Institutes
Refine
Year of publication
Document Type
- Article (300)
- Doctoral Thesis (172)
- Preprint (41)
- Book (1)
Language
- English (514) (remove)
Has Fulltext
- yes (514)
Is part of the Bibliography
- no (514)
Keywords
- SARS-CoV-2 (7)
- Podospora anserina (6)
- aging (6)
- Cyanobacteria (5)
- Membrane Proteins (5)
- Phylogeny (5)
- Acetogenesis (4)
- Ecology (4)
- Haloferax volcanii (4)
- Saccharomyces cerevisiae (4)
Institute
- Biowissenschaften (514) (remove)
The glidobactin-like natural products (GLNPs) glidobactin A and cepafungin I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881–1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
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.
Aim: Knowledge concerning species distribution is important for biodiversity conservation and environmental management. Fungi form a large and diverse group of species and play a key role in nutrient cycling and carbon storage. However, our understanding of fungal diversity and distribution remains limited, particularly at large spatial scales. Here, we predicted the diversity and distribution of ectomycorrhizal and saprotrophic macrofungi at relatively fine spatial resolution at a continental scale and examined the importance of variables that affect the distribution of these two functional groups. Location: Europe. Time period: 1990–2018. Major taxa studied: Macrofungi. Methods: From observations of 1,845 macrofungal species, we predicted the diversity and distribution of two functional groups of macrofungi at a resolution of 5 km across eight European countries based on 25 environmental variables using the MAXENT model. We determined the importance of variables that affect the distribution of these two functional groups of macrofungi using the built-in jackknife test in the model. Results: Analysis of the modelling results showed that eastern Denmark and southern Sweden are biodiversity hotspots for both functional groups of macrofungal species. Tree species and human disturbance (i.e., the human footprint index) were found to be the two most important predictor variables explaining the distribution of ectomycorrhizal and saprotrophic macrofungi. Main conclusions: Overall, our study demonstrates that tree species and human disturbance have played a more important role than climatic factors in determining the diversity and distribution of macrofungi at the continental scale. Our study suggests that fungal diversity and distribution might change considerably if the strongest predictors (i.e., tree species) were to be affected by climate change and/or human activity. Changes in fungal diversity might, in turn, influence other processes, because fungi are important in driving ecosystem processes, such as nutrient and carbon cycling.
One current goal in native mass spectrometry is the assignment of binding affinities to noncovalent complexes. Here we introduce a novel implementation of the existing laser-induced liquid bead ion desorption (LILBID) mass spectrometry method: this new method, LILBID laser dissociation curves, assesses binding strengths quantitatively. In all LILBID applications, aqueous sample droplets are irradiated by 3 µm laser pulses. Variation of the laser energy transferred to the droplet during desorption affects the degree of complex dissociation. In LILBID laser dissociation curves, laser energy transfer is purposely varied, and a binding affinity is calculated from the resulting complex dissociation. A series of dsDNAs with different binding affinities was assessed using LILBID laser dissociation curves. The binding affinity results from the LILBID laser dissociation curves strongly correlated with the melting temperatures from UV melting curves and with dissociation constants from isothermal titration calorimetry, standard solution phase methods. LILBID laser dissociation curve data also showed good reproducibility and successfully predicted the melting temperatures and dissociation constants of three DNA sequences. LILBID laser dissociation curves are a promising native mass spectrometry binding affinity method, with reduced time and sample consumption compared to melting curves or titrations.
Cancer is a disease characterized by uncontrolled cell growth and the capacity to disseminate to distant organs. The properties of cancers are caused by genetic and epigenetic alterations when compared to their normal counterparts. Genetic mutations occur in oncogenes and tumor suppressor genes and are the initial drivers of cellular transformation (Lengauer et al., 1998; Vogelstein and Kinzler, 2004). In addition, epigenetic alterations, which influence the expression of oncogenes and tumor suppressor genes independently from sequence alterations, are also involved in the transformation process (Esteller and Herman, 2001; Sharma et al., 2010). Genetic alterations and epigenetic regulatory signals cooperate in tumor etiology. Glioblastoma multiforme (GBM) is a frequent and aggressive malignant brain tumor in humans. The median survival of GBM patients is about 15 months after diagnosis. Like in other cancers, genetic and epigenetic alterations can be detected in GBM. Genetic alterations in GBM affect cell growth, apoptosis, angiogenesis, and invasion; however, epigenetic alterations in GBM also affect the expression of oncogenes or tumor suppresser genes that increase tumor malignancy (Nagarajan and Costello, 2009).
Reprogramming is a cellular process in which somatic cells can be induced to assume the properties of less differentiated stem cells. This process can be mediated through epigenetic modifications of the genome of somatic cells by the action of four defined transcription factors (Oct4, Sox2, Klf4 and Myc) or by the action of the miR 302/367 cluster (Anokye-Danso et al., 2011; Takahashi and Yamanaka, 2006; Takahashi et al., 2007) and result in the generation of induced pluripotent stem cells (iPS cells). Reprogramming of somatic cells by the miR 302/367 cluster can generate nontumorigenic iPS cells through the inhibition of the epithelial to mesenchymal transition (EMT), cell cycle regulatory genes and epigenetic modifiers (Lin and Ying, 2013).
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
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.