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Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications. CHIP increase with age and is also associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with chronic ischemic heart failure (CHF) in humans is unknown. Therefore, we analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by NGS to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. Forty-seven mutations with a VAF of at least 2% were found in 18.5% of 200 patients with CHF. The mutations most commonly occurred in the genes DNMT3A and TET2. During a median follow-up of 4.4 years, a significantly worse clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers was notable. Importantly, there was a significant dose-response association between VAF and clinical outcome. Our data suggest that somatic mutations in hematopoietic cells, may be significantly associated with the progression and poor prognosis of CHF.
ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
The functional and molecular role of transglutaminase 2 in hematopoietic stem and progenitor cells
(2023)
Long-term repopulating hematopoietic stem cells (LT-HSCs) that reside in the bone marrow (BM) give rise to all blood cell types including erythrocytes, leukocytes and platelets. LT-HSCs are mainly quiescent during steady state hematopoiesis. LT-HSCs can process self-renewal to expand and maintain stemness, or commit to differentiation into short-term (ST) repopulating HSC and multipotent progenitors (MPPs). MPPs differentiate into oligopotent lineagerestricted progenitors which eventually produce all mature blood cell lineages, and thereby regenerate hematopoietic system.
Previous studies have shown in transcription profiles and quantitative PCR (qPCR) analysis that transglutaminase 2 (Tgm2) is one of the most upregulated genes in quiescent LT-HSCs in comparison to active HSCs, mobilized HSCs, ST-HSCs, MPPs, as well as leukemic stem cells (LSC). However, the reason why Tgm2 is strongly upregulated in dormant mouse LTHSCs and what the role of Tgm2 is in LT-HSCs has not been investigated yet.
Tgm2, encoded by the Tgm2 gene, is a multi-functional protein within the transglutaminase family. It has been found to be widely expressed inside and outside the cells. It consists of four domains and two functionally exclusive forms that are regulated by the Ca2+ and GTP concentration. Besides the most well-known transglutaminase enzymatic activity for transamidation, deamidation and crosslinking, Tgm2 acts also as a GTPase/ATPase, kinase, adhesion/scaffold protein, as well as disulfide isomerase. The role of Tgm2 in hematopoiesis remains elusive. Accordingly, the aim of this dissertation is to investigate the role of Tgm2 in murine hematopoiesis, especially in murine LT-HSCs.
Firstly, the expression of Tgm2 was analyzed in highly purified murine hematopoietic stem and progenitor cell (HSPC) populations. Low input label-free mass spectrometric proteomics and WES protein analysis confirmed the highly specific expression of Tgm2 in LT-HSCs at protein level. Already at the state of MPPs, Tgm2 protein was almost absent with further decline towards oligopotent progenitors. These results indicated Tgm2 as a specific protein marker for LT-HSCs, justifying the future generation of a fluorescent reporter mouse line based on endogenous Tgm2 tagging.
To delineate the functional and molecular role of Tgm2 in LT-HSCs, a conditional Tgm2 knockout mouse model was generated using the Mx1-Cre/loxP system, with the loxP sites flanking the coding exons of the catalytic domain of Tgm2. After PolyIC-mediated induction, a more than 95% knockout efficiency was observed in purified LT-HSCs and the protein expression of Tgm2 was confirmed to be vanished in the purified LT-HSCs from conditional Tgm2-KO mice. Conditional knockout mice are viable and show no aberrant organ functions.
In steady state condition, the distribution of mature blood cell lineages and immunophenotypically-defined HSPC populations within the BM, the mitochondrial potential of HSPCs reflected by the non-invasive cationic dye JC-1, as well as the cell cycle status of HSPCs mirrored by the intracellular Ki67 staining did not show any significant variations upon loss of Tgm2. However, the in vitro continuous observation of prospectivly isolated LT-HSCs by time-lapse microscopy-based cell tracking revealed a delayed entry into cell cycle with a two fold increased apoptosis rate after knocking out Tgm2, indicating Tgm2 expression might be essential for survival of LT-HSCs. Moreover, while the absence of Tgm2 in LT-HSCs did not influence differentiation and lineage choice in vitro, overexpression of Tgm2 in LT-HSCs resulted in an increase of the most immature subpopulation upon cultivation. All these features were not observed in Tgm2-deleted MPPs, suggesting Tgm2 playing a specific function at the level of LT-HSCs. Upon stress hematopoiesis, induced by the administration of 5-fluorouracil (5-FU), there was a trend towards delayed recovery of LT-HSCs lacking Tgm2. Although Tgm2 express specificly in LT-HSCs, two rounds of competitive BM serial transplantation displayed an equal overall engraftment and multi-lineage reconstitution of LT-HSCs from Tgm2-WT and Tgm2-KO mice in peripheral blood (PB), BM and spleens. Interestingly, LT-HSCs from Tgm2-KO mice reconstituted to more myeloid cells and fewer B cells in the first four weeks after primary transplantation, which disappeared at later time points.
Gene expression profiling and simultaneous single cell proteo-genomic profiling indicated that HSPCs and LT-HSCs from Tgm2-KO mice were transcriptionally more active. A heterogeneity of Tgm2 expression within Tgm2-WT LT-HSCs was revealed by single cell data. Commonly up-regulated genes in Tgm2-KO LT-HSCs and MPPs were significantly involved in regulation of transcription from RNA polymerase II promoter in response to stress, positive regulation of cell death as well as negative regulation of mitogen-activated protein kinase (MAPK) signaling pathways. In Tgm2-KO LT-HSCs, 136 up-regulated genes demonstrated an enrichment of genes involved in apoptosis, as well as negative regulation of MAPK signaling pathway.
Taken together, this dissertation shows that Tgm2 protein is highly specifically expressed in LT-HSCs, but not in subsequent progenitor populations. However, Tgm2 is not essential for differentiation and maturation of myeloid lineages, the proliferation and the long-term multilineage reconstitution potential of LT-HSCs after transplantation. Tgm2 might be involved in accurate stress response of LT-HSCs and the transition from LT-HSCs into MPPs, meaning that the absence of Tgm2 results in poor survival, myeloid bias upon transplantation, as well as slower recovery upon chemotherapeutic treatment.
Gravitropism is a fundamental process in plants that allows shoots to grow upward and roots to grow downward. Protein phosphorylation has been postulated to participate in the intricate signaling cascade of gravitropism. In order to elucidate the underlying mechanisms governing the gravitropic signaling and unearth novel protein constituents, an exhaustive investigation employing microgravity-induced phosphoproteomics was undertaken. The significantly phosphorylated proteins unraveled in this study can be effectively divided into two groups through clustering analysis. Furthermore, the elucidation of Gene Ontology (GO) enrichment analysis disclosed the conspicuous overrepresentation of these clustered phosphoproteins in cytoskeletal organization and in hormone-mediated responses intimately intertwined with the intricate phenomenon of gravitropism. Motif enrichment analysis unveiled the overrepresentation of [-pS-P-] and [-R-x-x-pS-] motifs. Notably, the [-pS-P-] motif has been suggested as the substrate for the Casein kinase II (CK II) and Cyclin-dependent kinase (CDK). Kinase-inhibitor assays confirmed the pivotal role played by CK II and CDK in root gravitropism. Mutant gravitropism assays validated the functional significance of identified phosphoproteins, with some mutants exhibiting altered bending kinetics using a custom-developed platform. The study also compared phosphoproteomics data from different platforms, revealing variations in the detected phosphopeptides and highlighting the impact of treatment differences. Furthermore, the involvement of TOR signaling in microgravity-induced phosphorylation changes was uncovered, expanding the understanding of plant gravitropism responses.
To fulfill the large-scale verification of interesting candidates from the phosphoproteomics study, a novel root and hypocotyl gravitropism phenotyping platform was developed. This platform integrated cost-effective hardware, including Raspberry Pi, a high-quality camera, an Arduino board, a rotation stage (obtained from Prof. Dr. Maik Böhmer), and programmable green light (modified by Sven Plath). In addition, through collaboration with a software developer, machine-learning-based software was developed for data analysis. This platform tested the gravitropic response of candidate mutants identified in the phosphoproteomics study. Furthermore, the capabilities of this platform were expanded to investigate tropisms in other species and organs. To find novel proteins that might act as partners of a key protein that is involved in gravitropism signaling, ALTERED RESPONSE TO GRAVITY 1 (ARG1), immunoprecipitation coupled with Mass Spectrometry (IP-MS) was performed and identified ARG1-LIKE1 (ARL1) as a potential interacting protein with ARG1. This interaction was further confirmed through in vivo pull-down assays and bimolecular fluorescence complementation assays. In addition, the interaction between ARG1 and HSP70-1 was also validated.
Overall, this thesis sheds light on the molecular components and signaling events involved in plant gravitropism. It contributes to existing knowledge and opens up new ways to investigate this fascinating area of plant biology.
Polyploidie in Prokaryoten
(2018)
Diese Arbeit teilt sich in drei Teile auf, die sich mit der Regulation der Polyploidie sowie mit der Genkonversion als evolutionären Vorteil von Polyploidie in Haloferax volcanii beschäftigen.
Im ersten Teil dieser Arbeit, wurde der Einfluss der DNA-Replikationsinitiatorproteine Orc1/Cdc6 auf das Ploidielevel untersucht. Hierbei konnte anhand von Deletionsmutanten zunächst gezeigt werden, dass lediglich drei der 16 Orc1/Cdc6-Proteine in H. volcanii essentiell sind. Bestimmung des Ploidielevels mittels qPCR-Analyse ergab, dass jedes der 12 untersuchten Orc1/Cdc6-Proteine das Ploidielevel mindestens eines Replikons beeinflusst und dementsprechend sowohl die mit einem Replikationsursprung assoziierten als auch die „verwaisten“ Orc1/Cdc6-Proteine eine Funktion haben. Die mit einem Replikationsursprung assoziierten Orc1/Cdc6-Proteine hatten hierbei keinen größeren Einfluss auf das Ploidielevel als die „verwaisten“. Zusätzlich konnte durch Wachstumsanalysen in Mikrotiterplatten gezeigt werden, dass die meisten Deletionsmutanten unter allen getesteten Bedingungen ein mit dem Wildtyp vergleichbares oder besseres Wachstum zeigen. Eine Deletionsmutante eines Orc1/Cdc6-Proteins hingegen zeigte nur verbessertes Wachstum bei Glukose als Kohlenstoffquelle, was ein Hinweis auf die Verwendung verschiedener Orc1/Cdc6-Proteine unter verschiedenen Bedingungen sein könnte. Zusätzlich wurden zwei mit dem Replikationsursprung assoziierte Orc1/Cdc6-Proteine überexprimiert und via ihres N-terminalen His-Tag im Western-Blot nachgewiesen, sodass diese nun für Co-Affinitätsaufreinigungen zur weiteren Charakterisierung des komplexen Zusammenspiels der Orc1/Cdc6-Proteine zur Verfügung stehen.
Im Rahme des zweiten Teils der Arbeit wurde der Einfluss der in der 5‘-Region der der Replikationsursprünge ori1 und ori2 kodierten Proteine auf Wachstum und die Kopienzahl des Hauptchromosoms bestimmt. Zunächst wurde die Expression der drei in Haloarchaea hoch-konservierten oap-Gene upstream von ori1 mittels Nothern-Blot untersucht und es konnte gezeigt werden, dass das oap-Operon tatsächlich als Operon abgelesen wird. Um alle Gene in den 5‘-Regionen von ori1 und ori2 genauer zu charakterisieren, wurden induzierbare Überexpressionsmutanten im Wildtyp-Hintergrund angefertigt. Es konnte mittels Wachstumsversuchen in Mikrotiterplatten gezeigt werden, dass bei Induktion von Beginn an die Überexpression der Hef-Helikase und des oapB-Proteins zu einem starken Wachstumsdefekt führen, die von oapC und HVO_1724 zu einem moderaten Wachstumsdefekt, wohingegen für die Überexpressionsmutante von oapA vergleichbares Wachstum zum Wildtyp und für die Überexpression der Rad25d-Helikase verbessertes Wachstum beobachtet werden konnte. Es konnte darüber hinaus gezeigt werden, dass sowohl die Deletion als auch die Überexpression der Helikasen keinen Einfluss auf das Ploidielevel hat; die Deletion von oapC führt jedoch zu einer Reduktion der Genomkopienzahl in exponentieller und stationärer Phase, was ein erster Hinweis darauf ist, dass das oap-Operon eine Rolle bei der Regulation des Ploidielevels spielen könnte.
Im dritten Teil der Arbeit wurde eine Methode entwickelt, um Genkonversion farblich sichtbar zu machen. Hierbei wurde sich H. volcaniis Carotinoidbiosynthese zu Nutze gemacht. Es wurden zwei verschiedene, auxotrophe Elternstämme mittels Protoplastenfusion verschmolzen, um eine heterozygote Tochterzelle zu erzeugen. Ein Genkonversionsereignis wurde durch einen roten Keil angezeigt, der aus einer weißen Kolonie wuchs und durch die erfolgreiche Reparatur des Carotinoidbiosynthesegens entstand. Es wurden insgesamt 8525 Klone ausgestrichen und 0,14 % der Kolonien zeigten eine entsprechende rote Färbung. Das Proof-of-Principle dieser Methode ist in damit in dieser Arbeit gelungen. Um die Genkonversion in den weißen Kolonien auf genetischer Ebene genauer zu untersuchen, wurde PCR verwendet. Es konnte gezeigt werden, dass in den Zellen aller 135 untersuchten Kolonien Genkonversion stattgefunden hatte und zwar so effizient, dass nur in seltenen Fällen Heterozygotie vorlag. Unter Selektionsdruck stehende Loci hatten in beiden untersuchten Fällen eine starke Präferenz in Richtung Homozygotie und Erhalt der Prototrophie. Für nicht unter Selektionsdruck stehende Loci konnte gezeigt werden, dass die Hälfte der untersuchten Kolonien dem Elternstamm 1 glich, während die andere Hälfte dem Elternstamm 2 glich. Auch hier waren die Zellen nur in seltenen Fällen homozygot.
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Life-attenuated measles virus (MV) vaccines have revealed their capacity to routinely induce life-long immunity against MV after just a single or two low-dose injections. Moreover, MV vaccines have been shown to be extensively safe and well tolerated, in general. Thus, MV is a prime candidate for a recombinant vaccine platform to protect also against other pathogens after vaccination. For this purpose, foreign genes can be inserted into additional transcription units (ATU) in recombinant MV genomes so that the encoded foreign proteins are co-expressed with MV proteins in infected cells. These so-called bivalent MV should protect against infection by MV or the pathogen, which the encoded foreign protein had been derived from. Bivalent MVs have already been shown to be effective vaccines against e.g. dengue virus or hepatitis B virus infections by inducing humoral and sometimes also cellular immune responses. In most of these studies, soluble or soluble versions of the pathogens' antigens were used for generation of bivalent MVs.
We hypothesized that the form of the antigen expressed by bivalent MVs is crucial for the potency and constitution of the induced immune responses. Therefore, three different forms of an antigen expressed by bivalent MVs were analyzed, here. The model antigen chosen for this purpose has been the envelope protein (Env) of SIVsmmPBj1.9. In its natural mature form, Env is composed of the surface unit gp120 and the transmembrane unit gp41, which stay non-covalently linked after proteolytic processing of the common precursor protein gp160. However, gp120 can be shed by infected cells or virus particles. Therefore, natural gp160 antigen was used as shedding form. Furthermore, stabilized covalently-linked gp160 variants and soluble gp140 variants were used in this thesis. These different antigen forms were inserted either behind the P or behind the H expression cassettes into the MV genome. The respective bivalent MVs were rescued and characterized. Expression of SIVsmmPBj1.9 Env variants by the bivalent MVs was confirmed by immuno blot and in situ immunoperoxidase assays. Replication curves of bivalent MV showed that growth of MVs expressing the different Env variants was slightly delayed by approximately 24 h compared to control viruses.
For immunization of transgenic, MV-susceptible IFNAR-/--CD46Ge mice, which are the current standard to analyze MV vaccines in a small animal model, an optimal dose of 1x105 TCID50 was determined. For the evaluation of humoral immune responses in transgenic mice, two ELISA systems for the detection of total α-MV and α-SIV antibodies and neutralization assays for detection of neutralizing antibodies against MV and SIV in sera of immunized mice were established. Mice immunized with any of the bivalent MVs showed significant humoral immune responses against MV comparable to those elicited by the parental MV vaccine strain without further genetic modifications. Mice immunized with MVvac2-gp140(P) expressing the soluble gp140 variant revealed highest α-SIV titers with a maximal OD of up to 0.4. Second highest levels of α-SIV antibodies were detected in mice that were immunized with the shedding variants or soluble Env in other positions. MVs expressing the stabilized variants induced only very low α-SIV antibody titers. Neutralizing antibodies directed against SIV could be detected in sera of mice immunized with MVs expressing the soluble or shedding variants, but not in sera of mice immunized with MVs expressing the stabilized variants. In sera of control mice immunized with PBS no antibodies could be detected, as expected. Thus, soluble and shedding antigens induced humoral immune responses, whereas stabilized antigens induced only weak humoral immune responses but no neutralizing antibodies. Analysis of cellular immune responses is still ongoing.
Besides Env, further SIV antigens could be tested for their potency to induce humoral as well as cellular immune responses.
Besides being used as a vaccine platform, recombinant MVs are evaluated as future agent for cancer therapy due to their significant inherent tumor-lytic, so-called oncolytic activity. Currently, the anti-tumoral activity of MV is analyzed in clinical phase I trials. MV strains with high fusion activity are used as oncolytic agents. The fusion protein F of MV strain NSe is highly fusogenic, in contrast to e.g. F of MVwt323, a clone of the pathogenic strain IC-B. Sequence analysis of these two proteins identified one coding nucleotide difference at aa 94 in the F2 domain: a valine (V) in FNSe and a methionine (M) in Fwt323. To evaluate impact of this difference, residues at aa 94 were exchanged. After transient-transfection of MV F and H expression plasmids in receptor-positive cells, V94 in the F2 subunit of FNSe or Fwt323 led to about 6-fold higher fusion activity compared to F proteins with M94. The co-expressed H protein (HNSe or Hwt323) did not influence fusion activity, indicating that the receptor (CD46 or SLAM) bound by H does not quantitatively affect the F proteins' activation. Analysis of F and H showed that formation and transport of MV glycoprotein complexes are not altered by substitution in aa 94 of FNSe or Fwt323.
Furthermore, recombinant MVNSe, MVNSe-F-M94, MVwt323, or MVwt323-F-V94 were rescued. Viral replication revealed slightly higher titers for recombinant MVs expressing M94 in F after 96 h of replication, compared to MVs expressing V94. MVs expressing V94 in F2 showed 2.5-fold higher fusion activity on CD46- and SLAM-positive Vero-hSLAM cells and 2-fold higher fusion activity on B95a cells expressing only SLAM compared to MVs expressing F with M94. Fusion activity of recombinant MVs can thus be modulated by substituting a single aa. V94 in the F protein results in highly fusion active MVs with possibly increased direct cytotoxicity in infected tumors, whereas M94 in F could be associated with decreased fusion activity for therapies, where higher virus titers are required.
In allen drei Domänen des Lebens ist in der Translation die Initiation der geschwindigkeits-bestimmende Schritt. Die Effizienz der Translationsinitiation und ihre unterschiedliche Regula-tion ist von Translationsinitiationsfaktoren (IFs) abhängig. Bakterien enthalten nur drei IFs, während die Anzahl bei Archaeen (aIFs) und Eukaryoten (eIFs) deutlich höher ist.
Das Archaeon Haloferax volcanii beispielsweise besitzt 14 Gene, die für aIFs bzw. deren Untereinheiten kodieren. Eine Deletionsanalyse ergab, dass fünf aIFs essenziell und neun aIFs nicht essenziell sind. Um einen Einblick in die Funktions- und Interaktionsbereiche der aIFs in H. volcanii zu erhalten, wurden die aIFs mit einem His-Tag versehen und überexpri-miert. Die Überexpression erfolgte in der jeweiligen Deletionsmutante. Für essenzielle aIFs fand sie im Wildtyp statt. Durch Affinitätsaufreinigungen wurden die aIFs und ihre Bindungs-partner isoliert und mittels Massenspektrometrie (MS) identifiziert. Für den Ausschluss unspe-zifischer Proteine dienten zwei stringente Kontrollen als Referenz, das Reportergen Dihydro-folatreduktase (HVO_1279) mit His-Tag und das Expressionsplasmid ohne Gen.
Die ersten Arbeiten konzentrierten sich auf den heterotrimeren Faktor aIF2. Er bindet die Ini-tiator-tRNA und ist damit für die Bildung des Präinitiationskomplexes von zentraler Bedeu-tung. Der Faktor aIF2 besteht aus jeweils einer α-, β- und γ-Untereinheit. In H. volcanii existie-ren zwei Orthologe für aIF2β. Die Überexpressionen der α-, β1-, β2- und γ-Untereinheiten führten zur Co-Isolation der jeweils anderen Untereinheiten des aIF2 (α, β1/ β2, γ).
Die Strategie der Co-Affinitätsaufreinigung und MS wurde auf alle weiteren annotierten aIFs ausgedehnt, um mögliche Funktionen zu identifizieren und ein potenzielles Interaktionsnetz-werk der aIFs zu erstellen. Für alle aIFs konnte ein unterschiedliches Muster an co-gereinigten Proteinen festgestellt werden. Mitgereinigte Proteine waren aIFs, Proteine der Translation, Transkription, Replikation und ribosomale Proteine. Auch RNA-Polymerase-Untereinheiten (RNAPUs) konnten co-isoliert werden. Mit 13 der 14 aIFs konnten andere Ini-tiationsfaktoren co-gereinigt werden. Sechs aIFs konnten zu Beginn bei keinem weiteren Initi-ationsfaktor mitgereinigt werden. Einer dieser Faktoren war aIF2β-1, der jedoch in den Affini-tätsaufreinigungen mit nachfolgender FPLC von aIF2β-2 identifiziert werden konnte. Der Fak-tor aIF1 konnte nur in der stationären Phase von aIF2α mitgereinigt werden.
Die am häufigsten co-gereinigten Proteine waren aIF2Bδ-1 und aIF5B. Für aIF2Bδ-1 kam dies überraschend, da er bereits als Translationsinitiationsfaktor ausgeschlossen wurde. Mit dem Faktor aIF2Bδ-1 selbst konnten fünf aIFs co-gereinigt werden.
Da mit den aIFs auch RNAPUs co-gereinigt werden konnten, wurden sieben RNAPUs ebenfalls mit einem His-Tag versehen und überexprimiert. Auch mit den RNAPUs konnten aIFs, sowie weitere Proteine der Translation mitgereinigt werden.
Diese Umstände legen nahe, dass es möglicherweise eine engere Verbindung der Tran-skription und Translation in H. volcanii geben könnte, als bisher angenommen.
Das Zytolysin ClyA von Escherichia coli ist der Prototyp einer neuartigen Familie von porenbildenden, bakteriellen Zytolysinen, welche in verschiedenen Vertretern der Enterobacteriaceae vorkommen. Es handelt sich bei diesem Toxin um ein Protein von 34 kDa, das hämolytische und zytotoxische Aktivität aufweist und das in Zellmembranen stabile Poren einführt, indem es sich zu ringförmigen ClyA-Oligomeren zusammenlagert. Die vorliegende Dissertation sollte einen Beitrag zur funktionalen Charakterisierung des ClyA-Proteins von E. coli K12 liefern. Insbesondere sollte die Bedeutung der N-terminalen Region für den Sekretionsmechanismus, für die hämolytische und porenbildende Aktivität, für die Interaktion mit Zielmembranen und für die Fähigkeit zur Oligomerisierung des Proteins analysiert werden. Im Rahmen dieser Arbeit wurden hierzu ClyA-Derivate mit spezifischen Mutationen innerhalb des N-terminalen Bereiches hergestellt und auf ihre Funktions-tüchtigkeit überprüft. Diese Untersuchungen gingen von den bestehenden Erkenntnissen aus, dass die Sekretion von ClyA über eine periplasmatische Zwischenstufe verläuft und keine N-terminale Prozessierung des Toxins erfolgt. Es wurde festgestellt, dass bereits kurze, sukzessive Deletionen innerhalb der beginnenden N-terminalen Region, wie die Deletion der Aminosäuren an Position zwei bis fünf (ClyA∆2-5) und sechs bis zehn (ClyA∆6-10), einen deutlich hemmenden Effekt auf den Transfer von ClyA über die zytoplasmatische Membran zur Folge hatte, welcher durch die Deletion der Aminosäuren an Position zwei bis zehn (ClyA∆2-10) weiter verstärkt wurde. Größere N-terminale Mutationen, wie die Deletionen der Aminosäuren an Position zwei bis fünfzehn (ClyA∆2-15) und zwei bis zwanzig (ClyA∆2-20) sowie interne Deletionen, die Aminosäuren jenseits von Aminosäureposition zehn betrafen (ClyA∆6-15, ClyA∆11-15, ClyA∆11-20, ClyA∆16-20), führten zur fast vollständigen Inhibierung (ClyA∆6-15) oder totalen Blockierung (ClyA∆11-15, ClyA∆11-20, ClyA∆16-20, ClyA∆2-20, ClyA∆2-15) des Transports von ClyA in den periplas-matischen Raum. Während die hämolytische Aktivität der ClyA-Mutanten ClyA∆2-5, ClyA∆6-10 und ClyA∆2-10 zwar abnahm, jedoch im Vergleich zu der des wildtypischen ClyA-Proteins noch verhältnismäßig hoch war, beeinflussten die weiteren Deletionen (ClyA∆6-15, ClyA∆11-15, ClyA∆11-20, ClyA∆16-20, ClyA∆2-15, ClyA∆2-20) die hämolytische Eigenschaft des Toxins ganz erheblich. So verursachten diese Mutationen eine massive Abnahme (ClyA∆6-15) bzw. einen kompletten Verlust (ClyA∆11-15, ClyA∆11-20, ClyA∆16-20, ClyA∆2-20, ClyA∆2-15) der zytolytischen Aktivität des ClyA-Proteins gegenüber Erythrozyten. Untersuchungen an künstlichen Lipidmembranen ergaben, dass die Deletion der Aminosäuren an Position zwei bis fünf, sechs bis zehn und zwei bis zehn die porenbildende Aktivität des ClyA-Proteins nur unwesentlich beeinträchtigt. Dagegen störte die Deletion der Aminosäuren an Position zwei bis fünfzehn und zwei bis zwanzig die porenbildende Eigenschaft von ClyA so tiefgreifend, dass keine (ClyA∆2-20) oder nur Transmembranporen mit extrem geringem Innendurchmesser (ClyA∆2-15) erzeugt wurden. Daneben spiegelten instabile Membranporen, die durch die ClyA-Mutanten ClyA∆6-15, ClyA∆11-15, ClyA∆11-20 und ClyA∆16-20 im künstlichen Lipid-Bilayer gebildet wurden, die negativen Auswirkungen dieser Mutationen auf die porenbildende Aktivität des Toxins wider. Die gesammelten Daten unterstreichen somit nicht nur die essentielle Bedeutung der N-terminalen Region des Zytolysins ClyA von E. coli K12 für den Transport in den periplasmatischen Raum, sondern auch für die hämolytische und porenbildende Eigenschaft des Toxins. Allerdings wurde die hämolytische und porenbildende Aktivität des ClyA-Proteins erst durch Mutationen ab Aminosäureposition zehn signifikant beeinträchtigt. Demgegenüber verdeutlichten Untersuchungen zum Bindungsverhalten an Zielzellen sowie Crosslinking-Experimente, dass die N-terminale Aminosäuresequenz von Position zwei bis zwanzig von ClyA weder für die Bindung an Erythrozyten noch für die Oligomerisierung des Toxins einen wesentlichen Faktor darstellt.