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From features to functions : leveraging protein feature architectures in comparative genomics
(2024)
When analyzing genomic data, one of the key challenges is the annotation of new genes. The toolkit for incorporating newly discovered proteins into a comprehensive evolutionary and functional network is diverse. It includes search heuristics based on sequence similarity to identify significantly similar sequences. Additionally, it involves identifying orthologs, which are also used for preliminary functional annotation. However, since the function of gene can change if given enough time, it is necessary to consider other information to identify functional divergence between proteins.
As one complementary form of information, it is possible to annotate protein sequences with features such as functional domains, transmembrane domains, low complexity regions, secondary structure elements, or compositional properties. The sum of all features annotated onto a sequence, the feature architecture, can provide further information on a proteins function. To perform this task effectively, tools that can compare and classify feature architectures on a large scale are necessary However, multiple challenges arise when dealing with feature architecture. Many existing schemes for comparing feature architectures cannot cope with features arising from multiple annotation sources. Those that do, fall short in the resolution of overlapping and redundant feature annotations.
In this thesis, I present different approaches to leveraging feature architectures as a complementary information source for evolutionary and functional studies. First, we introduce the Feature Architecture Similarity, a tool to perform pairwise scoring of the similarity between two feature architectures. It uses a scoring method that considers the presence/absence of features, as well as positional information. It also allows the integration of features from multiple annotation sources into one feature architecture, while resolving overlapping feature annotations. A benchmark on more than 10,000 human-yeast ortholog pairs, architecture similarities assessed with FAS are consistently more plausible than those obtained using e-values to resolve overlaps or leaving overlaps unresolved. We then demonstrate the utility of FAS on feature architecture comparison tasks in three case studies.
In the second work package, we apply FAS in the assessment of the functional impact of alternative splicing. Here, we present SPICE, a tool that aids in understanding the functional variations within a proteome resulting from different protein isoforms expressed through alternative splicing. In this pipeline, we introduce a new measure, the Expression Weighted Feature Disturbance (EWFD), that combines the FAS score between protein isoforms with their relative transcript expression values. We demonstrate the use of SPICE with two datasets: First, we do an exemplary analysis using long-read sequencing data from the Long-read RNA-seq Genome Annotation Assessment Project and demonstrate how the results can be explored within the SPICE dashboard. Secondly, we explore how SPICE performs with the inclusion of novel, unannotated transcripts using a larger, more diverse dataset provided by the ENCODE consortium.
In the last part, we move away from pairwise comparisons of feature architectures and instead consider groups of functional equivalent proteins for machine learning. Here, we train AI models based on the feature architectures of a functional group of proteins to identify novel members. We propose a novel approach to encode feature architectures for later machine learning applications in two variants. In the first, we dynamically define, for each model, a feature space out of the features annotated with FAS. In the second, we consider a static feature space based on the DSSP 8-state secondary structure representation and disordered regions. In a test on 59 orthologous groups of the tricarboxylic acid cycle annotated in the Kyoto Encyclopedia of Genes and Genomes we confirm that both variants have a high recall rate.
With the three work packages introduced in this thesis, we extend the toolbox for studies on protein function with tools that effectively leverage feature architecture information on large-scale datasets and provide ideas for the future development of further methods for protein comparison and annotation.
Living in social groups and thereby maximizing both chance of survival and reproductive success is a phylogenetic old way of live and practiced by many different species. The evolution of social behavior optimized co-habitation of multiple animals and enhanced the effectiveness of its advantages. Oxytocin, a neuropeptide initially described in the context of labor and lactation, was correlated to different forms of social behavior since early 1990s, but the complexity of oxytocinergic signaling or function of oxytocin producing neurons in the perception of conspecifics is not fully understood yet.
In summary, this thesis project uncovered a pivotal role of oxytocin receptors in development and maintenance of social preference and shoaling behavior of subadult zebrafish, identified a subpopulation of oxytocinergic neurons with specific reaction to the visual presence of conspecifics and spawned a novel immobilization method, which enables scientists to reliably immobilize awake zebrafish at 2 – 4 wpf for future analyses of neuronal activity and eye movements.
Biodiversity is threatened globally and its decline is being accelerated mainly due to human activities. Halting the current rate of biodiversity loss requires protection and conservation of species at local as well as at global scales. Biological conservation strategies heavily rely upon the availability of good quality and real-time information about species distribution. Information on species distribution can also be utilized to address crucial ecological challenges including estimation of reintroduction success and biotic interactions. Traditionally, species distribution data is mainly collected through invasive monitoring approaches that relied upon capturing the species or observing them closely. While these invasive monitoring approaches are well-established, they may cause harm to rare and endangered species. Also, these monitoring approaches may not be efficient for certain cases, e.g., early detection of biological invasions by pathogenic or predatory species. Hence, there is a dire need for a non-invasive and robust monitoring technique to map species distributions. One potential solution is the establishment of environmental DNA (eDNA) techniques based on molecular species detection from DNA shed by organisms naturally in the environment. This non-invasive and robust monitoring technique is being increasingly applied in biodiversity conservation and applied ecology.
In this thesis, I aimed to develop eDNA based TaqMan assays for several species including one locally endangered and reintroduced fish species, Alburnoides bipunctatus. My main goal was to assess the reintroduction success of A. bipunctatus across the Federal German state of Hessen by integrating eDNA and species distribution modeling (SDM) in an iterative format. In addition to Global Biodiversity Information Facility (GBIF) data, I built SDM models with high-quality presence/absence data retrieved through eDNA to evaluate the improvement in the predictive power of SDM and used SDM predictions for an effective eDNA sampling design. I also utilized eDNA in a multi-species context to understand the dynamics of predator and prey interactions in a protected area. For this, I designed and established eDNA based TaqMan assays for two invasive predatory fish species, Pseudorasbora parva and Lepomis gibbosus. I related the presence of both predator and prey species to test for potential effects of predators on prey species occurrences.
My results highlight the potential of eDNA techniques beyond simple species detection for efficient biodiversity assessment and conservation. I successfully developed and validated highly specific and sensitive TaqMan assays for A. bipunctatus, P. parva and L. gibbosus. By using these TaqMan assays, I found that (i) eDNA can be utilized for the assessment of reintroduction success, to provide fast and reliable feedback to conservation management. Moreover, the combination of eDNA and SDM in an iterative approach can be useful to guide future reintroduction actions, (ii) eDNA can serve as an effective technique to explore biotic interactions between predator and prey species.
Overall, the results of my study underpin the usefulness of eDNA-based detection methods for biodiversity management and conservation. The great flexibility of eDNA to integrate with other techniques can give a tremendous boost to species monitoring and conservation. For example, I showed that the integration of eDNA and SDM can enhance the predictive accuracy of SDM. This enhancement facilitates the probability of species presence, serving as a tool for exploring the spread of reintroduced species. Moreover, this combined approach holds the potential to discover unrecognised populations of rare species. Furthermore, eDNA can be used for the assessment of biotic interactions, especially the effects of predator-prey interactions, which are considered as a crucial driver for shaping community structure.
For future research, I suggest implementing the combined eDNA-SDM approach to estimate the reintroduction success of other naturally extinct species, early detection, and monitoring of invasive or pathogenic species to guide conservation management actions. Also, the application of eDNA techniques can be extended to more complex systems to assess biotic interactions between species for long periods. In addition to these, several other scientific questions such as, exploring ancient community structure, niche separation, delineating pollinator networks, and also biosecurity issues including monitoring water quality and biological vectors of deadly pathogenic species can be addressed. In conclusion, eDNA-based species detection is a robust and cost-effective tool for applied ecology and biodiversity conservation.
A classic discussion of large mammalian herbivore population dynamics would focus on top-down and bottom-up drivers. Yet what is often forgotten, is that many of these species are also highly mobile, covering hundreds or thousands of kilometers in a year, and that this mobility can also influence population dynamics, although the mechanisms are still understudied. While the top-down and bottom-up drivers are more researched, global change will alter how all three drivers impact population dynamics. Of particular concern are habitat fragmentation, which alters movement patterns, and climate change through its direct impact on large herbivore physiology but also its indirect influence through impacts on vegetation dynamics. Understanding these potential effects remains challenging, so the goal of my dissertation was to show that one approach to understanding the effects of global change is by modeling both plant and herbivore ecophysiology. To do this I made new additions to a dynamic global vegetation model coupled to a physiological model of herbivores, using Mongolian gazelle in the steppes of eastern Mongolia as a case study.
To parameterize the ecophysiological model for Mongolian gazelle, I needed to better understand how gazelle move to select for forage during the growing season and snow cover during winter. I also wanted to understand if selection differs between the individual and population level. To do this I combined gazelle movement data with satellite data on vegetation greenness and snow cover and used resource and step-selection functions to test for selection. At the population level, gazelle selected for higher-than-average vegetation greenness during the growing season indicating that they select areas of higher forage cover in a landscape where forage cover is often sparse. In winter, at the population level, gazelle selected for intermediate snow cover, striking a balance between staying hydrated and being able to move through the snow. At the individual level, in both seasons and across various spatial scales, I was not able to detect selection for most individuals. This was likely because vegetation, even up to 35 km away, is still very similar to where a gazelle currently is. Therefore, once gazelle are in a good foraging patch, they can move within it for a long time before they must decide where to move to next. In such a landscape, random searches might be the best foraging strategy. For the ecophysiological herbivore-vegetation model, this meant using the ~45 x 45km grid cell size of the vegetation model and a simple random search movement pattern was adequate to describe gazelle movements.
Based on the results of the habitat selection study I added movement to an existing ecophysiological herbivore-vegetation model and adapted it to work for temperate ungulates. I used this model it to ask how movement drives the population dynamics of Mongolian gazelle. I did this by running the model once allowing gazelle to move freely within the landscape and once restricting movement to ~45 x 45km areas. Not only were gazelle more than two times more abundant when they were allowed to move, their population also increased more during years of abundant forage and decreased less during drought, indicating that movement also stabilized population dynamics. Restricting movement resulted in local extinctions because gazelle were vulnerable to boom-bust dynamics or harsh winters. The results suggest that for highly mobile species, protected areas are not an adequate conservation measure and that the focus must be on creating permeable landscapes.
For many arctic and temperate herbivores, including Mongolian gazelle, harsh winters decrease survival. Yet with warmer winters due to climate change this fundamental population control might change. Simultaneously, many areas are experiencing vegetation greening trends which have been linked to the plant-physiological effects of increased atmospheric CO2 concentrations (CO2 fertilization). Both changes could positively influence temperate herbivores but are not well studied and so I examined their effects with the ecophysiological herbivore-vegetation model by modifying it to better account for large herbivore energy expenditures like thermoregulation. I then ran with model with climate data for two contrasting socio-economic future scenarios. Gazelle abundance increased in both future scenarios, driven equally by increases in forage biomass and decreases in winter thermoregulation costs. Increases in forage biomass were due to increases in growing season length and CO2 fertilization effects. While ultimately negative consequences of climate change might cancel out these positive effects, the results show these positive effects are large and cannot be ignored like they currently are.
While there are detailed ecophysiological models of herbivores or vegetation, the combination is still rare. My PhD shows that the combination is key. Gazelle respond to environmental conditions by moving and these movements influence energy intake and expenditure, scaling up to influence population abundance and stability. Under climate change, accounting for both the physiological response of plants and herbivores, showed that both contribute equally to increases in gazelle abundance. Therefore, unlike most climate change studies which examine distribution, I was able to examine abundance. Most importantly, because the herbivore part of the model can simulate any terrestrial ungulate and the vegetation part works globally, I hope the model, whose code is freely available, will be applied to a variety of other questions and herbivore systems in the future.
Das Modell-Acetogen Acetobacterium woodii ist bisher das Acetogen, das physiologisch, biochemisch und bioenergetisch am besten studiert ist. Während A. woodii als Homoacetogen bekannt ist, das Acetat als einziges Endprodukt produziert, hat seine genetische Information das Potenzial für die natürliche Produktion anderer reduzierter Produkte wie Ethanol oder Laktat angedeutet. Darüber hinaus wurde die Umsetzung bestimmter Substrate in A. woodii nur begrenzt untersucht, und ihre Physiologie, Biochemie und Bioenergetik sind noch nicht ausreichend erforscht. Das Ziel dieser Arbeit war es, unentdeckte Stoffwechselwege bestimmter Substrate in A. woodii zu untersuchen und neue metabolische Eigenschaften durch gentechnisches Engineering zu entdecken.
Das erste Kapitel dieser Arbeit konzentrierte sich auf die Untersuchung der formatotrophen Acetogenese von A. woodii. Formiat, eine der vielversprechenden C1-Verbindungen, kann in A. woodii verstoffwechselt werden. Es wurde festgestellt, dass ein Gencluster von A. woodii für eine zweite Formyl-Tetrahydrofolat-Synthetase zusammen mit einem potenziellen Formiat-Transporter kodiert. Um die Rolle dieser Gene zu verstehen, wurden sie in A. woodii deletiert. Die Charakterisierung der daraus resultierenden Mutanten zeigte, dass dieses Gencluster eine wichtige Rolle beim Formiat-Stoffwechsel spielt.
Im zweiten Kapitel wurde die Ethanologenese von A. woodii untersucht. Die Wachstums- und Zellsuspensionsversuche zeigten, dass höhere Fruktosekonzentrationen, Na+-Limitierung und niedriger Bikarbonatgehalt die Ethanolbildung in A. woodii stimulierten. Anschließend wurden Expressionsanalysen und Mutationsstudien durchgeführt, um die Ethanol-bildende Alkohol-Dehydrogenase zu identifizieren.
Das dritte Kapitel konzentrierte sich auf die Charakterisierung der formatogenen Lebensweise von A. woodii während des methylotrophen Stoffwechsels mittels der hdcr-Deletionsmutante. In der ∆hdcr-Mutante wurden die Methylgruppen in Formiat und Acetat umgewandelt, und die Zugabe eines alternativen Elektronenakzeptors, Kaffeat, ermöglichte die Umrichtung des Stoffwechsels auf Homoformatogenese.
Im vierten Kapitel wurde eine Mutante hergestellt und charakterisiert, die Methylene-Tetrahydrofolat-Reduktase (MTHFR) nicht mehr vorhanden ist. In der ∆metVF-Mutante, bei der zwei Untereinheiten von MTHFR genetisch deletiert wurden, wurde ein bisher unbekannter fermentativer Stoffwechsel von A. woodii, gemischte Säuregärung, während des Wachstums auf Fruktose beobachtet. Darüber hinaus wurden durch Transkriptomanalysen die mit diesem neuen fermentativen Stoffwechsel assoziierten Enzyme aufgedeckt.
Im letzten Kapitel wurde die Laktogenese in A. woodii unter Verwendung eines genetisch veränderten Stammes untersucht. A. woodii hat das Potenzial, Laktat durch die bifurkierende Laktatdehydrogenase, LDH/ETF-Komplex, zu produzieren. Die ∆metVF-Mutante war zwar in der Lage, Laktat zu produzieren, aber es wurde eine erhebliche Menge an Elektronen für die H2-Produktion verbraucht. Um die Laktatproduktion zu verbessern, wurde eine Doppeldeletionsmutante, ∆hydBA/hdcr, ausgewählt und die Laktogenese aus C1-Verbindungen in ruhenden Zellen der ∆hydBA/hdcr-Mutante untersucht.
In the present studies, phylogenetic methodologies were used to study patterns and processes of diversification among freshwater pulmonate gastropods, the Basommatophora, which are important organisms of virtually all aquatic ecosystems. It was anticipated to identify monophyla of different hierarchical rank and investigate datasets of morphological and anatomical characters and their utility in phylogenetic reconstructions. Global biogeographic patterns of the limnic Basommatophora are studied using the new phylogenetic hypotheses as basic assumptions of relationships among the taxa of concern. Finally, a case study deals with microevolutionary patterns and processes from an ancient lake system on the island of Sulawesi, Indonesia. First, the composition and position of basommatophoran gastropods among the Euthyneura was investigated using multi-locus DNA sequencing and employing maximum parsimony, maximum likelihood, and Bayesian inference methodologies. According to these analyses, Pulmonata are a monophyletic unit. Within pulmonates, Stylommatophora and Archaeopulmonata are monophyletic, whereas Basommatophora s.l. are paraphyletic and branch into several differently supported groups. Siphonaria is independent of the archaeopulmonate clade. Hygrophila, a clade comprising the superfamilies Lymnaeoidea, Planorboidea, Physoidea, Acroloxoidea, and Latia is monophyletic and the superfamilies themselves are well supported. Parsimonious and maximum likelihood reconstructions of ancestral habitat types occupied by the taxa studied suggest that starting from the ancestral marine habitat, the invasion of the land happened two times independently in the monophyletic Stylommatophora and within the archaeopulmonates. However, the freshwater was invaded only ones by the freshwater pulmonates (Hygrophila). Contrary to previous assumptions, this pathway was entirely aquatic with no trace of terrestrial intermediate stages. The phylogeny of Basommatophora s.str. was studied using a larger taxon set. Besides the previously recognized superfamilies of Hygrophila, the families Acroloxidae, Physidae, Bulinidae, and Lancidae are monophyletic and supported as well. The Ancylidae and Planorbidae, however, were not monophyletic and cluster within the Planorboidea, a clade that is well supported. A sister-group relationship of Physoidea and Lymnaeoidea was well supported in both maximum likelihood and Bayesian inference analyses. Within Acroloxidae, Acroloxus lacustris is basal to the clade comprising lake Baikal endemic genera. Physidae consist of two clearly distinct well supported groups that do not correspond to the subfamilies commonly accepted. Although in Lymnaeoidea, relationships between groups are not well resolved, Lancidae appear as sister-group of Lymnaeidae. A rapid, possibly simultaneous, radiation event (hard polytomy) of the splits between Acroloxoidea, Planorboidea, Physoidea plus Lymnaeoidea is suggested. The diversification patterns of lineages through time was analyzed with two molecular clock approaches. A fossil calibrated tree and the constant rate molecular clock tree were almost identical in their temporal branching pattern. Two phases of accelerated lineage splitting could be recognized in both approaches. Fitting a geological time scale to the molecular trees showed that accelerated lineage splitting periods coincide with recovery phases in the aftermath of the well known pulse mass extinction event at the Cretaceous-Tertiary (K-T) boundary 65 MYA and the biotic crisis at the boundary from Cenomanian-Turonian (C-T) 93.5 MYA, an anoxic event. It appears as if impacts on the evolutionary history of the Basommatophora due to mass extinctions were world-wide and not restricted to a particular biogeographic region. While recovery from mass extinctions and subsequent radiations have been repeatedly demonstrated for marine and terrestrial taxa, the mass extinction events also triggered evolutionary bursts in this major freshwater taxon, supporting the general importance of these global bioevents for the evolution of freshwater ecosystems. A comprehensive phylogenetic hypothesis was inferred for the most diverse taxon of freshwater basommatophorans, the Planorboidea. Besides the distinct Burnupia clade, two major clades were recovered that correspond to subfamily level taxa. All ancylid taxa (except Burnupia) cluster within Bulininae. This ancylid clade is most advanced within Bulininae and consists of two subclades. Sister to this clade is a monophyletic group comprising highspired (buliniform) Australian planorbids and the patelliform endemic Protancylus from Sulawesi. Planorbinae fell into two major groups. Clades recognized by current classification that are supported by the recent analysis are Planorbinae, Bulinini, Planorbini, Segmentinini. The present study found that Ancylidae, as traditionally understood, is paraphyletic. Monophyly of Planorboidea was demonstrated. Previous hypotheses of shell shape evolution were not supported by this study; limpet-shaped taxa are most basal within Planorboidea. Although many taxa still remain to be studied, these results, have strengthened our understanding of planorbid evolutionary history. The utility of morphological and anatomical characters in phylogenetic reconstructions of the Basommatophora was tested. A maximum parsimony analysis of 47 shell characters demonstrated monophyly for Physidae and Ancylidae, whereas Bulinidae, Lymnaeidae and Planorbidae are non-monophyletic according to this dataset. The analysis of 81 morphological and anatomical characters resulted in monophyly for the Lymnaeoidea, Physidae with good supported, whereas the monophylum Acroloxidae was only weakly supported. 128 combined characters were analyzed and showed Acroloxidae, Lymnaeoidea, Ancylidae, and Physidae to be monophyletic. However, considerable bootstrap support was obtained for Physidae and Ancylidae only. Apomorphies inferred for the supported monophyletic taxa are discussed. The inclusion of more taxa and new characters did improve the phylogenetic inferences as compared to previous studies based on morphological and anatomical datasets. The low resolution and absence of support in large parts of the obtained trees must be attributed to an extraordinarily high degree of homoplasy among the taxa of concern. Comparing all the morphological trees and particularly speaking about the absence of resolution and/or support, it could well be that the mosaic-pattern found among many of the characters is an indication of a rapid initial radiation event of lineages leading to the major superfamily taxa that are extant. “Hotspots” or centers of diversity among world-wide Basommatophora are identified as eastern North America, the European–Mediterranean region and the Australian region. There is a general trend of more generic diversity in the northern hemisphere. The lowest diversity was found in South America, Sahara Africa and Madagascar. A parsimony analysis of endemicity (PAE) with taxa-as-characters (generic dataset) yielded a tree characterized by a fairly broad basal polytomy. From this analysis, the relationship between certain geographic superregions can not be resolved by the recent taxa occurring there. Within the superregions, many neighboring biogeographic (sub-) regions cluster together. Brooks Parsimony Analysis (BPA) and Dispersal-Vicariance Analysis (DIVA) yielded complex, partly conflicting reconstruction of the biogeographic history of the world-wide Basommatophora s.str. involving multiple vicariance and dispersal events. An initial vicariance event separated the Acroloxidae (basal lineage to lake Baikal endemics), while in the Palaearctic region both the Physidae and Lymnaeidae and the widespread genus Acroloxus arose independently. The Palaearctic region became separated from eastern and western North America, South Asia, Africa, the Malagassian region, the Guayana-Brazilian subregion, the Australian region, and Indo-westpacific region. Patterns and processes of dispersal and vicariance are discussed for the taxa studied and the geographic subregions involved. It is apparent from this study that no clear-cut pattern can be drawn of either vicariance or dispersal as predominant components in the long history of word-wide distribution of Basommatophora s.str. Rather, the picture is very complex with both processes and additionally strong extinction events at times being in force probably throughout the taxons existence. Comparatively recent and repeated longdistance (cross-continent) dispersals in post-drift times obviously represents a major contribution to the present wide range of several genera and species, particularly in Lymnaeidae and Planorbidae. Thus, the results of this study point to dispersal as a valid counterpart and addition to vicariance theory in explaining patterns found in biogeography. Freshwater molluscs and the Basommatophora s.str. in particular would provide a prime model group for a new methodological framework that places dispersal into the place it deserves. Macroecological rules were tested using the global dataset of distribution patterns of Basommatophora. Bergmann’s rule could not be confirmed for basommatophoran gastropods on a global scale. These findings are in concert with the growing evidence that body size in ectotherms is not at all related to latitude or even follow the converse to Bergmann’s rule. The analysis of generic ranges revealed no indication of a Rapoport effect, which states that a species’ geographical extent decreases from higher to lower latitudes, on a global scale either. Therefore, this is another example of the growing number of studies that raise doubts on the generality of a latitudinal gradient. Among the potential factors limiting the species ranges of freshwater pulmonates on a worldwide scale, two explaining variables were retained in multiple regressions. A climate variable was composed of climatic factors related to the water regime a given species experiences and, therefore, to species’ intrinsic factors and generally to physiological ecology of freshwater pulmonates. Differential adaptations may explain differences in tolerances to temporally changing environmental conditions. These adaptations involve temperature tolerance and life history characteristics. Other factors responsible for obtaining larger range sizes are resistence of a given species to anoxia and desiccation tolerance. The second variable found in this study explaining range size variation was the potential area of different biogeographic regions that were actually occupied by a species. Once again, dispersal capabilities were identified to be crucial in range size evolution. Current ranges are not an adequate reflection of historical ranges in Basommatophora. It can indirectly be concluded that pronounced dispersal by passive means is crucial in obtaining a certain range size. Additionally, it becomes clear from this study, that large scale environmental variation figures prominently in the possession of a particular range size by a particular taxon. Microevolutionary patterns and processes among Basommatophora were studied using a model of endemic limpet-like gastropods from the ancient lakes on Sulawesi (Indonesia), which exhibit an interesting case of diversification in a generally species-poor group of freshwater gastropods. Within these lakes, at least three species of Protancylus evolved allopatrically. These species are characterized by low morphological variation and a specific life history living as epizoa on the caenogastropod Tylomelania spp. They possess an unique reproduction strategy involving the only known case of brood care among Basommatophora. Phylogenetic relationships, phylogeographic pattern, morphological differentiation, species delimitation, life history parameters, and ecology are discussed in the context of evolutionary history. Among the potential modes of speciation, drift-based speciation in the lakes of the Malili Lake system and lake Poso was identified as most likely. Altogether, the studies summarized in this thesis contribute to the understanding of patterns and processes of diversification, and thus evolution, of the Basommatophora. Hopefully, they can provide the basis and trigger more interest in this fascinating model group of pulmonate gastropods.
Drug action is predicated on target engagement in cells and tissues. Historically, pure proteins or protein fragments have been used to query engagement and guide medicinal chemistry efforts toward optimized small molecule inhibitors. However, pure protein assays may fail to comprise the protein complexes and conformations relevant to the disease state. Thus, drug pharmacology observed in a defined, isolated setting may not correlate with pharmacology observed using a biomarker of target activity in the milieu of a living cell. Assessments of target vulnerability are ideally evaluated in live cells, in a microenvironment representing where drug action would occur. Kinases are among the most diverse classes of intracellular enzymes and are implicated in the pathophysiology of numerous diseases. With over 80 FDA approved kinase inhibitors, many kinases are intrinsically vulnerable to enzymatic inhibition via small molecule engagement in the ATP co-substrate binding pocket. Although such vulnerability is readily observed in an isolated setting, achieving selective intracellular engagement without collateral engagement to off-target kinases can be a major challenge. In cells, kinase engagement can be impacted by a number of factors, including ATP competition, multi-protein complexes, target activation state, and drug partitioning. Such factors can be difficult to simulate in a defined system, and methods to interrogate critical binding parameters in cells represents an unmet need. Therefore, intracellular engagement data and selectivity profiles for novel chemical matter is not generally reported in the literature, even for clinical-stage inhibitors. For many kinase inhibitors, cell- free data represents a vast over-estimate of the polypharmacology occurring in a native setting. Thus, providing a more physiologically-accurate assessment of compound selectivity and affinity would enable chemical probe initiatives and pharmaceutical discovery efforts. The RAS/RAF/MEK/ERK pathway (so called MAPK pathway) is among the most highly studied kinase-related pathways in human disease. These MAPK signaling components naturally function in a multi-protein complex (i.e., the RAS signalosome). Dysregulation in this pathway is known driver of tumorigenesis, and mutations in RAS or RAF are observed in >30% of human cancers. For example, RAF kinase is commonly mutated in melanoma, and the GTPase KRAS is the most mutated proto- oncogene in solid tumors. Despite ongoing multidisciplinary drug discovery efforts, this signaling complex has proven undruggable for several decades. As this multiprotein complex cannot currently be reconstituted in a biochemical setting, demonstrating vulnerability at protomers within the RAS/RAF complex has represented a significant challenge. Although potent drug binding or enzyme inhibition can be observed using isolated fragments of RAS or RAF kinase, the measured potencies are generally not commensurate with pathway inhibition in disease-relevant cells. For instance, despite sub-nM potency observed with clinical-stage RAF kinase inhibitors in enzymatic assays, potency of intracellular inhibition of MAPK signaling may be >100-1000-fold weaker. This opens the possibility that underappreciated drug escape mechanisms may be evident in cells that are not observed using purified components. Such observations cast doubt on the assumed mechanism of action of such clinical-stage drugs. Thus, new methods are needed to quantify drug engagement at these critical signaling components to support more accurate assessments of drug vulnerability. Herein are reported advancements in the Bioluminescence Resonance Energy Transfer (BRET) method to accurately query target engagement at monomeric and multiprotein complexes in cells. The prototype target engagement method leveraged an intracellular reporter system comprising NanoLuc (NLuc) tagged proteins in a BRET complex with cell-permeable fluorescent drug tracers.
Engagement of unmodified compounds results in competitive displacement of the BRET complex in live cells. When the BRET tracers are applied at concentrations approximating their apparent affinity, the resulting IC50 of the test compound is within 2-fold of a constant value. Among all reported target engagement methods, BRET is the only method that offers quantitation in live cells. This quantitative accuracy allows for assessments of selectivity within target families. Furthermore, since the method operates with intact live cells, both affinity and binding kinetics can be measured. Using broad-spectrum BRET tracers, entire target families can be evaluated in a single experiment. Herein, a novel workflow is described, to enable improved broad-spectrum kinome profiling in live cells against nearly 200 full length protein kinases.
Understanding the functional roles of cells in neuronal circuits and behavior requires the ability to control neuronal activity in an acute and precise way. The field of optogenetics offers a variety of molecular tools to excite or inhibit neurons with light. In the last decade, several strategies have been proposed for reversible silencing of neurotransmission. These tools vary widely in their mechanisms: ranging from opsin-based light-driven ion pumps or anion channels, which are known to hyperpolarize the cell, to alter ionic gradients or cellular biochemistry; over metabotropic receptors, to tools damaging the neurotransmitter release machinery, that allow only long-term silencing as their recovery requires de novo synthesis of targeted proteins. Therefore, the optogenetic toolbox still lacks tools that combine fast activation and fast reversibility with the ability of long-term silencing.
In this study, the optogenetic tool optoSynC (optogenetic synaptic vesicle clustering) was characterized in depth. optoSynC utilizes the light-induced homo-oligomerization of Arabidopsis thaliana cryptochrome 2 (CRY2) for silencing synaptic transmission via clustering of synaptic vesicles (SVs). CRY2 was targeted to the SV membrane by fusion to the SV transmembrane protein synaptogyrin-1 (SNG-1). The silencing kinetics of optoSynC were determined with analyses of swimming and crawling behavior in Caenorhabditis elegans (C. elegans). Pan-neuronally expressed optoSynC reduced swimming locomotion by 80% within 30 s following photo-stimulation (τon ~7.2 s). Locomotion recovered within 15 min in darkness (τoff ~6.5 min). Analysis of crawling behavior indicated even faster activation within 2 s and an almost complete inhibition of the LITE-1-mediated escape response. This escape response occurs at high blue light intensities and results in an increased velocity, which was not detected after optoSynC activation. However, optoSynC can exert its full effect at significantly lower intensities (25 µW/mm²) and is so light-sensitive that it can even be activated at 1.4 µW/mm². optoSynC could be fully recovered and reactivated at least twice without decreased efficiency. With a combination of pharmacological analysis and optogenetics, it has been demonstrated that optoSynC can inhibit neurotransmitter release for several hours. To realize its full potential, optoSynC should be expressed in an sng-1 mutant background. Expression along with endogenous SNG-1 decreases the effectiveness of optoSynC by 50%. Specific expression of optoSynC in cholinergic or GABAergic neurons could robustly inhibit swimming behavior by 55% and 30%, respectively. Moreover, optoSynC can selectively inhibit individual neurons like the nociceptive neuron pair PVD. When PVD is photoactivated by Chrimson, a red-light activatable channelrhodopsin, forward locomotion increases. Activation of optoSynC reduced this behavior by 50%. Besides my experiments in C. elegans, my cooperation partners Dr. Holger Dill and Yilmaz Arda Ateş demonstrated silencing of neurotransmission with optoSynC in zebrafish, and Dr. Shigeki Watanabe and Brady D. Goulden in murine hippocampal neurons.
The clustering of SVs as the mode of action of optoSynC could be confirmed using transmission electron microscopy. Analysis of micrographs of cholinergic synapses stimulated with and without light revealed that distances of neighboring synaptic vesicles in the cytosol were reduced by around 14% after photoactivation. Distances of docked SVs at the plasma membrane remained unaffected. However, near the dense projection, docked SVs accumulated, while other docked SVs were depleted after optoSynC activation. Activation of optoSynC increased the appearances of SVs and dense core vesicles (DCVs) in micrographs. It is unclear whether sizes became physically larger due to lateral pressure by CRY2 aggregates in the SV membrane or if oligomer formation only altered their appearance in micrographs. optoSynC did not accumulate in the plasma membrane to the extent that abnormal structures at the plasma membrane or dense projection were observed. Recycling of SVs remained unaffected by optoSynC as no unusual number of large vesicles was determined. Therefore, optoSynC inhibits neuronal activity mainly by clustering of cytosolic SVs.
To study the precise sequence and timing of events of vesicle mobilization, it is necessary to introduce known stops in the synaptic vesicle cycle which can be achieved by optoSynC. The C. elegans mutation dyn-1(ts-) is a temperature-sensitive dynamin mutation that blocks the recycling of SVs from the plasma membrane and early endosomes at temperatures exceeding 25 °C. By expressing optoSynC in dyn-1(ts-) animals, a novel assay was established, enabling the transfer of SVs between different stages in the SV cycle. Cluster formation of reserve pool SVs blocks the SV cycle before the process of docking and priming begins, while the SV cycle is blocked after the fusion of SVs at high temperatures. It could be demonstrated that behavior returned 15 min after optoSynC activation while animals without optoSynC remained immobile. Unfortunately, the time scale of the recovery from inhibition by optoSynC is too long to effectively study vesicle mobilization.
Die Charakterisierung reaktiver Intermediate ist experimentell äußerst anspruchsvoll und häufig nicht eindeutig möglich. Die chemische Fachliteratur ist daher durchzogen von Arbeiten, in denen Intermediate anhand chemischer Intuition oder aufgrund spekulativer Interpretationen experimenteller Daten postuliert werden. Auch wenn es die Chemie wie kaum eine andere wissenschaftliche Disziplin geschafft hat, naturgegebene Zusammenhänge intuitiv erfassbar zu machen und es Chemikern somit möglich ist, mit Zettel und Stift Moleküle mit gewünschten Eigenschaften zu designen und komplexe Reaktionen zu planen, so ist doch davon auszugehen, dass zahlreiche in der Literatur postulierte Intermediate nicht korrekt zugeordnet wurden. Durch die massive Steigerung der Leistungsfähigkeit von Computern und Software in den vergangenen Jahrzehnten lassen sich quantenchemisch immer größere Systeme mit hinreichender Genauigkeit behandeln, sodass es in vielen Fällen möglich ist, experimentelle Untersuchungen theoretisch zu evaluieren und auch experimentell nicht nachweisbare Moleküle detailliert zu untersuchen. Insbesondere durch kombinierte experimentelle und quantenchemische Studien können komplexe chemische Zusammenhänge detailliert verstanden werden. Dadurch lassen sich neue Konzepte entwickeln, die eine Weiterentwicklung chemischer Intuition ermöglichen. Anhand dieses Leitbilds wurde in dieser Arbeit die Stoffklasse der Metallopniktinidene sowie daraus abgeleitete Verbindungen quantenchemisch untersucht.
K + is the most abundant cytosolic cation in bacteria, and its homeostasis is vital for bacterial survival, playing roles in many essential processes like pH homeostasis, protein synthesis and osmoregulation. When surrounding K + concentrations become very low, bacteria require an active high-affinity uptake system to ensure sufficient cellular K + levels. In many prokaryotes, this system is the K + pump KdpFABC. Peculiarly, KdpFABC forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB), and for a long time, the mechanism of how transport and ATP hydrolysis between these subunits are coordinated remained unclear. By applying a combination of cryo-EM, biochemical assays, and MD simulations, we have been able to shed light on a unique transport mechanism that combines both the channel and P-type ATPase subunits.
At high K + levels, KdpFABC needs to be inhibited to prevent excessive K + accumulation. This is achieved by a phosphorylation of the serine residue in the TGES 162 motif in the A domain of the pump subunit KdpB, which was shown to stall the complex in the E1P intermediate. Using cryo-EM studies under turnover conditions, we illuminated how stalling in this high-energy intermediate is possible.
Furthermore, we identify a previously uncharacterized atypical serine kinase domain in the sensor histidine kinase KdpD as the responsible kinase for KdpB phosphorylation, giving it a dual role in transcriptional and post-translational regulation of the Kdp system.
