, isoelektischer Punkt und Stabilität sind nicht in der Lage dies hinreichend zu erklären. Zusammenfassend kann gesagt werden, dass die Verwendung von Immunglobulinen und FcRn-abhängigem Recycling durch IgBDs und Fc Fusionsproteine rekombinante Fusionsproteine mit vorteilhaften pharmakokinetischen Eigenschaften schaffen kann

Utilization of KNX Technology for Operational and Technical Functions Control in Smart Home

Tato bakalářská práce se zabývá řízením provozních funkcí v inteligentních domech. V teoretické části se podrobně zaměřuje na sběrnicový systém KNX, popis systému, topologii, adresování, způsob komunikace a softwarový nástroj ETS k programování jednotlivých komponentů. Dále se zaměřuje na komunikaci a spolupráci jednotlivých komponentů pro ovládání provozně technických funkcí se zaměřením na snížení provozních nákladů. V praktické části se zabývá návrhem projektu pro řízení provozně technických funkcí s ohledem na snížení provozních nákladů.This bachelor thesis deals with the management of operational functions in smart homes. The theoretical part focuses in detail on the KNX bus system, system description, topology, addressing, communication method and software tool ETS for programming of individual components. It also focuses on communication and cooperation of individual components for the control of operational and technical functions with a focus on reducing operating costs. The practical part deals with the design of a project for the management of operational and technical functions with regard to the reduction of operating costs.450 - Katedra kybernetiky a biomedicínského inženýrstvívelmi dobř

Measurement of the CKM matrix element |V_ts|²

Diese Arbeit beschreibt die erste direkte Messung des CKM-Matrixelements |V_ts|. Sie basiert auf Daten, die im Jahr 2012 mit dem ATLAS Detektor bei √s = 8 TeV pp-Kollisionen und einer integrierten Luminosität von 20.3 fb⁻¹ aufgezeichnet wurden. Insgesamt sind 112171 mögliche tt̅-Ereignisse im Lepton+Jets-Kanal mit einer Reinheit von 90.0 % rekonstruierbar, die für die Hauptanalyse zur Verfügung stehen. Laut Vorhersage zerfallen hiervon 183 Ereignisse über tt̅→WWbs̅ (inkl. Ladungskonjugation) und können für die Bestimmung des Betragsquadrats |V_ts|² verwendet werden. Für eine Identifikation dieser seltenen Zerfälle werden verschiedenste Observablen untersucht, wie z.B. die Eigenschaften von Jets und Tracks sowie von b-Quark Identifikationsmethoden. Darüber hinaus werden Hadronen von s-Quarks betrachtet, die K0s Teilchen, die über einen kinematischen Fit rekonstruiert werden. Die Observablen mit den besten Eigenschaften werden anschließend in einer multivariaten Analyse ("Boosted decision trees") zu einer einzigen zusammengefasst. Die dazugehörigen Monte-Carlo-Simulationen werden dann als Template für eine Beschreibung der Daten verwendet, was als Ergebnis zu einem Signifikanz-Wert von 0.7σ für t→s+W Zerfälle führt. Insgesamt ergibt sich ein oberes Limit von |V_ts|² < 1.74 %. Dieses wurde bezüglich eines Vertrauensbereiches von 95 % bestimmt, unter Berücksichtigung sämtlicher systematischen und statischen Unsicherheiten. Diese Arbeit, basierend auf einer direkten Messung des CKM-Matrixelements |V_ts|², führt somit zum bisher besten direkten Limit für |V_ts|².This is the first direct measurement of the CKM matrix element |V_ts|, using data collected by the ATLAS detector in 2012 at √s=8 TeV pp-collisions with a total integrated luminosity of 20.3 fb⁻¹. The analysis is based on 112171 reconstructed tt̅ candidate events in the lepton+jets channel, having a purity of 90.0 %. 183 tt̅→WWbs̅ decays are expected (charge conjugation implied), which are available for the extraction of the CKM matrix element |V_ts|². To identify these rare decays, several observables are examined, such as the properties of jets, tracks and of b-quark identification algorithms. Furthermore, the s-quark hadrons K0s are considered, reconstructed by a kinematic fit. The best observables are combined in a multivariate analysis, called "boosted decision trees". The responses from Monte Carlo simulations are used as templates for a fit to data events yielding a significance value of 0.7σ for t→s+W decays. An upper limit of |V_ts|² < 1.74 % at 95 % confidence level is set, including all systematic and statistical uncertainties. So this analysis, using a direct measurement of the CKM matrix element |V_ts|², provides the best direct limit on |V_ts|² up to now

Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches

Structure of the 26S proteasome with ATP-gamma S bound provides insights into the mechanism of nucleotide-dependent substrate translocation

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-gamma S induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of inter-subunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6

Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasomes regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover

Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-gamma S to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATP(h)) and ATP-gamma S conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATP(h) conformation, whereas the lid is more similar to the ATP-gamma S bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle