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Neurleptic drugs, e.g., aripiprazole, targeting the dopamine D2S and D3 receptors (D2SR and D3R) in the central nervous system are widely used in the treatment of several psychotic and neurodegenerative diseases. Therefore, a new series of benzothiazole-based ligands (3-20) was synthesized by applying the bioisosteric approach derived from the selective D3Rs ligand BP-897 (1) and its structurally related benz[d]imidazole derivative (2). Herein, introduction of the benzothiazole moiety was well tolerated by D2SR and D3R binding sites leading to antagonist affinities in the low nanomolar concentration range at both receptor subtypes. However, all novel compounds showed lower antagonist affinity to D3R when compared to that of 1. Further exploration of different substitution patterns at the benzothiazole heterocycle and the basic 4-phenylpiperazine resulted in the discovery of high dually acting D2SR and D3R ligands. Moreover, the methoxy substitution at 2-position of 4-phenylpiperazine resulted in significantly (22-fold) increased D2SR binding affinity as compared to the parent ligand 1, and improved physicochemical and drug-likeness properties of ligands 3-11. However, the latter structural modifications failed to improve the drug-able properties in ligands having un-substituted 4-phenylpiperazine analogs (12-20). Accordingly, compound 9 showed in addition to high dual affinity at the D2SR and D3R [Ki (hD2SR) = 2.8 ± 0.8 nM; Ki (hD3R) = 3.0 ± 1.6 nM], promising clogS, clogP, LE (hD2SR, hD3R), LipE (hD2SR, hD3R), and drug-likeness score values of −4.7, 4.2, (0.4, 0.4), (4.4, 4.3), and 0.7, respectively. Also, the deaminated analog 10 [Ki (hD2SR) = 3.2 ± 0.4 nM; Ki (hD3R) = 8.5 ± 2.2 nM] revealed clogS, clogP, LE (hD2SR, hD3R), LipE (hD2SR, hD3R) and drug-likeness score values of −4.7, 4.2, (0.4, 0.4), (3.9, 3.5), and 0.4, respectively. The results observed for the newly developed benzothiazole-based ligands 3-20 provide clues for the diversity in structure activity relationships (SARs) at the D2SR and D3R subtypes.
Paramyxo- and pneumoviruses include many pathogens with great relevance for human and animal health. To identify common host factors involved in the Paramyxo- and Pneumoviridae life cycle as a basis for new insights in the biology of these viruses and the development of rationally designed therapeutics, genome scale siRNA screens with wild-type measles, mumps, and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line, were performed. A comparative bioinformatics analysis yielded different members of the coatomer complex I, the translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. The strongest common hit, ABCE1, an ATP-binding cassette transporter member, was chosen for further study. We found that ABCE1 supports replication of all three viruses, confirming its importance for both virus families. While viral protein kinetics showed that ABCE1 knockdown resulted in a drastic decrease of MeV protein expression, viral mRNA kinetics are not directly affected by a reduction of ABCE1.
The impact of ABCE1 on viral and global cellular translation was investigated using both 35S metabolic labelling and non radioactive fluorescent protein labelling. ABCE1 knockdown strongly inhibited the production of MeV proteins, while only modestly affecting global cellular protein synthesis and showed that ABCE1 is specifically required for efficient viral, but not general cellular, protein synthesis, indicating that paramyxoand pneumoviral mRNAs may exploit specific translation mechanisms.
In a second approach the efficacy of the small-molecule polymerase inhibitor ERDRP-0519 against MeV was assessed in squirrel monkeys. Animals treated with the drug experienced less severe clinical disease compared to untreated controls, and this effect correlated with the onset of drug treatment.
We observed a reduction of levels of PBMC-associated viremia and virus release in the upper airways, illustrating effective inhibition of virus replication by the drug treatment. ERDRP-0519 drug treatment also alleviated MeV-induced immunosuppression. In addition to providing proof-of-concept for the support of MeV eradication efforts by preventing disease and transmission with a small-molecule polymerase inhibitor, this dissertation provides a novel perspective on cellular proteins that impact the replication of MeV, MuV and HRSV and highlights the role of ABCE1 as host factor that is required for efficient paramyxo- and pneumovirus translation.
ATP-binding cassette (ABC) transporters constitute an omnipresent superfamily of integral membrane proteins, which catalyze the translocation of a multitude of chemically diverse substrates across biological membranes. In humans, ABC transporters typically act as highly promiscuous exporters, responsible for many physiological processes, multi-drug resistance, and severe diseases, such as hypercholesterolemia, lipid trafficking disorders, and immune deficiency. In all ABC transporters, ATP-driven movements within two highly conserved nucleotide-binding domains (NBDs) are coupled to conformational changes of two transmembrane domains (TMDs), which provide a framework for substrate binding and release on the opposite side of the membrane and enable the transporter to cycle between inward-facing and outward-facing orientations. Several structures of ABC transporters determined either by X-ray crystallography or single-particle electron cryo-microscopy (cryo-EM) have been reported, mostly exhibiting a variation of the inward-facing state, which highlights their dynamic behavior. However, for a complete understanding of the conformational dynamics, further structural information on intermediates is needed – especially for heterodimeric ABC transporters, which are predominant in humans and for which only limited structural information is available.
One prime example of such human heterodimeric ABC transport complexes is the transporter associated with antigen processing (TAP). TAP is a key player of the adaptive immune response, because it translocates proteasomal degradation products into the ER lumen for loading of MHC I molecules. Many functional aspects of TAP have been disclosed in recent years. However, structural information is lacking far behind and a major challenge in the field of medical relevant transporters. Recently, the heterodimeric ABC export system TmrAB (Thermus thermophilus multidrug resistance proteins A and B) was identified as an ortholog of TAP, by sharing structural homology with TAP and, intriguingly, being able to restore antigen presentation in human TAP-deficient cells. Thus, TmrAB is a biochemically well-characterized ABC exporter that can be regarded as a functional ortholog of TAP and serves as a model system for (heterodimeric) ABC export systems in general.
Thus, to illuminate the molecular basis of substrate translocation by single-particle cryo-EM, one of the main objectives of this work was the generation of stabilizing chaperones (synthetic antibodies, nanobodies, cyclic peptides) to reduce the conformational heterogeneity of TAP and TmrAB. Selected antibodies were analyzed with respect to stable complex formation, conformational trapping, and the ability to serve as alignment tools for structural studies by single-particle cryo-EM. Both antibody types were shown to form sufficiently stable complexes to serve as a rigid body for EM analyses. However, all selected antibodies bound to the inward-facing state exclusively.
Hence, for EM studies, various ligands were added to elucidate the full spectrum of conformational states during the catalytic cycle. For TAP, first attempts by negative-stain EM revealed a homogenous distribution of particles on the grid. Surprisingly, no transporter-like features were observed although various attempts were applied to increase the overall sample quality.
For TmrAB, in contrast, the complete conformational space in a native-like lipid environment under turnover conditions was mapped. Cryo-EM analysis of TmrAB incubated with ATP-Mg2+ and substrate revealed two distinct inward-facing conformations (IFwide and IFnarrow) as well as two asymmetric conformations with dimerized NBDs, which were markedly different from all previously reported structures. Here, the catalytically active site was slightly wider and contained ADP, while ATP was still bound at the catalytically-inactive site within the NBDs, demonstrating an asymmetric post-hydrolysis state. Intriguingly for the inward-facing conformations, a weak additional density close to residues M139TmrB and W297TmrB was observed in the inward-facing conformation, which displayed a higher degree of cytosolic gate opening (IFwide) indicating the presence of substrate. To verify that this density corresponds to substrate, single alanine mutations of M139TmrB and W297TmrB were introduced, leading to a strong reduction in substrate binding and transport. Since substrate release requires the opening of the extracellular gate, the absence of an outward-facing open conformation indicated that the opening must be highly transient. In order to explore the outward-facing open conformation, a cryo-EM analysis of the catalytically-inactive TmrAE523QB mutant upon incubation with ATP-Mg2+ was performed. Remarkably, within the same dataset, two different outward-facing conformations (occluded and open) were resolved, both in an ATP-bound state, which indicated that binding of ATP is sufficient to drive the large-scale conformational transition from inward-facing to outward-facing open. To explore the effect of nucleotide hydrolysis, TmrAB was trapped by vanadate. Again, two populations were observed, representing the outward-facing open and outward-facing occluded conformation.
Based on several structures of key intermediates, determined under turnover conditions or trapped in the pre-hydrolysis and hydrolysis transition state, for the first time the complete description of the ATP hydrolysis and translocation cycle of a heterodimeric ABC transport complex was elucidated in one single study. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates, fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters were resolved and a general template for (heterodimeric) ABC exporter-catalyzed substrate translocation was provided.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review.
Living matter is defined by metastability, implying a tightly balanced synthesis and turnover of cellular components. The first step of eukaryotic protein degradation via the ubiquitin-proteasome system (UPS) leads to peptides, which are subsequently degraded to single amino acids by an armada of proteases. A small fraction of peptides, however, escapes further cytosolic destruction and is transported by ATP-binding cassette (ABC) transporters into the endoplasmic reticulum (ER) and lysosomes. The ER-resident heterodimeric transporter associated with antigen processing (TAP) is a crucial component in adaptive immunity for the transport and loading of peptides onto major histocompatibility complex class I (MHC I) molecules. Although the function of the lysosomal resident homodimeric TAPL-like (TAPL) remains, until today, only loosely defined, an involvement in immune defense is anticipated since it is highly expressed in dendritic cells and macrophages. Here, we compare the gene organization and the function of single domains of both peptide transporters. We highlight the structural organization, the modes of substrate binding and translocation as well as physiological functions of both organellar transporters.
Die Lebensfunktion der Zelle beruht unter anderem auf der Funktion und Wechselwirkung der Nukleinsäuren DNA (2’-Desoxyribonukleinsäure) und RNA (Ribonukleinsäure). Mit Hilfe von PDS (engl. ’pulsed dipolare spectroscopy’)-Techniken, basierend auf der EPR (engl. ’electron paramagnetic resonance’)-Spektroskopie, können Abstände in einem Bereich von 2-10 nm zwischen zwei markierten Positionen einer Nukleinsäure bestimmt werden. Daneben kann mit der Abstandsverteilung auf die Flexibilität des Moleküls geschlossen werden. Durch PDS-Messungen eröffnet sich die Möglichkeit, Bewegungen und Zustandsänderungen zu untersuchen. Die Messungen beruhen auf der dipolaren Kopplung von Radikalen (Spinlabel). Da die gemessenen dipolaren Kopplungen eine anisotrope Wechselwirkung sind, können an starren Systemen neben den Abstandsinformationen auch die Orientierungen der beiden Spinlabel zueinander bestimmt werden. Diese zusätzliche Information ermöglicht es, mittels orientierungsselektiver PDS-Messungen noch genauer die Geometrie und Flexibilität des Systems zu untersuchen. Klassischerweise werden alle Messungen mit der Doppelfrequenztechnik PELDOR (engl. ’pulsed electron-electron double resonance’) durchgeführt. Einzelfrequenzmethoden basieren dagegen auf Breitbandanregung, die mit den technischen Gegebenheiten l nge nicht möglich war. Eine solche Sequenz ist 2D-SIFTER.ImmRahmen dieser Arbeit von PELDOR ausgehende, weiterentwickelte Simulationsprozedur etabliert. Eine große Herausforderung ist die eindeutige Interpretation der sensitiven orientierungsselektiven PELDOR-Messungen. Sie mittels MD (Moleküldynamik)-Simulationen zu beschreiben war bisher nur qualitativ möglich. Allerdings wurden mehrere neue Kraftfelder publiziert. Mit einem quantitativen Vergleich mit orientierungsselektiven PELDOR-Daten kann sichergestellt werden, dass die Flexibilität des Systems durch Kraftfelder richtig beschrieben ist. PELDOR-Zeitspuren, gemessen bei Raumtemperatur und 50 K, unterscheiden sich besonders in ihrer Dämpfung. Der physikalische Unterschied beider Messungen konnte durch MD-Simulationen qualitativ nachvollzogen worden. Eine Schwierigkeit für speziell orientierungsselektive PELDOR-Messungen ist die aufwendige Synthese von mit dem starren Ç-Label markierten Nukleinsäuren. Als Alternative wurde in der Sigurdsson-Gruppe das halbstarre IMU-Label entwickelt. Die Analyse der orientierungsselektiven Daten ergab ein klares Bild der Dynamik dieses Labels. Ein weiterer interessanter Spinlabel ist der `G. Dieser Label ist nicht kovalent gebunden, sondern interkaliert in eine Stelle der Nukleinsäure, in der eine Guanin- Base fehlt. MD-Simulationen im quantitativen Vergleich mit orientierungsselektiven PELDOR-Messungen an verschiedenen Magnetfeldern haben eine hohe Übereinstimmung. Dabei konnte gezeigt werden, dass der Label, interkaliert in eine dsDNA, flippen kann, was zu einer Ausmittelung der Anisotropie führt, allerdings zu keiner Verbreiterung der Abstandsverteilung. Dagegen wird in der dsRNA dieses Flippen um die Einfachbindung sterisch gehindert, so dass neben dem Abstand auch die Orientierung des Labels bestimmt werden kann. Kurze dsRNA-Bausteine tendieren dazu, Oligomere zu bilden, was zu Multispineffekten führte. Zusätzlich beeinflusst diese Aggregation die Dynamik der einzelnen RNAs. Daher musste dieses ’end-to-end’-Stacking verhindert werden. Eine Nukleobasean einem Ende der dsRNA führt zu einer Dimerisierung, während eine Nukleobase an beiden Seiten dieses Stacking vollständig verhindert. Messungen mit unterschiedlichen Salzkonzentrationen konnten zusätzlich zeigen, dass die Interaktion zweier dsRNAs bei höheren Salzkonzentrationen zunimmt.
The three major autoimmune diseases (ADs) of the liver are primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). All of those diseases show an aggressive immune reaction resulting in the destruction of liver tissue and finally to the development of hepatic fibrosis.
PSC is an autoimmune mediated disease of unknown etiology. It is characterized by inflammation of intra- and extrahepatic bile ducts. The progressive destruction of the bile ducts can lead to liver cirrhosis and finally to liver failure. Clinical signs for PSC are increased alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) levels, presence of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and bile ducts with characteristic strictures and dilations of the biliary tree as well as onion skin fibrosis surrounding the damaged bile ducts. Currently, there is no established treatment for PSC patients. The administration of ursodeoxycholic acid (UDCA) is being use as a therapy. However, it merely serves a symptomatic treatment to reduce serum AP and GGT as well as the formation of gallstones. In the advanced stage of PSC, liver transplantation is the last therapeutic option. Mdr2-/- mice are an excepted mouse model for human PSC. Such mice show lymphocytes infiltration into the liver, bile duct lesions, as well as the presence of the typical onion skin-like pericholangitis and periductal fibrosis.
AIH is a rare chronic autoimmune disease of the liver that results from the loss of self-tolerance to hepatocytes and leads to destruction of the hepatic parenchyma with the onset of cirrhosis. Clinical signs for AIH are elevated alanine aminotransferase (ALT) and aspartate transaminase (AST) levels, hypergammaglobulinemia and different types of autoantibodies. In addition, interphase hepatitis with lymphocytic and plasmacellular infiltrates in the periportal field are characteristic for AIH. Two different subtypes of AIH exist and depending on their autoantibody profile they can be distinguished into AIH type 1 which is characterized by the presence of anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies, and AIH type 2 showing liver/kidney microsomal autoantibodies (LKM-1). LKM-1 recognizes the major autoantigen, the 2D6 isoform of the cytochrome P450 enzyme family (CYP2D6). One mouse model for AIH is the CYP2D6 model in which the injection of Ad-2D6 leads to a breakdown of the immune tolerance by the destruction of hepatocytes.
There are some patients with autoimmune diseases of the liver who have both cholestatic and hepatic liver enzymes and histological features suggestive of two different liver diseases. These patients are diagnosed with an overlap syndrome (OS).
In my thesis I generated an animal model with characteristics of both diseases, which would mimic features of human PSC-AIH OS. Mdr2-/- mice which spontaneously develop PSC were infected with Ad-2D6 to trigger the autoimmune-driven hepatic injury. Pathogenesis of PSC-AIH OS mice was compared to mice with solitary PSC or AIH. Naïve FVB wild type mice have been used as healthy controls. The characterization of the PSC-AIH OS model was done by analyzing serological parameters like ALT, AP, different antibodies like pANCA, LKM-1 like CYP2D6 and total IgG. Additionally, fibrosis and cholangitis were analyzed by immunohistochemistry and Western blotting. Moreover, cellular infiltrations of CD4+ and CD8+ T cells, dendritic cells (DCs), monocytes/macrophages and neutrophils were determined with immunohistochemistry. Finally, the overall immune balance in the liver and the frequency of CYP specific T cells were analyzed via flow cytometry. Our new mouse model indeed represents the characteristics of both PSC and AIH and mimics features of the human PSC-AIH OS. It allows studying the development of a PSC-AIH OS and how the two overlapping diseases are influencing one another. In a second approach I wanted to induce CYP2D6-specific tolerance in AIH mice. Therefore, I tried four different approaches, namely intranasal peptide administration, injection of tolerogenic DCs, antigen-coupled splenocytes, and Ag-coupled nanoparticles (NP) and evaluated their potential to induce CYP2D6 specific Treg with the capacity to prevent AIH in mice. Unfortunately, the intranasal peptide administration and also the injection of tolerogenic DCs did not increase the amount of CYP2D6 specific Treg which would lead to a reduction of the frequency of inflammatory T cells. Surprisingly, the injection of antigen-coupled splenocytes showed the opposite effect characterized by a very strong cytokine secretion in the tolerized mice. The use of NPs led to an increase in CYP2D6 specific Treg as well as in decrease in the frequency of inflammatory T cells and finally has the potential for a therapeutic approach.
In summary, the generated PSC-AIH OS model represents many clinical signs which can also be observed in PSC-AIH OS patients. This model can be used to study the etiology of this overlap syndrome and further to test potential therapeutic approaches. The different immune tolerance induction pathways which I tried in the AIH model show that NPs have to potential to induce immune tolerance but this approach has to be refined and the outcome has to be characterized in more detail.
Die CXCR4-CXCL12-Signalachse gilt als eines der bislang am besten studierten Signalsysteme in der Hämatopoese. Allerdings stammt unser Wissen über diesen kritischen Signalweg maßgeblich aus subtraktiven Studien, wie z.B. knock-out Modellen oder pharmakologischer Inaktivierung. Zwar können aus diesen Modellen wichtige Erkenntnisse über die physiologische Rolle dieses Signalwegs abgeleitet werden, aber dennoch bleiben einige Phänomene ungeklärt. So konnte gezeigt werden, dass es sowohl bei CXCR4-Defizienz als auch bei Patienten mit dem WHIM-Syndrom (ausgelöst durch eine überaktive CXCR4-Mutante) zu einer ausgeprägten B-Zellaplasie kommt. Dies scheint intuitiv nicht vereinbar. Daher wurde in der vorliegenden Arbeit ein Modell mit einer überaktiven CXCR4- Mutante (CXCR41013/1013) hinsichtlich der (un)reifen Hämatopoese systematisch untersucht.
Zunächst wurden hämatopoetische Stamm- und Vorläuferzellen (HSPC) hinsichtlich der aberranten CXCR4-Signalweiterleitung ex vivo analysiert. Die CXCR4-Überaktivierung konnte sowohl in frühen Effekten nach Aktivierung des Rezeptors (F-Aktinpolymerisierung, Aktivierung des MAPK- Signalweges), als auch in späten, zellfunktionellen Effekten (Migrationsassay) nachgewiesen werden. Die veränderte CXCR4 Signalintegration hatte auch bereits in der Homöostase organismische Konsequenzen im Mausmodell. So konnte eine massiv vergrößerte HSPC-Population in der Milz von CXCR41013/1013-Tieren detektiert werden, im Sinne einer extramedullären Hämatopoese. Knochenmarks-HSPC aus CXCR41013/1013-Tiere zeigten ein massiv eingeschränktes (serielles) Repopulationspotenzial. Kombiniert mit der oben genannten ausgeprägten extramedullären Hämatopoese in diesen Tiere interpretieren wir diese Beobachtung als starken Hinweis auf eine dysfunktionelle Interaktion der Stammzellen mit der hämatopoetischen Stammzellnische im Knochenmark. In diesem Zusammenhang besonders interessant ist die Tatsache, dass auch ein Kompetitorknochenmark das Überleben einer Sekundärtransplantation nicht sichert. Dabei ist zu diskutieren, ob dieser Effekt durch eine effizientere Besetzung von Stammzellnischen durch CXCR41013/1013-Zellen, eine Akkumulation von CXCL12 in der Knochenmarkflüssigkeit (siehe unten) oder eventuell sogar ein vesikelabhänginger Transport von mutiertem CXCR4 in Kompetitorzellen ausgelöst wird. Ein weiteres Merkmal dieser Dysfunktion könnte ebenfalls die gezeigte Akkumulation von CXCL12 in der Knochenmarkflüssigkeit von CXCR41013/1013-Tiere darstellen. Diese Akkumulation könnte die Suppression co-transplantierter wildtypischer Hämatopoese sowie die verminderte Effizienz der G-CSF-induzierten Stammzellmobilisierung funktionell erklären. Zusätzlich konnte gezeigt werden, dass die Mobilisierung von Stammzellen aus dem Knochenmark durch einen CXCR4- Inhibitor in CXCR41013/1013-Tieren ebenfalls erheblich hinter der in Wildtypmäusen zurückbleibt.
Analog zu Patienten mit WHIM-Syndrom zeichnen sich CXCR41013/1013-Mäuse weiterhin durch eine ausgeprägte Leukopenie, insbesondere durch einen schweren B-Zell-Mangel, aus. Aus diesem Grund wurde die B-Lymphopoese und humorale Immunfunktion genauer analysiert. Eine grundsätzliche humorale Immunkompetenz von CXCR41013/1013-Tieren konnte nachgewiesen werden, jedoch ist die B-Memory-Funktion erheblich eingeschränkt. Durchflusszytometrisch und funktionell konnte eine reduzierte preB/pro-B Population im Knochenmark bei einer gleichzeitig vergrößerten preB/pro-B Population in der Milz (vgl. extramedulläre Hämatopoese) nachgewiesen werden. Ebenfalls konnten wir in dieser Zellpopulation eine stark erhöhte CXCR4-Oberflächenexpression im Vergleich zu wildtypischen Zellen nachweisen. Da diese unreifen B-Zellen keine verstärke Apoptoserate aufweisen, gehen wir derzeit davon aus, dass der Differenzierungsstopp nicht durch selektiven Zelltod, sondern durch aberrante Retention der preB/proB-Zellen in einer primitiven B-Vorläufer- Nische im Knochenmark zustande kommt, beziehungsweise durch eine gestörte Migration in differenzierende Nischen im Knochenmark. Alternativ könnte die Überdosis CXCR4-Signal differenzierenden Signalen entgegenstehen. Beide Hypothesen können das eingangs erwähnte Paradoxon bezüglich einer B-Zellaplasie in CXCR4-defizienten und CXCR4-überaktiven Zellen hinreichend erklären.