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Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
The aim of this work was to establish a new way of predicting novel dual active compounds by combining classical fingerprint representation with state-of-the-art machine learning algorithms. Advantages and disadvantages of the applied 2D- and 3D-fingerprints were investigated. Further, the impact of various machine learning algorithms was analyzed. The new method developed in this work was used to predict compounds, which inhibit two different targets (LTA4H and sEH) involved in the same disease pattern (inflammation). The development of multitarget drugs has become more important in recent years. Many widespread diseases like metabolic syndrome, or cancer are of a multifactorial nature, which makes them hard to be treated effectively with a single drug. The new in silico method presented in this work can help to accelerate the design and development of multitarget drugs, saving time and efforts.
The nowadays readily available access to a large number of 3D-structures of biological targets and published activity data of millions of synthesized compounds enabled this study and was used as a starting point for this work. Four different data sets were compiled (crystalized ligands from the PDB, active and inactive compounds from ChEMBL23, newly designed compounds using a combinatorial library). Those data sets were collected and processed using an automated KNIME workflow. This automation has the advantage of allowing easy change and update of compound sources and adapted processing ways.
In a next step, the compounds from the compiled data sets were represented using a variety of well-established 2D- and 3D-fingerprints (PLIF, AtomPair, Morgan, FeatMorgan, MACCS). All those fingerprints share the same underlying bit string scheme but vary in the way they describe the molecular structure. Especially the difference between 2D- and 3D-fingerprints was investigated. 2D-fingerprints are solely based on ligand information. 3D-fingerprints, on the other hand, are based on X-ray structure information of protein-ligand complexes. One major difference between 2D- and 3D-fingerprints usage is the need for a 3D-conformation (pose) of the compound in the targets of interest when using 3D-fingerprints. This additional step is time-consuming and brings further uncertainties to the method.
Based on the calculated fingerprints state-of-the-art machine learning algorithms (SVC, RF, XGB and ADA) were used to predict novel dual active compounds. The models were evaluated by 10-fold cross validation and accuracy as the primary measure of model performance was maximized. Second, individual parameters of the four machine learning algorithms were optimized in a grid search to achieve maximal accuracy using the optimized partitioning scheme. Overall accuracies, regardless of fingerprint and machine learning algorithm, are slightly better for LTA4H than for sEH.
The goal to predict dual active compounds was realized by comparing the set of predicted to be active compounds for LTA4H and sEH. For the 3D-fingerprint PLIF the machine learning algorithm Random Forest was chosen, from which compounds for synthesis and testing were selected. Of 115 predicted to be active compounds, six compounds were cherry picked. Two compounds showed very good/moderate dual inhibitory activity. Of the 2D-fingerprints, the AtomPair fingerprint in combination with the machine learning algorithm Random Forest was chosen from which compounds were selected for synthesis and testing. 116 compounds were predicted to be dual active against LTA4H and sEH. One of those compounds showed good dual inhibitory activity.
In this work it was possible to show advantages and disadvantages of using 2D- and 3D-fingerprints in combination with machine learning algorithms. Both strategies (2D: ligand-based, 3D: structure-based) lead to the prediction of novel dual active compounds with moderate to very good inhibitory activity. The method developed in this work is able to predict dual active compounds with very good inhibitory activity and novel (previously unknown) scaffolds inhibiting the targets LTA4H and sEH. This contribution to in silico drug design is promising and can be used for the prediction of novel dual active compounds. Those compounds can further be optimized regarding binding affinity, solubility and further pharmacological and physicochemical properties.
Introduction: Gastropoda are guided by several sensory organs in the head region, referred to as cephalic sensory organs (CSOs). These CSOs are innervated by distinct nerves. This study proposes a unified terminology for the cerebral nerves and the categories of CSOs and then investigates the neuroanatomy and cellular innervation patterns of these cerebral nerves, in order to homologise them. The homologisation of the cerebral nerves in conjunction with other data, e.g. ontogenetic development or functional morphology, may then provide insights into the homology of the CSOs themselves.
Results: Nickel-lysine axonal tracing (“backfilling”) was used to stain the somata projecting into specific nerves in representatives of opisthobranch Gastropoda. Tracing patterns revealed the occurrence, size and relative position of somata and their axons and enabled these somata to be mapped to specific cell clusters. Assignment of cells to clusters followed a conservative approach based primarily on relative location of the cells. Each of the four investigated cerebral nerves could be uniquely identified due to a characteristic set of soma clusters projecting into the respective nerves via their axonal pathways.
Conclusions: As the described tracing patterns are highly conserved morphological characters, they can be used to homologise nerves within the investigated group of gastropods. The combination of adequate number of replicates and a comparative approach allows us to provide preliminary hypotheses on homologies for the cerebral nerves. Based on the hypotheses regarding cerebral nerve homology together with further data on ultrastructure and immunohistochemistry of CSOs published elsewhere, we can propose preliminary hypotheses regarding homology for the CSOs of the Opisthobranchia themselves.
An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.
Urwald relict species – Saproxylic beetles indicating structural qualities and habitat tradition
(2005)
On the basis of the list of saproxylic beetles of Germany, the authors present a definition and list of “Urwald relict species”, comprising 115 beetles that are considered to be associated with primeval forest (“Urwald”) structures and features. We use the term “habitat tradition” to describe a continuity in supply of old growth dead wood and forest structures. The selection of species is made on behalf of the following criteria: relict records in Central Europe; attachment to continuity of deadwood resources and habitat tradition; continuity of old growth stand features like tree and deadwood maturity and di-versity; absence from cultivated Central European forest.
Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.
Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.
Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.
Background: The alpha-7 nicotinic acetylcholine receptor (alpha 7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the alpha 7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings: This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the alpha 7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of alpha 7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance: The results reported here demonstrate a hitherto unknown relationship between the alpha 7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration.
A widespread application of 3D bioprinting in basic and translational research requires accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithography apparatus (SLA) printer is developed. This SLA bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a parallelized tissue production. To showcase the capability of the bioprinter, constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel are produced. A thorough characterization of different GelMA/PEGDA ratios reveals that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirm that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.
With which political developments is BiKF confronted as a research centre as well as concerning its research and transfer efforts? Are there any hints for emerging research questions that meet practical needs? This paper gives an overview – as of June 2010 – on priority issues in the run-up to CBD’s COP-10, the 10th Conference of the Parties to the Convention on Biological Diversity (CBD), which will take place in Nagoya/Japan in October 2010. Highlighted discourse threads are: (1) the state of negotiations for an Access and Benefit Sharing (ABS) regime within CBD, (2) European and international preparations for renewing the political objectives for protecting biodiversity (Post-2010 Targets) and (3) the recent decision on an Intergovernmental Science-Policy Platform for Biodiversity and Ecosystem Services (IPBES). These three threads are selected against the background of an in depth analysis of the discourse field which was carried out in 2008/09 for BiKF. They show how the field progresses and which developments are worth being incorporated into BiKF’s further work. This Knowledge Flow Paper documents the talk given by the author during the second BiKF Retreat, 17–18 July 2010.
Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation.
Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl(2) (10 microM) application (controls: 2.4 +/- 1.6%; 5 h: +5.1 +/- 2.3%, 20 h: +5.7 +/- 2.5%, 48 h: +3.3 +/- 1.0% and 72 h: +2.1 +/- 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells ( approximately 4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein +/- the mdr1 inhibitor PSC833 (0.5 microM). When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 +/- 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 +/- 1.4%; p < or = 0.001). Cadmium-induced apoptosis and mdr1 up-regulation depended on ROS, since co-incubation with the ROS scavengers N-acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses. Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, induced by cadmium. The data show that 1) cadmium-mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-kappaB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis. These data suggest that mdr1 up-regulation, at least in part, provides anti-apoptotic protection for PT cells against cadmium-mediated stress.
Highlights
• High resolution profile of C. pipiens' sugar diet has been obtained using UHPLC-MS.
• Artificial feeding using ornamental plants provides similar sugar profiles as observed in field collected mosquitoes.
• Metabolomic profiling found secondary metabolites and pollutants of anthropogenic use.
Abstract: Culex pipiens (Linnaeus, 1758) mosquitoes search plant sources of sugars to cope with the energetic demand of various physiological processes. The crop as part of the digestive system is devoted to the storage of sugar-based meal obtained from various nectars sources. The profiling of sugars and metabolites in the Culex pipiens’ crop is scarce, and only few studies used Liquid Chromatography – Mass Spectrometry (LC-MS), which provides broad detection for biomonitoring environmental substances and even contaminants in the sugar diet of mosquitoes populations.
Therefore, sugar and metabolite profiling were performed on crops obtained from mosquitoes exposed to plant nectar under laboratory or natural conditions by Ultra High-Performance LC-MS (UHPLC-MS). This method allowed us a precise quantitative and qualitative identification of sugar diet and associated environmental compounds in the crop of the mosquito C. pipiens. Under laboratory condition, mosquitoes were allowed to feed on either glucose solution, commercially-available flowers or field collected flowers. In addition, we collected mosquitoes from the field to compare those crop metabolomes with metabolome patterns occurring after nectar feeding in the lab.
The sugar quantities and quality obtained from the crops of mosquitoes collected in the field were similar to those crops obtained from mosquitoes that fed on commercially-available flowers and from field collected flowers with a limit of detection of 10 μg/L for sucrose, glucose and sucrose. Next to sugar compounds, we identified 2 types of amino acids, 12 natural products, and 9 pesticides.
Next to the diversity of sugar compounds, we could confirm that secondary metabolites and environmental pollutants are typically up taken from floral nectar sources by C. pipiens. The in-depth knowledge on mosquito–plant interactions may inspire the development and further optimization of mosquito trap systems and arboviral surveillance systems.
DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.
The opportunistic human pathogen Acinetobacter baumannii is one of the leading causes of nosocomial infections. The high prevalence of multidrug‐resistant strains, a high adaptability to changing environments and an overall pronounced stress resistance contribute to persistence and spread of the bacteria in hospitals and thereby promote repeated outbreaks. Altogether, the success of A. baumannii is mainly built on adaptation and stress resistance mechanisms, rather than relying on ‘true’ virulence factors. One of the stress factors that pathogens must cope with is osmolarity, which can differ between the external environment and different body parts of the human host. A. baumannii ATCC 19606T accumulates the compatible solutes glutamate, mannitol and trehalose in response to high salinities. In this work, it was found that most of the solutes vanish immediately after reaching stationary phase, a very unusual phenomenon. While glutamate can be metabolized, mannitol produced by MtlD is excreted to the medium in high amounts. First results indicate that A. baumannii ATCC 19606T undergoes a rapid switch to a dormant state (viable but non‐culturable) after disappearance of the compatible solutes. Resuscitation from this state could easily be achieved in PBS or fresh medium.
The thesis entitled „Investigations on the significance of nucleo-cytoplasmic transport for the biological function of cellular proteins" aimed to unreveal molecular mechanisms in order to improve our understanding of the impact of nucleo-cytoplasmic transport on cellular functions. Within the scope of this work, it could be shown that regulated nucleo-cytoplasmic transport of a subfamily of homeobox transcription factors controlled their intra- and intercellular transport, and thereby influencing also their transcriptional activity. This study describes a novel regulatory mechanism, which could in general play an important role for the ordered differentiation of complex organisms. Besides cis-active transport Signals, also post-translational modifications can influence the localization and biological activity of proteins in trans. In addition to the known impact of phosphorylation on the transport and activity of STAT1, experimental evidence was provided demonstrating that acetylation affected the interaction of STAT1 with NF-kB p65, and subsequently modulated the expression of apoptosis-inducing NF-kB target genes. The impact of nucleo-cytoplasmic transport on the regulation of apoptosis was underlined by showing that the evolutionary conservation of a NES within the anti-apoptotic protein survivin plays an essential role for its dual function in the inhibition of apoptosis and ordered cell division. Since survivin is considered a bona fide cancer therapy target, these results strongly encourage future work to identify molecular decoys that specifically inhibit the nuclear export of survivin as novel therapeutics. In order to further dissect the regulation of nuclear transport and to efficiently identify transport inhibitors, cell-based assays are urgently required. Therefore, the cellular assay Systems developed in this work may not only serve to identify synthetic nuclear export and Import inhibitors but may also be applied in systematic RNAi-screening approaches to identify novel components of the transport machinery. In addition, the translocation based protease- and protein-interaction biosensors can be applied in various biological Systems, in particular to identify protein-protein interaction inhibitors of cancer relevant proteins. In summary, this work does not only underline the general significance of nucleo-cytoplasmic transport for cell biology, but also demonstrates its potential for the development of novel therapies against diseases like cancer and viral infections.
Cercosporoid fungi (Mycosphaerellaceae, Mycosphaerellales, Ascomycota) are one of the largest and most diverse groups of hyphomycetes causing a wide range of diseases of economically important plants as well as of plants in the wild. Although more than 6000 species are known for this group, the documentation of this fungal group is far from complete. Especially in the tropics, the diversity of cercosporoid fungi is poorly known. The present study aims to identify and characterise cercosporoid fungi collected on host plants belonging to Fabaceae in Benin, West Africa. Information on their morphology, host species and DNA sequence data (18S rDNA, 28S rDNA, ITS and tef1) is provided. DNA sequence data were obtained by a simple and non-culture-based method for DNA isolation which has been applied for cercosporoid fungi for the first time in the context of the present study. Among the loci used for the phylogenetic analysis, tef1 provided the best resolution together with the multigene dataset. Species delimitation in many cases, however, was only possible by combining molecular sequence data with morphological characteristics. Based on forty specimens recently collected in Benin, 18 species are presented with morphological descriptions, illustrations and sequence data. Among these, six species in the genus Cercospora and two species in Pseudocercospora are proposed as species new to science. The newly described species are Cercospora (C.) beninensis on Crotalaria macrocalyx, C. parakouensis on Desmodium tortuosum, C. rhynchophora on Vigna unguiculata, C. vignae-subterraneae on Vigna subterranea, C. tentaculifera on Vigna unguiculata, C. zorniicola on Zornia glochidiata, Pseudocercospora sennicola on Senna occidentalis and Pseudocercospora tabei on Vigna unguiculata. Eight species of cercosporoid fungi are reported for Benin for the first time, three of them, namely C. cf. canscorina, C. cf. fagopyri and C. phaseoli-lunati are new for West Africa. The presence of two species of cercosporoid fungi on Fabaceae previously reported from Benin, namely Nothopassalora personata and Passalora arachidicola, is confirmed.
Unraveling the activation mechanism of taspase1 which controls the oncogenic AF4–MLL fusion protein
(2015)
We have recently demonstrated that Taspase1-mediated cleavage of the AF4–MLL oncoprotein results in the formation of a stable multiprotein complex which forms the key event for the onset of acute proB leukemia in mice. Therefore, Taspase1 represents a conditional oncoprotein in the context of t(4;11) leukemia. In this report, we used site-directed mutagenesis to unravel the molecular events by which Taspase1 becomes sequentially activated. Monomeric pro-enzymes form dimers which are autocatalytically processed into the enzymatically active form of Taspase1 (αββα). The active enzyme cleaves only very few target proteins, e.g., MLL, MLL4 and TFIIA at their corresponding consensus cleavage sites (CSTasp1) as well as AF4–MLL in the case of leukemogenic translocation. This knowledge was translated into the design of a dominant-negative mutant of Taspase1 (dnTASP1). As expected, simultaneous expression of the leukemogenic AF4–MLL and dnTASP1 causes the disappearance of the leukemogenic oncoprotein, because the uncleaved AF4–MLL protein (328 kDa) is subject to proteasomal degradation, while the cleaved AF4–MLL forms a stable oncogenic multi-protein complex with a very long half-life. Moreover, coexpression of dnTASP1 with a BFP-CSTasp1-GFP FRET biosensor effectively inhibits cleavage. The impact of our findings on future drug development and potential treatment options for t(4;11) leukemia will be discussed.
Background: Through the rapid development in DNA sequencing methods and tools, microbiome studies on a various number of species were performed during the last decade. This advance makes it possible to analyze hundreds of samples from different species at the same time in order to obtain a general overview of the microbiota. However, there is still uncertainty on the variability of the microbiota of different animal orders and on whether certain bacteria within a species are subject to greater fluctuations than others. This is largely due to the fact that the analysis in most extensive comparative studies is based on only a few samples per species or per study site. In our study, we aim to close this knowledge gap by analyzing multiple individual samples per species including two carnivore suborders Canoidea and Feloidea as well as the orders of herbivore Perissodactyla and Artiodactyla held in different zoos. To assess microbial diversity, 621 fecal samples from 31 species were characterized by sequencing the V3–V4 region of the 16S rRNA gene using Illumina MiSeq.
Results: We found significant differences in the consistency of microbiota composition and in fecal microbial diversity between carnivore and herbivore species. Whereas the microbiota of Carnivora is highly variable and inconsistent within and between species, Perissodactyla and Ruminantia show fewer differences across species boundaries. Furthermore, low-abundance bacterial families show higher fluctuations in the fecal microbiota than high-abundance ones.
Conclusions: Our data suggest that microbial diversity is significantly higher in herbivores than in carnivores, whereas the microbiota in carnivores, unlike in herbivores, varies widely even within species. This high variability has methodological implications and underlines the need to analyze a minimum amount of about 10 samples per species. In our study, we found considerable differences in the occurrence of different bacterial families when looking at just three and six samples. However, from a sample number of 10 onwards, these within-species fluctuations balanced out in most cases and led to constant and more reliable results.
Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development
(2011)
Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature.
The tremendous diversity of life in the ocean has proven to be a rich source of inspiration for drug discovery, with success rates for marine natural products up to 4 times higher than other naturally derived compounds. Yet the marine biodiscovery pipeline is characterized by chronic underfunding, bottlenecks and, ultimately, untapped potential. For instance, a lack of taxonomic capacity means that, on average, 20 years pass between the discovery of new organisms and the formal publication of scientific names, a prerequisite to proceed with detecting and isolating promising bioactive metabolites. The need for “edge” research that can spur novel lines of discovery and lengthy high-risk drug discovery processes, are poorly matched with research grant cycles. Here we propose five concrete pathways to broaden the biodiscovery pipeline and open the social and economic potential of the ocean genome for global benefit: (1) investing in fundamental research, even when the links to industry are not immediately apparent; (2) cultivating equitable collaborations between academia and industry that share both risks and benefits for these foundational research stages; (3) providing new opportunities for early-career researchers and under-represented groups to engage in high-risk research without risking their careers; (4) sharing data with global networks; and (5) protecting genetic diversity at its source through strong conservation efforts. The treasures of the ocean have provided fundamental breakthroughs in human health and still remain under-utilised for human benefit, yet that potential may be lost if we allow the biodiscovery pipeline to become blocked in a search for quick-fix solutions.
Dendrites form predominantly binary trees that are exquisitely embedded in the networks of the brain. While neuronal computation is known to depend on the morphology of dendrites, their underlying topological blueprint remains unknown. Here, we used a centripetal branch ordering scheme originally developed to describe river networks—the Horton-Strahler order (SO)–to examine hierarchical relationships of branching statistics in reconstructed and model dendritic trees. We report on a number of universal topological relationships with SO that are true for all binary trees and distinguish those from SO-sorted metric measures that appear to be cell type-specific. The latter are therefore potential new candidates for categorising dendritic tree structures. Interestingly, we find a faithful correlation of branch diameters with centripetal branch orders, indicating a possible functional importance of SO for dendritic morphology and growth. Also, simulated local voltage responses to synaptic inputs are strongly correlated with SO. In summary, our study identifies important SO-dependent measures in dendritic morphology that are relevant for neural function while at the same time it describes other relationships that are universal for all dendrites.
Unique features of a global human ectoparasite identified through sequencing of the bed bug genome
(2016)
The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.
Autophagosome biogenesis requires a localized perturbation of lipid membrane dynamics and a unique protein-lipid conjugate. Autophagy-related (ATG) proteins catalyze this biogenesis on cellular membranes, but the underlying molecular mechanism remains unclear. Focusing on the final step of the protein-lipid conjugation reaction, ATG8/LC3 lipidation, we show how membrane association of the conjugation machinery is organized and fine-tuned at the atomistic level. Amphipathic α-helices in ATG3 proteins (AHATG3) are found to have low hydrophobicity and to be less bulky. Molecular dynamics simulations reveal that AHATG3 regulates the dynamics and accessibility of the thioester bond of the ATG3∼LC3 conjugate to lipids, allowing covalent lipidation of LC3. Live cell imaging shows that the transient membrane association of ATG3 with autophagic membranes is governed by the less bulky- hydrophobic feature of AHATG3. Collectively, the unique properties of AHATG3 facilitate protein- lipid bilayer association leading to the remodeling of the lipid bilayer required for the formation of autophagosomes.
During animal development, it is crucial that cells can sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex can simultaneously respond to and create forces during cytokinesis is not well understood. Here we show that under mechanical stress, cortical actomyosin flow switches its polarization during cytokinesis in the C. elegans embryo. In unstressed embryos, longitudinal cortical flows contribute to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos. Rotational cortical flow is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and thereby contributes to furrowing in loaded embryos. A targeted screen for factors required for rotational flow revealed that actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.
Increasing trends in global trade make it extremely difficult to prevent the entry of all potential invasive species (IS). Establishing early detection strategies thus becomes an important part of the continuum used to reduce the introduction of invasive species. One part necessary to ensure the success of these strategies is the determination of priority survey areas based on invasion pressure. We used a pathway-centred conceptual model of pest invasion to address these questions: what role does global trade play in invasion pressure of plant ecosystems and how could an understanding of this role be used to enhance early detection strategies? We concluded that the relative level of invasion pressure for destination ecosystems can be influenced by the intensity of pathway usage (import volume and frequency), the number and type of pathways with a similar destination, and the number of different ecological regions that serve as the source for imports to the same destination. As these factors increase, pressure typically intensifies because of increasing a) propagule pressure, b) likelihood of transporting pests with higher intrinsic invasion potential, and c) likelihood of transporting pests into ecosystems with higher invasibility. We used maritime containerized imports of live plants into the contiguous U.S. as a case study to illustrate the practical implications of the model to determine hotspot areas of relative invasion pressure for agricultural and forest ecosystems (two ecosystems with high potential invasibility). Our results illustrated the importance of how a pathway-centred model could be used to highlight potential target areas for early detection strategies for IS. Many of the hotspots in agricultural and forest ecosystems were within major U.S. metropolitan areas. Invasion ecologists can utilize pathway-centred conceptual models to a) better understand the role of human-mediated pathways in pest establishment, b) enhance current methodologies for IS risk analysis, and c) develop strategies for IS early detection-rapid response programs.
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.
The compound class of the fabclavines was described as secondary or specialized metabolites (SM) for Xenorhabdus budapestensis and X. szentirmaii. Their corresponding structure was elucidated by NMR and further derivatives could be identified in both strains. Biochemically, fabclavines are hybrid SMs derived from two non-ribosomal-peptide-synthetases (NRPS), one type I polyketide-synthase (PKS) and polyunsaturated fatty acid (PUFA) synthases. In detail, a hexapeptide is connected via partially reduced polyketide units to an unsual polyamine. Structurally, they are related to the (pre-)zeamines, described for Serratia plymuthica and Dickeya zeae. Fabclavines exhibit a broad-spectrum bioactivity against a variety of different organisms like Grampositive and Gram-negative bacteria, fungi, protozoa but also against eukaryotic celllines.
In this work, the fabclavine biosynthesis was elucidated and assigned to two independently working assembly lines. The NRPS-PKS-pathway is initiated by the first NRPS FclI via generation of a tetrapeptide, which is elongated by the second NRPS FclJ, leading to a hexapeptide. Alternatively, FclJ can also act as direct start of the biosynthesis, resulting in the final formation of shortened fabclavine derivatives with a diinstead of a hexapeptide. In both cases, the peptide moiety is transferred to the iterative type I PKS FclK, leading to an elongation with partially reduced polyketide units. The resulting NRPS-PKS-intermediate is still enzyme-bound. The PUFA-homologues FclC, FclD and FclE in combination with FclF, FclG and FclH belong to the polyamine-forming pathway. Briefly, repeating decarboxylative Claisen thioester condensation reactions of acyl-coenzym A building blocks lead to the generation of an acyl chain in a PKS- or fatty acid biosynthesis-like manner. The corresponding β-keto-groups are either completely reduced or transaminated in a specific and repetitive way, resulting in the concatenation of so-called amine-units. The final β-keto-group is reduced to a hydroxy-group and the intermediate is reductively released by the thioester reductase FclG. A subsequent transamination step leads to the final polyamine. The NRPS-PKS- as well as the polyamine-pathway are connected by FclL. This condensation domain-like protein catalyzes the condensation of the polyamine with the NRPS-PKS-part, which results in the release of the final fabclavine. The results are described in detail in the first publication (first author).
Fabclavine biosynthesis gene cluster (BGC) are widely spread among the genus Xenorhabdus and Photorhabdus. In Xenorhabdus strains a high degree of conservation regarding the BGC synteny as well as the identity of single proteins can be observed. However, Photorhabdus strains harbor only the PUFA-homologues. While in Photorhabdus no product could be detected, our analysis revealed that the Xenorhabdus strains produce a large chemical diversity of different derivatives. Briefly, the general backbone of the fabclavines is conserved and only four chemical moieties are variable: The second and last amino acids of the NRPS-part, the number of incorporated polyketide units as well as the number of amine units in the polyamine. In combination with the elucidated biosynthesis, these variables could be assigned to single biosynthesis components as diversity mechanisms. Together with the 10 already described derivatives, a total of 32 derivatives could be detected. Interestingly, except for taxonomic closely related strains, all analyzed strains produce their own set of derivatives. Finally, we could confirm that the fabclavines are the major bioactive compound class in the analyzed strains under laboratory conditions. The results are described in detail in the second publication (first author).
Together with our collaboration partner Prof. Selcuk Hazir a potent bioactivity against Enterococcus faecalis, which is associated with endodontic infections, could be contributed to X. cabanillasii. Here, we could confirm that this bioactivity can be assigned to the fabclavines. The results are described in detail in the third publication(co-author).
Among the genus Xenorhabdus, X. bovienii represents an exception as its NRPS and PKS genes of the fabclavine BGC are missing or truncated, resulting in the exclusive production of polyamines. Furthermore, its PUFA-homologue FclC harbors an additional dehydratase (DH) domain. Upon extensive analysis a yet unknown deoxy-polyamine was identified and assigned to this additional domain. Finally, the DH domain was transferred into other polyamine pathways. Regardless of an in cis or in trans integration, the chimeric pathways produced deoxy-derivatives of its naturally occurring polyamines, suggesting that this represents another diversification mechanism. The results are described in detail in the attached manuscript (first author).
Proteins are biological macromolecules playing essential roles in all living organisms.
Proteins often bind with each other forming complexes to fulfill their function. Such protein complexes assemble along an ordered pathway. An assembled protein complex can often be divided into structural and functional modules. Knowing the order of assembly and the modules of a protein complex is important to understand biological processes and treat diseases related to misassembly.
Typical structures of the Protein Data Bank (PDB) contain two to three subunits and a few thousand atoms. Recent developments have led to large protein complexes being resolved. The increasing number and size of the protein complexes demand for computational assistance for the visualization and analysis. One such large protein complex is respiratory complex I accounting for 45 subunits in Homo sapiens.
Complex I is a well understood protein complex that served as case study to validate our methods.
Our aim was to analyze time-resolved Molecular Dynamics (MD) simulation data, identify modules of a protein complex and generate hypotheses for the assembly pathway of a protein complex. For that purpose, we abstracted the topology of protein complexes to Complex Graphs of the Protein Topology Graph Library (PTGL). The subunits are represented as vertices, and spatial contacts as edges. The edges are weighted with the number of contacts based on a distance threshold. This allowed us to apply graph-theoretic methods to visualize and analyze protein complexes.
We extended the implementations of two methods to achieve a computation of Complex Graphs in feasible runtimes. The first method skipped checks for contacts using the information which residues are sequential neighbors. We extended the method to protein complexes and structures containing ligands. The second method introduced spheres encompassing all atoms of a subunit and skipped the check for contacts if the corresponding spheres do not overlap. Both methods combined allowed skipping up to 93 % of the checks for contacts for sample complexes of 40 subunits compared to up to 10 % of the previous implementation. We showed that the runtime of the combined method scaled linearly with the number of atoms compared to a non-linear scaling of the previous implementation We implemented a third method fixing the assignment of an orientation to secondary structure elements. We placed a three-dimensional vector in each secondary structure element and computed the angle between secondary structure elements to assign an orientation. This method sped up the runtime especially for large structures, such as the capsid of human immunodeficiency virus, for which the runtime decreased from 43 to less than 9 hours.
The feasible runtimes allowed us to investigate two data sets of MD trajectories of respiratory complex I of Thermus thermophilus that we received. The data sets differ only by whether ubiquinone is bound to the complex. We implemented a pipeline, PTGLdynamics, to compute the contacts and Complex Graphs for all time steps of the trajectories. We investigated different methods to track changes of contacts during the simulation and created a heat map put onto the three-dimensional structure visualizing the changes. We also created line plots to visualize the changes of contacts over the course of the simulation. Both visualizations helped spotting outstandingly flexible or rigid regions of the structure or time points of the simulation in which major dynamics occur.
We introduced normalizations of the edge weights of Complex Graphs for identi-fying modules and predicting the assembly pathway. The idea is to normalize the number of contacts for the number of residues of a subunit. We defined five different normalizations.
To identify structural and functional modules, we applied the Leiden graph clustering algorithm to the Complex Graphs of respiratory complex I and the respiratory supercomplex. We examined the results for the different normalizations of the weights of the Complex Graphs. The absolute edge weight produced the best result identifying three of four modules that have been defined in the literature for respiratory complex I.
We applied agglomerative hierarchical clustering to the edges of a Complex Graph to create hypotheses of the assembly pathway. The rationale was that subunits with an extensive interface in the final structure assemble early. We tested our method against two existing methods on a data set of 21 proteins with reported assembly pathways. Our prediction outperformed the other methods and ran in feasible runtimes of a few minutes at most.
We also tested our method on respiratory complex I, the respiratory supercomplex and the respiratory megacomplex. We compared the results for the different normalizations with an assembly pathway of respiratory complex I described in the literature. We transformed the assembly pathways to dendrograms and compared the predictions to the reference using the Robinson-Foulds distance and clustering information distance. We analyzed the landscape of the clustering information distance by generating random dendrograms and showed that our result is far better than expected at random. We showed in a detailed analysis that the assembly prediction using one normalization was able to capture key features of the assembly pathway that has been proposed in the literature.
In conclusion, we presented different applications of graph theory to automatically analyze the topology of protein complexes. Our programs run in feasible runtimes even for large complexes. We showed that graph-theoretic modeling of the protein structure can be used to analyze MD simulation data, identify modules of protein complexes and predict assembly pathways.
Understanding the diverging opinions of academic experts, stakeholders and the public is important for effective conservation management. This is especially so when a consensus is needed for action to minimize future risks but the knowledge upon which to base this action is uncertain or missing. How to manage non-native, invasive species (NIS) is an interesting case in point: the issue has long been controversial among stakeholders, but publicly visible, major disagreement among experts is recent. To characterize the multitude of experts’ understanding and valuation of non-native, NIS we performed structured qualitative interviews with 26 academic experts, 13 of whom were invasion biologists and 13 landscape experts. Within both groups, thinking varied widely, not only about basic concepts (e.g., non-native, invasive) but also about their valuation of effects of NIS. The divergent opinions among experts, regarding both the overall severity of the problem in Europe and its importance for ecosystem services, contrasted strongly with the apparent consensus that emerges from scientific synthesis articles and policy documents. We postulate that the observed heterogeneity of expert judgments is related to three major factors: (1) diverging conceptual understandings, (2) lack of empirical information and high scientific uncertainties due to complexities and contingencies of invasion processes, and (3) missing deliberation of values. Based on theory from science studies, we interpret the notion of an NIS as a boundary object, i.e., concepts that have a similar but not identical meaning to different groups of experts and stakeholders. This interpretative flexibility of a concept can facilitate interaction across diverse groups but bears the risk of introducing misunderstandings. An alternative to seeking consensus on exact definitions and risk assessments would be for invasive species experts to acknowledge uncertainties and engage transparently with stakeholders and the public in deliberations about conflicting opinions, taking the role of honest brokers of policy alternatives rather than of issue advocates.
My PhD work employed genetic and pharmacological manipulations, coupled with highresolution live imaging, to understand intercellular communications during zebrafish cardiovascular development. The heart is the first organ to form, and it is composed of several tissues, among which interactions are crucial. I identified two important interactions between muscular and non-muscular tissues in poorly characterized contexts, and the molecules required for the signalling. First, I discovered an important cellular and molecular crosstalk orchestrating the development of the cardiac outflow tract (i.e., the aortic root in mammals).
Endothelial-derived TGF-beta signalling controls the generation of the local extracellular matrix (ECM). The ECM in turn affects endothelial proliferation as well as smooth muscle cell organization (Boezio et al, 2020; Bensimon-Brito*, Boezio* et al, 2020). In my second project, I investigated the crosstalk between the epicardial layer and the myocardial wall. By generating epicardial-impairment models, I identified a novel role for the epicardium in regulating cardiomyocyte volume during heart development (Boezio et al, 2021). Ultimately, this research contributed to our understanding of how paracrine signalling controls the multicellular interactions integral to organogenesis.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
Anthropogenic interventions have altered all ecosystems around the world. One of those ecosystems are forests, the main resource for timber. They have been strongly transformed in their structure with large consequences on forest biodiversity. Especially the decrease in dead-wood volume due to the timber extraction and alternation of natural forest structures with even-aged stands of less diverse tree species composition has put especially saproxylic, i.e., dead-wood dependent species, under threat, which comprise about 20% of all forest species. Beetles, fungi and bacteria are three functional important groups for decomposition processes but we still lack much information about their sampling and the drivers of their diversity, thus it is difficult to comprehensively protect their diversity. Saproxylic fungi are a highly diverse species group and the main drivers of dead-wood decomposition; hence they play a major role in the global carbon cycle. Due to their cryptic lifestyle, many species are still unknown, but the recent advances in environmental DNA barcoding methods (metabarcoding) shed light on the formerly underestimated diversity. Yet, this method's accuracy and suitability in detecting specific species have not been assessed so far, limiting its current usefulness for species conservation. On the other hand, these methods are a convenient tool to study highly diverse areas with high numbers of unknown species, enabling the study of global diversity and its drivers, which are unknown for saproxylic fungi, but important to assess to predict the future impacts of global change. Since nature conservation concepts are usually not applied on a global scale, the drivers of diversity must also be assessed on smaller scales. Besides understanding the drivers of diversity, to identify focus scales to create comprehensive, evidence-based conservation concepts must utilize multi-taxonomic studies since saproxylic species are differently sensitive towards environmental variables and closely interact with each other. Filling these knowledge gaps is utterly needed to protect the high saproxylic diversity and ensure the functional continuity of decomposition processes, especially regarding the global change.
To address the usefulness of metabarcoding for fungal species conservation, I compared the traditional method of fruit body sampling with metabarcoding and their efficiency in detecting threatened fungal species in the first chapter of this thesis. Both methods have advantages and disadvantages. Their ability to detect threatened saproxylic fungal species and their dependencies on detecting specific fungal groups have not been compared, albeit they are important to inform species conservation like Red Lists properly. I found metabarcoding to generally detect more threatened fungal species than fruit body sampling with a higher frequency than fruit body sampling. Moreover, fruit body sampling detected a unique set of species, while fruit body sampling missed large parts of fungal diversity due to species-specific fruiting characteristics. Metabarcoding with high sampling intensity is thus a viable method to assess threatened saproxylic fungal diversity and inform nature conservation like Red Lists about distribution and abundances. Nevertheless, a complementary approach with fruit body sampling is indispensable for assessing all threatened fungal species.
In order to analyse the global diversity of saproxylic fungi and its drivers, I examined whether fungal species richness increases from the poles towards the equator and thus follows the latitudinal diversity gradient already found in many other species groups. I further investigated whether such an increase is caused by increasing ecological specialisation, i.e., niche partitioning, or local tree diversity, i.e., niche space. Gamma diversity per biome increased from the boreal, over the temperate to the tropics and thus confirmed the latitudinal diversity for saproxylic fungi. Contrastingly, alpha diversity at the log level did not significantly increase towards the tropics, suggesting a grain size dependency of the observed pattern and an equal niche space within dead-wood across latitudes. Ecological specialisation on the plot level was globally on a high level but did not increase significantly towards the equator. Additionally, I found local tree species richness to drive plot-based fungal diversity. Further analysis of gamma diversity against the total number of sampled tree species strengthened the assumption that tree species diversity and not increased ecological specialisation was the main driver of the latitudinal diversity gradient, as there was no significant difference between the gamma diversity of the temperate and tropical biome. Nonetheless, as the gamma diversity of the boreal biome was still significantly smaller, my results do not allow a complete neglection of the ecological specialisation hypothesis. The overall results indicate a strong dependency of saproxylic fungi diversity with host tree species diversity and that the global loss of tree species threatens saproxylic fungi with an unpredictable impact on carbon and nutrient cycling.
To support saproxylic conservation, I conducted two analyses. First, I compared the beta diversity of the three main decomposer groups (beetles, fungal fruit bodies, mycelial fungi (metabarcoding), and bacteria (metabarcoding)) across different scales to assess the impact of different environmental variables on their overall diversity. I used an experimental design to disentangle two different spatial scales, influenced by differences in macroclimate, forest microclimate and spatial distance, and two host scales, driven by differences between tree lineages and tree species. I set these beta diversities in relation to the gamma diversity of the three main decomposer groups to identify whether a unified conservation concept could be applied to one scale to optimally protect the diversity of all three species groups. Second, I identified whether diversity and community composition of fungi and bacteria differed among climate and land use gradients. Further I explored whether specialisation and niche packing could explain the expected pattern. To do so I used an experimental design disentangling climate and land use across a large gradient in Germany. The results differed among the species groups, denying a unified conservation concept focusing on one scale. Saproxylic beetle and fruit body beta diversity was equally high on each scale, as they are more sensitive towards environmental factors like macro- and microclimate. On the other hand, mycelial fungi and bacteria beta diversity was highest on the host scale, especially the host tree scale, indicating a high host specificity of the two groups. The second study also identified tree species as the main driver of diversity and community composition of these two study groups. Specialisation of fungi was not influenced by land use or climate. Bacterial specialisation and diversity were under a strong influence of mean precipitation. Comprehensive conservation of multi-taxonomic diversity across regions thus requires the integration of several scales. Within different macroclimatic regions, forests of varying microclimates, i.e., forest management, must be implemented. In these forests, dead-wood of different tree lineages, i.e., angio- and gymnosperms and tree species, must be provided.
Taken together, I could demonstrate that metabarcoding is an efficient method to sample threatened fungal species and identify differing drivers of fungal diversity present as fruit bodies or mycelium. Its usefulness will further increase due to the ongoing improvement of sequencing databases and thus better inform conservation concepts. Using metabarcoding, I could demonstrate that high host specialisation of saproxylic fungi is not a European but a global phenomenon and identify tree species loss under global change as one major concern for saproxylic diversity. My dissertation further highlighted the importance of multi-taxonomic studies for evidence-based nature conservation, as different species groups require varying concepts. These results were especially important for saproxylic bacteria as the drivers of their diversity are still largely unknown. Howbeit, large research gaps still exist regarding the impacts of global change on species and processes. Moreover, the spatial coverage of studies is needed to confirm or neglect the generality of current research especially concerning the highly diverse tropical areas. An increased focus on the drivers of diversity in these areas is crucial to ensure a globally comprehensive saproxylic conservation and the various ecosystem functions they control.
Quantitative models have several advantages compared to qualitative methods for pest risk assessments (PRA). Quantitative models do not require the definition of categorical ratings and can be used to compute numerical probabilities of entry and establishment, and to quantify spread and impact. These models are powerful tools, but they include several sources of uncertainty that need to be taken into account by risk assessors and communicated to decision makers. Uncertainty analysis (UA) and sensitivity analysis (SA) are useful for analyzing uncertainty in models used in PRA, and are becoming more popular. However, these techniques should be applied with caution because several factors may influence their results. In this paper, a brief overview of methods of UA and SA are given. As well, a series of practical rules are defined that can be followed by risk assessors to improve the reliability of UA and SA results. These rules are illustrated in a case study based on the infection model of Magarey et al. (2005) where the results of UA and SA are shown to be highly dependent on the assumptions made on the probability distribution of the model inputs.
The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins.
Synechococcus (Anacystis nidulans, strain L 1402-1) were grown at + 37 °C in an atmosphere of 0.04 vol.% CO2 using different light conditions. Changing the culture conditions caused alterations in pigment ratios and ultrastructure of Synechococcus. In comparison to the low white and red light grown cells under strong white light the number of thylakoids decreased and an accumulation of storage carbohydrates could be observed. The number of the polyhedral bodies also varied with culture conditions. The results are discussed with reference to the pigment composition and the function of the polyhedral bodies.
The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore’s isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.
Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.
Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins.
In the last twenty years, there has been splendid progress in energy conversion technologies to have sustainable energy sources. For example, solar cells contribute significantly to energy production as the sun is an enormous source for renewable energy. Currently, the most common commercialized photovoltaic devices are silicon-based. The scientists' main targets are high efficiency, low cost, environmentally friendly, and easy to synthesize new semiconductor materials to replace silicon. Furthermore, understanding the photophysical properties of these materials is very important for designing high efficient photoconversion systems.
This thesis investigates the photophysics of lead-based wide-bandgap perovskites with different dimensionality (2D, 3D) and how they can be optimized for optoelectronic applications. In chapter 1, we present the background and progress in perovskite research. The basic concepts of semiconductor and spectroscopic methods of the applied techniques in this work are discussed in chapter 2.
In the first project (chapter 3.1), we used our time-resolved techniques to study the ultrafast dynamics of energy transfer from the inorganic to the organic layer in a series of three lead-based mixed-halide 2D perovskites containing benzyl ammonium (BA), 1-naphthyl methyl ammonium (NMA), and 1-pyrene methyl ammonium (PMA) thin films.
In the second project (chapter 3.2), we used time-resolved spectroscopic techniques to study the effect of adding 5% of Cs on the dynamics of a mixed-cation wide bandgap bromide-based 3D perovskite.
In another side project (chapter 4), we present the photophysics properties of newly synthesized new Schiff bases containing indole moieties using piperidine as an organic base catalyst and Au@TiO2 as a heterogeneous catalyst. Finally, the results of this work are summarized in Chapter 5 with an outlook and a discussion of open questions for further research.
In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.
The inhibitory glycine receptor (GlyR) in developing spinal neurones is internalized efficiently upon antagonist inhibition. Here we used surface labeling combined with affinity purification to show that homopentameric α1 GlyRs generated inXenopus oocytes are proteolytically nicked into fragments of 35 and 13 kDa upon prolonged incubation. Nicked GlyRs do not exist at the cell surface, indicating that proteolysis occurs exclusively in the endocytotic pathway. Consistent with this interpretation, elevation of the lysosomal pH, but not the proteasome inhibitor lactacystin, prevents GlyR cleavage. Prior to internalization, α1 GlyRs are conjugated extensively with ubiquitin in the plasma membrane. Our results are consistent with ubiquitination regulating the endocytosis and subsequent proteolysis of GlyRs residing in the plasma membrane. Ubiquitin-conjugating enzymes thus may have a crucial role in synaptic plasticity by determining postsynaptic receptor numbers.
In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.
Rho GTPases control fundamental cellular processes and Cdc42 is a well-studied member of the family that controls filopodia formation and cell migration. Although the regulation of Cdc42 activity by nucleotide binding is well documented, the mechanisms driving its proteostasis are not clear. Here, we demonstrate that the highly conserved, RING domain containing E3 ubiquitin ligase XIAP controls the protein stability of Cdc42. XIAP binds to Cdc42 and directly conjugates poly ubiquitin chains to the Lysine 166 of Cdc42 targeting it for proteasomal degradation. Depletion of XIAP led to an increased protein stability and activity of Cdc42 in normal and tumor cells. Consistently, loss of XIAP enhances filopodia formation in a Cdc42-dependent manner and this phenomenon phenocopies EGF stimulation. Further, XIAP depletion promotes lung colonization of tumor cells in mice in a Cdc42-dependent manner. These observations shed molecular insights into ubiquitin-dependent regulation of Cdc42 and that of actin cytoskeleton.
Ubiquitination is a widespread post-translational modification that controls multiple steps in autophagy, a major lysosome-mediated intracellular degradation pathway. A variety of ubiquitin chains are attached as selective labels on protein aggregates and dysfunctional organelles, thus promoting their autophagy-dependent degradation. Moreover, ubiquitin modification of autophagy regulatory components is essential to positively or negatively regulate autophagy flux in both non-selective and selective pathways. We review the current findings that elucidate the components, timing, and kinetics of the multivalent role of ubiquitin signals in control of amplitude and selectivity of autophagy pathways as well as their impact on the development of human diseases.
By adopting a variety of shapes, proteins can perform a wide number of functions in the cell, from being structural elements or enabling communication with the environment to performing complex enzymatic reactions needed to sustain metabolism. The number of proteins in the cell is limited by the number of genes encoding them. However, several mechanisms exist to increase the overall number of protein functions. One of them are post-translational modifications, i.e. covalent attachment of various molecules onto proteins. Ubiquitin was the first protein to be found to modify other proteins, and, faithful to its evocative name, it is involved in nearly all the activities of a cell. Ubiquitylation of proteins was believed for a long time only to be responsible for proteasomal degradation of modified proteins. However, with the discovery of various types of ubiquitylation, such as mono-, multiple- or poly-ubiquitylation, new functions of this post-translational modification emerged. Mono-ubiquitylation has been implicated in endocytosis, chromatin remodelling and DNA repair, while poly-ubiquitylation influences the half-life of proteins or modulates signal transduction pathways. DNA damage repair and tolerance are example of pathways extensively regulated by ubiquitylation. PCNA, a protein involved in nearly all types of DNA transaction, can undergo both mono- and poly-ubiquitylation. These modifications are believed to change the spectrum of proteins that interact with PCNA. Monoubiquitylation of PCNA is induced by stalling of replication forks when replicative polymerases (pols) encounter an obstacle, such as DNA damage or tight DNA-protein complexes. It is believed that monoubiquitylation of PCNA stimulates the exchange between replicative pols to one of polymerases that can synthesize DNA across various lesions, a mechanism of damage tolerance known as translesion synthesis (TLS). Our work has helped to understand why monoubiqutylation of PCNA favours this polymerase switch. We have identified two novel domains with the ability to bind Ub non-covalently. These domains are present in all the members of Y polymerases performing TLS, and were named Ub-binding zinc finger (UBZ) (in polη and polκ) and Ub-binding motif (UBM) (in polι and Rev1). We have shown that these domains enable Y polymerases to preferentially gain access to PCNA upon stalling of replication, when the action of translesion polymerases is required. While the region of direct interaction between Y pols and PCNA had been known (BRCT domain in Rev1 and PIP box motif (PIP) in three others members), we propose that Ub-binding domains (UBDs) in translesion Y pols enhance the PIP- or BRCT-domain-mediated interaction between these polymerases and PCNA by binding to the Ub moiety attached onto PCNA. Following these initial studies, we have also discovered that Y polymerases themselves undergo monoubiquitylation and that their UBDs mediate this modification. This auto-ubiquitylation is believed to lead to an intramolecular interaction between UBD and Ub attached in cis onto the UBD-containing protein. We have mapped monoubiquitylation sites in polη in the C-terminal portion of the protein containing the nuclear localization signal (NLS) and the PIP box. Beside PIP, the NLS motif is also involved in direct interaction of polη with PCNA. Based on these findings, we propose that monoubiquitylation of either NLS or PIP masks them from potential interaction with PCNA. Lastly, using several functional assays, we have demonstrated the importance of all these three motifs in the C-terminus of polη (UBZ, NLS and PIP) for efficient TLS. We have also constructed a mimic of monoubiquitylated polη by genetically fusing polη with Ub. Interestingly, this chimera is deficient in TLS as compared to the wild-type protein. Altogether, these studies demonstrate that the C-terminus of polη constitutes a regulatory module involved in multiple-site interaction with monoubiquitylated PCNA, and that monoubiquitylation of this region inhibits the interaction between polη and PCNA. Our work has also revealed that the UBDs of Y pols as well as of other proteins implicated in DNA damage repair and tolerance, such as the Werner helicase-interacting protein 1 (Wrnip1), are required for their proper sub-nuclear localization. All these proteins localize to discrete focal structures inside the nucleus and mutation of their UBDs results in inability to accumulate in these foci. Interestingly, by exchanging UBDs between different proteins we have learned that each UBD seems to have a distinct functional role, surprisingly not limited to Ubbinding ability. In fact, swapping the UBZ of Wrnip1 with the UBM of polι abolished the localization of Wrnip1 to foci despite preserving the Ub-binding ability of the chimeric protein. In summary, this work provides an overview of how post-translation modification of proteins by Ub can regulate several DNA transactions. Firstly, key regulators (e.g. PCNA) can be differentially modified by Ub. Secondly, specialized UBDs (e.g. UBM, UBZ) embedded only in a subset of proteins act as modules able to recognize these modifications. Thirdly, by means of mediating auto-ubiquitylation, UBDs can modulate the behaviour of host proteins by allowing for either in cis or in trans Ub-UBD interactions.
Ubiquitin ligases and beyond
(2012)
First paragraph (this article has no abstract): In a review published in 2004 [1] and that still repays reading today, Cecile Pickart traced the evolution of research on ubiquitination from its origins in the proteasomal degradation of proteins through the revelation that it has a central role in cell cycle regulation and the recognition of regulatory roles for ubiquitin in intracellular membrane transport, cell signalling, transcription, translation, and DNA repair.
Ubiquitylation is a three-step process, which results in the attachment of the small protein ubiquitin (Ub) to lysine residues on a substrate protein. SUMO proteins are ubiquitin (Ub)-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. During my PhD I isolated and characterized SUMO1 and SUMO2 binding motifs. Using Yeast Two Hybrid system, bioinformatics and NMR spectroscopy we defined a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a β-strand that could bind in parallel or anti-parallel orientation to the β2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions. Mutation of the SUMO interacting motif of TTRAP (TRAFS and TNF receptor associated protein) influences both its localization and dynamic behaviour in living cells. Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitylated targets and control numerous biological processes including receptor trafficking, DNA repair, virus budding and gene transcription. They themselves undergo UBD-dependent monoubiquitylation, which promotes intramolecular binding of the UBD to the attached Ub and consequently leads to their functional inhibition. During the second part of my PhD I could show that, in contrast to the established ubiquitylation pathway, the presence of UBDs allows the monoubiquitylation of host protein independently of classical E3 ligases. UBDs of different types including UBA, UIM, UBM, NFZ and UBZ, can directly cooperate with E2 Ub-conjugating enzymes to promote monoubiquitylation of their host proteins. Using FRET technology I verified that the E2 enzyme and the substrate directly interact in cells. Moreover, UBD-containing proteins Stam2 and Sts2 promote self-ubiquitylation and not ubiquitylation of other targets or form polyUb chains from free Ub. Our study revealed a yet unappreciated role of E2 enzymes in ubiquitylation reactions of UBD containing proteins.
Legionella pneumophila, a Gram-negative intracellular bacterium, is one of the major causes of Legionnaires’ disease, a specific type of atypical pneumonia. Despite intensive research efforts that elucidated many relevant structural, molecular and medical insights into Legionella’s pathogenicity, Legionnaires’ disease continues to present an ongoing public health concern. Legionella’s virulence is based on its ability to simultaneously hijack multiple molecular pathways of the host cell to ensure its fast replication and dissemination. Legionella usurps the host ubiquitin system through multiple effector proteins, using the advantage of both conventional and unconventional (phosphoribosyl-linked) ubiquitination, thus providing optimal conditions for its replication. In this review, we summarize the current understanding of L. pneumophila from medical, biochemical and molecular perspectives. We describe the clinical disease presentation, its diagnostics and treatment, as well as host-pathogen interactions, with the emphasis on the ability of Legionella to target the host ubiquitin system upon infection. Furthermore, the interdisciplinary use of innovative technologies enables better insights into the pathogenesis of Legionnaires’ disease and provides new opportunities for its treatment and prevention.
NADH: ubiquinone oxidoreductase (complex I) is the first enzyme complex of the respiratory chain. Complex I is a redox-driven proton pump that contributes to the proton motive force that drives ATP synthase. The structure of complex I has been analyzed by x-ray crystallography and electron cryo-microscopy and is now well-described. The ubiquinone (Q) reduction site of complex I is buried in the peripheral arm and a tunnel-like structure is thought to provide access for the hydrophobic substrate from the membrane. Several intermediate binding positions for Q in the tunnel were identified in molecular simulations. Structural data showed the binding of native Q molecules and short chain analogs and inhibitors in the access pathway and in the Q reduction site, respectively. We here review the current knowledge on the interaction of complex I with Q and discuss recent hypothetical models for the coupling mechanism.
Poster presentation: NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the beta1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, co-immunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme.