Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

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Background In silico methods for toxicity prediction have increased significantly in recent years due to the 3Rs principle. This also applies to predicting reproductive toxicology, which is one of the most critical factors in pesticide approval. The widely used quantitative structure–activity relationship (QSAR) models use experimental toxicity data to create a model that relates experimentally observed toxicity to molecular structures to predict toxicity. Aim of the study was to evaluate the available prediction models for developmental and reproductive toxicity regarding their strengths and weaknesses in a pesticide database. Methods The reproductive toxicity of 315 pesticides, which have a GHS classification by ECHA, was compared with the prediction of different in silico models: VEGA, OECD (Q)SAR Toolbox, Leadscope Model Applier, and CASE Ultra by MultiCASE. Results In all models, a large proportion (up to 77%) of all pesticides were outside the chemical space of the model. Analysis of the prediction of remaining pesticides revealed a balanced accuracy of the models between 0.48 and 0.66. Conclusion Overall, predictions were only meaningful in rare cases and therefore always require evaluation by an expert. The critical factors were the underlying data and determination of molecular similarity, which offer great potential for improvement.

Attenuated poxviruses like modified vaccinia virus Ankara (MVA) are promising vectors for vaccines against infectious diseases and cancer. However, host innate immune responses interfere with the viral life cycle and also influence the immunogenicity of vaccine vectors. Sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a phosphohydrolase and reduces cellular deoxynucleoside triphosphate (dNTP) concentrations, which impairs poxviral DNA replication in human dendritic cells (DCs). Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) encode an accessory protein called viral protein X (Vpx) that promotes proteasomal degradation of SAMHD1, leading to a rapid increase in cellular dNTP concentrations. To study the function of SAMHD1 during MVA infection of human DCs, the SIV vpx gene was introduced into the MVA genome (resulting in recombinant MVA-vpx). Infection of human DCs with MVA-vpx led to SAMHD1 protein degradation and enabled MVA-vpx to replicate its DNA genome and to express genes controlled by late promoters. Late gene expression by MVA-vpx might improve its vaccine vector properties; however, type I interferon expression was unexpectedly blocked by Vpx-expressing MVA. MVA-vpx can be used as a tool to study poxvirus-host interactions and vector safety. IMPORTANCE SAMHD1 is a phosphohydrolase and reduces cellular dNTP concentrations, which impairs poxviral DNA replication. The simian SIV accessory protein Vpx promotes degradation of SAMHD1, leading to increased cellular dNTP concentrations. Vpx addition enables poxviral DNA replication in human dendritic cells (DCs), as well as the expression of viral late proteins, which is normally blocked. SAMHD1 function during modified vaccinia virus Ankara (MVA) infection of human DCs was studied with recombinant MVA-vpx expressing Vpx. Infection of human DCs with MVA-vpx decreased SAMHD1 protein amounts, enabling MVA DNA replication and expression of late viral genes. Unexpectedly, type I interferon expression was blocked after MVA-vpx infection. MVA-vpx might be a good tool to study SAMHD1 depletion during poxviral infections and to provide insights into poxvirus-host interactions.

Within toxicology, reproductive toxicology is a highly relevant and socially particularly sensitive field. It encompasses all toxicological processes within the reproductive cycle and therefore includes many effects and modes of action. This makes the assessment of reproductive toxicity very challenging despite the established in vivo studies. In addition, the in vivo studies are very demanding both in terms of their conduct and interpretation, and there is scope for decision‐making on both aspects. As a result, the interpretation of study results may vary from laboratory to laboratory. For the final classification, the assessment of relevance for men is decisive. The problem here is that relatively little is known about the species differences between men and the usual test animals (rat and rabbit). The rabbit in particular has hardly been researched in molecular biology. The aim of the dissertation was to develop approaches for a better assessment of reproductive toxicity, with two different foci:The first aim was to investigate species differences, focusing on the expression of xenobiotic transporters during ontogeny. Xenobiotic transporters, of the superfamily of ATP‐binding cassette transporters (ABC) or solute carriers (SLC), are known to transport exogenous substances in addition to their endogenous substrates and therefore play an important role in the absorption, distribution and excretion of xenobiotics. Species differences in kinetics can in turn have a major impact on toxic effects. In the study, the expression of 20 xenobiotic transporters during ontogeny was investigated at the mRNA level in the liver, kidney and placenta of rats and rabbits and compared with that of men. This revealed major differences in the expression of the transporters between the species. However, further studies on the functionality and activity of the xenobiotic transporters are needed to fully assess the kinetic impact of the observed species differences. Overall, the study provides a valid starting point for further systematic investigations of species differences at the protein level. Furthermore, it provides previously unavailable data on the expression of xenobiotic transporters during ontogeny in rabbits, which is an important step in the molecular biological study of this species. The second part focused on investigating the predictive power of in silico models for reproductive toxicology in relation to pesticides. Both the commercial and the freely available models did not perform adequately in the evaluation. Three reasons could be identified for this: 1. many pesticides are outside the chemical space of the models, 2. different definition/assessment of reproductive toxicity and 3. problems in detecting similarity between molecules. To solve these problems, an extension of the databases on reproductive toxicity in relation to pesticides, respecting a uniform nomenclature, is needed. Furthermore, endpoint‐specific models should be developed which, in addition to the usual structure‐based fingerprints, use descriptors for, for example, biological activity. Overall, the dissertation shows how essential it is to further research the modes of action of reproductive toxicity. This knowledge is necessary to correctly assess in vivo studies and their relevance to men, as well as to improve the predictive power of in silico models by incorporating this information.