iNClusive: a database collecting useful information on non-canonical amino acids and their incorporation into proteins for easier genetic code expansion implementation.

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique.

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Dimeric Lectin Chimeras as Novel Candidates for Gb3-Mediated Transcytotic Drug Delivery through Cellular Barriers.

Receptor-mediated transcytosis is an elegant and promising strategy for drug delivery across biological barriers. Here, we describe a novel ligand-receptor pair based on a dimeric, engineered derivative of the Pseudomonas aeruginosa lectin LecA, here termed Di-LecA, and the host cell glycosphingolipid Gb3. We characterized the trafficking kinetics and transcytosis efficiencies in polarized Gb3-positive and -negative MDCK cells using mainly immunofluorescence in combination with confocal microscopy. To evaluate the delivery capacity of dimeric LecA chimeras, EGFP was chosen as a fluorescent model protein representing macromolecules, such as antibody fragments, and fused to either the N- or C-terminus of monomeric LecA using recombinant DNA technology. Both LecA/EGFP fusion proteins crossed cellular monolayers in vitro. Of note, the conjugate with EGFP at the N-terminus of LecA (EGFP-LecA) showed a higher release rate than the conjugate with EGFP at the C-terminus (LecA-EGFP). Based on molecular dynamics simulations and cross-linking studies of giant unilamellar vesicles, we speculate that EGFP-LecA tends to be a dimer while LecA-EGFP forms a tetramer. Overall, we confidently propose the dimeric LecA chimeras as transcytotic drug delivery tools through Gb3-positive cellular barriers for future in vivo tests.

Designed membrane protein heterodimers and control of their affinity by binding domain and membrane linker properties.

Many membrane proteins utilize dimerization to transmit signals across the cell membrane via regulation of the lateral binding affinity. The complexity of natural membrane proteins hampers the understanding of this regulation on a biophysical level. We designed simplified membrane proteins from well-defined soluble dimerization domains with tunable affinities, flexible linkers, and an inert membrane anchor. Live-cell single-molecule imaging demonstrates that their dimerization affinity indeed depends on the strength of their binding domains. We confirm that as predicted, the 2-dimensional affinity increases with the 3-dimensional binding affinity of the binding domains and decreases with linker lengths. Models of extended and coiled linkers delineate an expected range of 2-dimensional affinities, and our observations for proteins with medium binding strength agree well with the models. Our work helps in understanding the function of membrane proteins and has important implications for the design of synthetic receptors.

Production of Phytochromes by High-Cell-Density E. coli Fermentation.

Phytochromes are important photoreceptors of plants, bacteria, and fungi responsive to light in the red and far-red spectrum. For increasing applications in basic research, synthetic biology, and materials sciences, it is required to recombinantly produce and purify phytochromes in high amounts. An ideal host organism for this purpose is E. coli due to its widespread use, fast growth, and ability for high-cell-density fermentation. Here, we describe the development of a generic platform for the production of phytochromes in E. coli that is compatible with high-cell-density fermentation. We exemplify our approach by the production of the photosensory domains of phytochrome B (PhyB) from A. thaliana and of the cyanobacterial phytochrome 1 (Cph1) from Synechocystis PCC 6803 in the multigram scale per 10 L fermentation run.

A Monoallelic Two-Hit Mechanism in PLCD1 Explains the Genetic Pathogenesis of Hereditary Trichilemmal Cyst Formation.

Trichilemmal cysts are common hair follicle-derived intradermal cysts. The trait shows an autosomal dominant mode of transmission with incomplete penetrance. Here, we describe the pathogenetic mechanism for the development of hereditary trichilemmal cysts. By whole-exome sequencing of DNA from the blood samples of 5 affected individuals and subsequent Sanger sequencing of a family cohort including 35 affected individuals, this study identified a combination of the Phospholipase C Delta 1 germline variants c.903A>G, p.(Pro301Pro) and c.1379C>T, p.(Ser460Leu) as a high-risk factor for trichilemmal cyst development. Allele-specific PCRs and cloning experiments showed that these two variants are present on the same allele. The analysis of tissue from several cysts revealed that an additional somatic Phospholipase C Delta 1 mutation on the same allele is required for cyst formation. In two different functional in vitro assays, this study showed that the protein function of the cyst-specific 1-phosphatidylinositol 4, 5-bisphosphate phosphodiesterase delta-1 protein variant is modified. This pathologic mechanism defines a monoallelic model of the two-hit mechanism proposed for tumor development and other hereditary cyst diseases.

Disordered Conformation with Low Pii Helix in Phosphoproteins Orchestrates Biomimetic Apatite Formation.

The interplay between noncollagenous proteins and biomineralization is widely accepted, yet the contribution of their secondary structure in mineral formation remains to be clarified. This study demonstrates a role for phosvitin, an intrinsically disordered phosphoprotein, in chick embryo skeletal development, and using circular dichroism and matrix least-squares Henderson-Hasselbalch global fitting, unravels three distinct pH-dependent secondary structures in phosvitin. By sequestering phosvitin on a biomimetic 3D insoluble cationic framework at defined pHs, access is gained to phosvitin in various conformational states. Induction of biomimetic mineralization at near physiological conditions reveals that a disordered secondary structure with a low content of PII helix is remarkably efficient at promoting calcium adsorption, and results in the formation of biomimetic hydroxyapatite through an amorphous calcium phosphate precursor. By extending this finding to phosphorylated full-length human recombinant dentin matrix protein-1 (17-513 AA), this bioinspired approach provides compelling evidence for the role of a disordered secondary structure in phosphoproteins in bone-like apatite formation.

Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes.

The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosan-stimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalized phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalized phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.

Differential effects of flavonols on inactivation of alpha1-antitrypsin induced by hypohalous acids and the myeloperoxidase-hydrogen peroxide-halide system.

Alpha1-antitrypsin is well known for its ability to inhibit human neutrophil elastase. Pretreatment of alpha1-antitrypsin with hypohalous acids HOCl and HOBr as well as with the myeloperoxidase-hydrogen peroxide-chloride (or bromide) system inactivated this proteinase. The flavonols rutin, quercetin, myricetin, and kaempferol inhibited the inactivation of alpha1-antitrypsin by HOCl and HOBr with rutin having the most pronounced effect. In contrast, these flavonols did not remove the proteinase inactivation by the myeloperoxidase-hydrogen peroxide-halide system. Taurine did not protect against the inactivation of alpha1-antitrypsin by HOCl, HOBr, or the myeloperoxidase-hydrogen peroxide-halide system, while methionine was efficient in all systems. A close association between myeloperoxidase and alpha1-antitrypsin was revealed by native gel electrophoresis and in-gel peroxidase staining. In addition, alpha1-antitrypsin binds to the myeloperoxidase components transferred after SDS-PAGE on a blotting membrane. With this complex formation, myeloperoxidase overcomes the natural antioxidative protective system of plasma and prevents the inactivation of alpha1-antitrypsin.