Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

Selenocysteine (Sec) is the 21st amino acid. Unlike other amino acids incorporation of Sec into proteins is more complex by far as it is encoded by the opal stop codon UGA. A complex and highly sophisticated incorporation mechanism is required in order to assure the correct co-translational incorporation of Sec into the nascent polypeptide chain. Even after years of intensive research the question as to why selenoproteins are important for mammalian life has not been fully understood. This study aimed to provide answers to this question using gluthathione peroxidase 4 (GPx4) as a model enzyme. It has been shown that GPx4 is essential for early embryogenesis, neuro- and retina-protection and male fertility in mice. But the role of selenothiol- versus thiol-based GPx4 catalysis in cells and mice, as well as the role of the catalytic tetrad in GPx4 catalysis and the subcellular localisation of the different GPx4 isoforms and their impact on cell protection have remained unclear. Therefore, a series of mutant variants of GPx4 were generated by site-directed mutagenesis, stably expressed in tamoxifen-inducible GPx4 knockout cells (Seiler et al., Cell Metab 2008), and functionally analysed. GPx4 mutant variants included those of the peroxidatic Sec, the catalytic tetrad, the mitochondrial leader sequence (Mls) and the non-peroxidatic cysteines. These studies revealed that Cys can functionally replace Sec in the active centre of GPx4 to a large extent in cells, whereas some amino acids of the catalytic site are necessary for GPx4 function. None of the non-peroxidatic Cys were shown to be required for the cell-protective function of GPx4, ruling out a possible resolving Cys in the catalytic cycle of GPx4 unlike homologues in other organisms that use a resolving Cys. The fuction of a resolving Cys is to start a nucleophilic attack upon the intermolecular bond between the peroxidatic Cys and the substrate in order to create an intramolecular disulfidbride. Apparently subcellular localization of GPx4 in the extramitochondrial/cytosolic compartment is crucial for its cell death preventing qualities as overexpression of mitochondrial GPx4 in the knockout cells failed to rescue cell death induced by endogenous GPx4 disruption.

Serine proteases in cytoplasmic granules of neutrophils (NSPs), namely neutrophil elastase, cathepsin G (CG) and proteinase 3, have been under intense investigation for several decades. They are mainly known for their role in intracellular killing of pathogens and are also increasingly recognized as key regulators of innate immune responses. In 2009, I identified a fourth serine protease in neutrophils that has been completely overlooked and neglected so far. The aim of this thesis was an in-depth biochemical and functional characterization of this novel serine protease 4 (NSP4) of human neutrophils. Using monoclonal antibodies to NSP4, the distribution of NSP4 in normal human tissues was studied. NSP4 was observed only in neutrophils and neutrophil precursors of the bone marrow. The content of NSP4 in neutrophil lysates was about 20-fold lower compared to CG. Nevertheless, NSP4 was found to be released into the supernatant upon neutrophil activation. NSP4 could be further identified as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions. For the functional analysis, the production and yield of recombinant NSP4 was clearly improved using different expression systems and DNA construct modifications. The proteolytic specificity was analyzed using E. coli peptide libraries, mass spectrometry and several synthetic peptide libraries. All these analyses clearly revealed an arginine specificity for NSP4. Consistent with this, NSP4 was strongly inhibited by heparin-accelerated antithrombin and C1 inhibitor and, with lower efficacy, by α1-proteinase inhibitor (α1PI). The data allowed me to generate an NSP4-specific α1PI variant that was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils. This finding strongly indicated that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. In addition, dipeptidyl peptidase I (DPPI) was identified as the activator of NSP4 in vivo, as DPPI deficiency resulted in complete absence of NSP4 in a Papillon-Lefèvre patient. Analysis of cell-based calcium assays revealed that proteinase-activated receptor-2 may represent a potential natural substrate of NSP4. So far, NSP4-deficient mice did not show an abnormal phenotype under clean housing conditions. Activation of isolated neutrophils by phorbol esters or immune complexes was also not impaired. This study establishes NSP4 as the only arginine-specific pre-activated serine protease stored in azurophil granules of neutrophils that may fullfil a quite distinct, supportive role in neutrophil responses to tissue damage and bacterial infections.

Complex phenotypes are the result of a complex interplay between genes and environmental factors. Extensive linkage, candidate and genome-wide association studies (GWASs) have been carried out to unravel genetic risk variants for human diseases. The identification of genes, involved in the pathomechanism of a disease, might be beneficial for its diagnosis, treatment and prognosis. While GWASs allowed the identification of a large number of common variants robustly associated with common complex diseases, the heritability, which can be explained by these variants, is small. The discrepancy between the estimated heritability from twin, family and adoption studies and the heritability obtained from GWAS was termed “missing heritability” and led to the investigation of additional factors that might also contribute to disease susceptibility, including gene-environment interactions, gene-gene interactions, structural variants and rare variants. In this thesis, the role of less common and rare variants in susceptibility to common complex diseases was investigated. In order to accomplish this, a candidate gene for panic disorder (PD) and a possible risk gene for major depressive disorder (MDD) were screened for the presence of common and rare variants using next-generation sequencing in a pooled approach. In a previously published GWAS, a haplotype containing two common intronic variants in the transmembrane protein 132D (TMEM132D) gene was associated with PD. Another GWAS identified solute carrier family 6 member 15 (SLC6A15), which encodes an amino acid transporter, as a risk gene for MDD. A common intergenic variant about 600 kilobase downstream of this gene was shown to decrease SLC6A15 gene expression in lymphoblastoid cell lines and hippocampus. Susceptibility genes for complex diseases, identified in GWAS, are promising candidates for the search of rare variants as genes harbouring common variants are likely to contain also rare variants. Pooled targeted re-sequencing of the exonic regions of TMEM132D in 300 anxiety disorder patients, mostly suffering from PD (84.7%), and 300 healthy controls allowed the detection of 371 genetic variants. Of these variants, 24.0% were common (minor allele frequency (MAF) > 5.0%), whereas the vast majority was less common (MAF 1.0 – 5.0%) to rare. 247 variants had not been reported before, including 12 novel non-synonymous variants leading to an amino acid exchange in the protein. While common variants associated with PD were not identified, an overrepresentation of non-synonymous variants and variants with predicted changes on splicing in healthy controls compared to PD patients was observed. These putatively functional relevant variants were distributed along a broad MAF spectrum, ranging from 0.17 to 30.0%. In addition, a higher rate of private non-synonymous variants, which were only present in either cases or controls in this study, but not in over 7,500 individuals with different ethnic backgrounds from other publicly available re-sequencing datasets, in patients compared to controls was seen. Combined with the data from the previous GWAS study in which the association with PD was carried by common variants, this pooled re-sequencing study suggests that not only common or rare variants alone, but a combination of both contributes to the development of anxiety-related phenotypes. Re-sequencing the whole SLC6A15 locus in 400 MDD patients and 400 healthy controls, 405 genetic variants were identified, including twelve non-synonymous variants. Only 15.0% of the detected variants were common. While none of the non-synonymous variants was significantly associated with MDD, two rare non-synonymous variants were identified to influence protein function. In contrast to the TMEM132D protein whose molecular function has still to be discovered, SLC6A15 is known to transport neutral amino acids into predominantly neuronal cells. The cellular uptake of neutral amino acids such as proline is thus a measurable property that associates with function. The uptake experiments identified two rare variants to be associated with a significant increase in proline uptake in HEK cells. This result suggests that rare variants in SLC6A15 might influence the biochemical function of its amino acid transporter and thus downstream neuronal function and possibly the risk for MDD and other stress-related psychiatric disorders. In addition, this study highlights that functional exploration of genetic variants might be promising to identify putatively disease-relevant variants as statistically significant associations for rare variants might only be achieved in extremely large samples.

New vectors for successful transgene delivery in patients are more than needed. Current gene therapeutic vectors are mostly based on virus genomes. A main reason is the very efficient administration of the vector into the target cells. The viral background of these systems also has severe side effects like e.g. immunological reactions of the patient or the activation of oncogenes due to insertional mutagenesis, which can result in cancer development. A possibility to overcome these problems is the establishment of extrachromosomally maintained, autonomously replicating and non-viral vectors. The goal of this work was to establish such a novel system with the help of the CENH3 protein. The centromere-specific histone 3 variant CENH3 (H.sapiens: CENH3CENP-A, D.melanogaster: CENH3CID) is sufficient for centromere formation. It confers a stable and epigenetically heritable mark at the centromeric region and does this also on plasmids. With the help of CENH3 I changed the passive piggyback segregation mechanism of Epstein-Barr virus-derived vectors into an active segregation mechanism for plasmid DNA (pDNA) vectors. I demonstrated that the new and active pDNA vector segregation mechanism prolongs pDNA vector retention in cells. The information gained from basic research might have great impact in the field of gene-therapeutic research, as this mechanism might be a helpful tool in future gene therapeutic vectors.

The long-term success of an introduced population depends on the ecological conditions in its new environment, but is also influenced by stochasticity. This is particularly clear in the first stage of an invasion when the population is still small and either goes extinct quickly or establishes a self-sustaining population. Once established, some populations grow and spread spatially, with potential impacts on native communities and ecosystems. The role of stochasticity during these later invasion stages remains unclear. Furthermore, little is known about the population genetic and evolutionary consequences of stochastic invasion trajectories. With this dissertation, I would like to contribute to a stochastic eco-genetic theory of the entire invasion process—from the first introduction up to potential impacts. The overarching questions in this dissertation are: a) How does a population’s movement through the invasion process depend on ecological factors influencing its average growth rate? b) How does it depend on factors influencing the stochastic variability in the population dynamics? c) How much genetic diversity do introduced populations harbor on average upon reaching a certain point in the invasion process? d) To what extent can the population-genetic consequences of invasion trajectories feed back onto the population dynamics? Together with my advisors and coauthors, I have conducted four studies, each addressing two or more of these questions for specific ecological scenarios. We employ several types of stochastic models: Markov chains, Markov processes, their diffusion approximations, and coalescent-like genealogy simulations. In Chapter 1 (Wittmann et al., 2013a, appeared in Theoretical Population Biology), we focus on a factor influencing the introduced population’s average growth rate: the intensity of competition with an ecologically similar native species. Our results indicate that the expected time until the introduced species drives the native competitor to extinction is smallest for intermediate competition intensity. This phenomenon results from the opposing effects of competition intensity at different points of the invasion process: On the one hand, intense competition renders the establishment of the introduced population more difficult; on the other hand, it facilitates the later exclusion of the native species. In Chapter 1, we also investigate to what extent the native species’ extinction is accelerated if a reduction in population size entails a reduction in genetic diversity and thus a reduced ability to adapt to a changing environment. We find this eco-genetic feedback to be particularly strong at small competition intensities. In Chapter 2 (Wittmann et al., 2013b, in press at Oikos), we compare introduction regimes with the same average number of individuals introduced per time unit, but with a different temporal distribution. Relative to regimes with many small introduction events, regimes with few large introduction events generate more variability in population-size trajectories. We show that this variability helps introduced populations to overcome difficult stages in the invasion process (those with a negative average growth rate), but is disadvantageous during easy stages (those with a positive average growth rate). In the light of our results, we can reinterpret three published data sets on invasion success under different introduction regimes. In Chapters 3 and 4 (Wittmann et al., 2013c,d), we examine levels of genetic diversity in populations that have successfully overcome a strong demographic Allee effect. In this ecological scenario, the average population growth rate is negative below a certain critical population size and positive above, such that the first stage in the invasion process is difficult and the second one easy. In Chapter 3, we assume Poisson-distributed offspring numbers. We show that compared to successful populations without an Allee effect, successful Allee-effect populations are expected to harbor either more or less genetic diversity, depending on the magnitude of typical founder population sizes relative to the critical population size. Part of the explanation is that, counter-intuitively, successful Allee-effect populations escape particularly fast from the range of small population sizes where genetic drift is strongest. In Chapter 3, we also identify conditions under which the critical population size can be estimated from genetic data. In Chapter 4, we consider a range of offspring-number models leading to either more or less variability in population dynamics than the Poisson model. For a fixed founder population size, we observe that the Allee effect has a negative influence on genetic diversity for small amounts of variability, but a positive influence for large amounts of variability. We show that the differences between our various offspring-number models are so substantial that they cannot be resolved by rescaling the parameters of the Poisson model. Taken together, these results offer some general conclusions with respect to the four main questions raised above. a) How fast an introduced population completes the invasion process is mainly determined by the presence and severity of difficult stages. Therefore, an ecological change promotes invasion success if it lessens such difficult stages. b) From the perspective of the introduced population, variability is advantageous during difficult but not during easy stages of the invasion process. c) Because the strength of genetic drift depends on population size, a key to understanding the population genetic consequences of invasion trajectories is to consider how much time the population of interest spends in different population-size ranges. d) Feedbacks between a reduction in population size and a loss of genetic diversity are strongest in ecological scenarios where the population of interest spends considerable time at small population sizes. Some of the most striking results in this dissertation cannot be understood from a deterministic point of view, but only when considering stochasticity. Thus, stochasticity does not just add “noise” to some average outcome, but can qualitatively change the behavior of biological systems.

Es gibt zahlreiche Bemühungen, neue Immuntherapien zur Behandlung von malignen Tumoren zu entwickeln, da die Prognose vieler Krebserkrankungen immer noch schlecht ist. Dazu muss das Verständnis, wie die verschiedenen Immunzellen innerhalb des Tumormilieus miteinander interagieren, verbessert werden. Mausmodelle für Spontantumoren spiegeln die klinische Situation besser wider als transplantierbare Tumoren und stellen somit eine gute Möglichkeit dar, die während der Tumorentwicklung stattfindenden Immunreaktionen zu analysieren. In der vorliegenden Dissertation wurde mit Mäusen gearbeitet, in deren B-Zellen das Onkogen c-MYC konstitutiv exprimiert wird und die nach einigen Wochen spontan B-Zell-Lymphome entwickeln. Frühere Arbeiten haben bereits gezeigt, dass natürliche Killerzellen (NK-Zellen) aus c-MYC-Tumoren im Vergleich zu Organen aus Wildtyp (wt)-Mäusen kaum Interferon-gamma (IFN-) produzierten und die Fähigkeit, Zielzellen zu töten, stark reduziert war. Dementsprechend sezernierten Tumor-infiltrierende dendritische Zellen (TIDZ) geringere Mengen Interleukin-12 (IL-12), das für die Induktion einer Th1 (T-Helferzelle 1) / ZTL (zytotoxischer T-Lymphozyt)-Antwort essenziell ist, aber vermehrt das immuninhibierende Zytokin IL-10. Für die Aktivierung von naiven T-Zellen ist die Hilfe von NK-Zellen und DZ notwendig, die in c-MYC-Mäusen offenbar nicht gegeben war. Es wurde daher erwartet, dass intratumorale T-Zellen nicht bzw. nur ungenügend aktiviert werden. Überraschenderweise wiesen Tumor-infiltrierende T-Zellen jedoch einen aktivierten Phänotyp auf, zeigten zum Teil sogar eine erhöhte IFN--Produktion, konnten degranulieren und proliferierten in vivo. Trotzdem übernahmen intratumorale T-Zellen im Gegensatz zu NK-Zellen keine immunüberwachende Funktion über die Lymphomentstehung, da c-MYC-Mäuse nach Depletion der T-Zell-Population nicht früher erkrankten als unbehandelte Tiere. Offenbar wurden T-Zellen in c-MYC-Tumoren Antigen-spezifisch aktiviert, was dazu führte, dass sie aufgrund der langanhaltenden Stimulation in einen Erschöpfungszustand geraten sind. Dies spiegelte sich in einer hohen Expression des Erschöpfungsmarkers PD-1 (Programmed Cell Death 1) wider. Die Interaktion des koinhibitorischen Rezeptors PD-1 mit dem entsprechenden Liganden PD-L1, der vor allem auf DZ in c-MYC-Tumoren detektiert wurde, führt in T-Effektorzellen zur Inhibierung des aktivierenden T-Zell-Rezeptor (TZR)-Signals. So konnten T-Zellen aus c-MYC-Tumoren im Vergleich zu T-Zellen aus normalen Mäusen in vitro über den TZR tatsächlich nicht mehr stimuliert werden, was ebenfalls für eine Erschöpfung sprach. Zudem exprimierten intratumorale T-Zellen neben PD-1 die koinhibitorischen Moleküle CTLA-4 (Cytotoxic T-Lymphocyte-Associated Antigen 4) und LAG-3 (Lymphocyte Activation Gene 3). Die Stimulierbarkeit der T-Zellen in vitro konnte durch Zugabe blockierender Antikörper (AK) gegen PD-1 und CTLA-4 verbessert werden. Aufgrund dieser Beobachtung wurden junge, noch gesunde c-MYC-Mäuse mit diesen AK behandelt. Durch die PD-1/CTLA-4-Blockade in vivo zeigten die Tiere ein signifikant verlängertes Überleben. Neben CD4+ T-Helferzellen und CD8+ zytotoxischen T-Lymphozyten wurde in c-MYC-Tumoren innerhalb der CD4+ T-Zellpopulation ein sehr hoher Anteil Foxp3+ regulatorischer T-Zellen (Treg) detektiert. Diese waren aktiviert, proliferierten in vivo und produzierten IL-10. Die koinhibitorischen Moleküle, welche auch auf erschöpften T-Zellen zu finden waren (PD-1, LAG-3 und CTLA-4), wurden ebenfalls von Treg exprimiert, was bekanntlich zur Verstärkung ihrer suppressiven Aktivität führt. Es wurden vor allem Helios-exprimierende natürliche Treg (nTreg) detektiert. Doch auch IL-10-produzierende, regulatorische Foxp3- Tr1-Zellen (T Regulatory Type 1 Zelle), die zu den induzierten Treg (iTreg) zählen, waren in c-MYC-Tumoren zu finden. Schließlich fanden sich auch T-Zellen, die sowohl IFN- als auch IL-10 produzierten. Diese könnten aus Th1-Zellen entstanden sein, welche die Zytokinexpression umgestellt haben. Treg unterdrückten in c-MYC-Tieren anscheinend eine effektive Antitumor-Immunantwort. Daher wurden Foxp3+ Treg in DEREG/c-MYC-Mäusen, die einen transgenen Diphtherietoxin (DT)-Rezeptor tragen, spezifisch durch DT-Injektionen in vivo depletiert. Erstmals wurde der DEREG-Mechanismus zur Treg-Depletion in einem endogenen Tumormodell angewandt. Die Depletion der Foxp3+ Treg verschaffte den DEREG/c-MYC-Mäusen einen leichten Überlebensvorteil. Diese Ergebnisse sind relevant für die Entwicklung neuer immuntherapeutischer Verfahren zur Behandlung maligner Erkrankungen in der Klinik.