Evolutionary and functional characterization of lagomorph guanylate-binding proteins: a story of gain and loss and shedding light on expression, localization and innate immunity-related functions.

Guanylate binding proteins (GBPs) are an evolutionarily ancient family of proteins that are widely distributed among eukaryotes. They belong to the dynamin superfamily of GTPases, and their expression can be partially induced by interferons (IFNs). GBPs are involved in the cell-autonomous innate immune response against bacterial, parasitic and viral infections. Evolutionary studies have shown that GBPs exhibit a pattern of gene gain and loss events, indicative for the birth-and-death model of evolution. Most species harbor large GBP gene clusters that encode multiple paralogs. Previous functional and in-depth evolutionary studies have mainly focused on murine and human GBPs. Since rabbits are another important model system for studying human diseases, we focus here on lagomorphs to broaden our understanding of the multifunctional GBP protein family by conducting evolutionary analyses and performing a molecular and functional characterization of rabbit GBPs. We observed that lagomorphs lack GBP3, 6 and 7. Furthermore, Leporidae experienced a loss of GBP2, a unique duplication of GBP5 and a massive expansion of GBP4. Gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and transcriptome data revealed that leporid GBP expression varied across tissues. Overexpressed rabbit GBPs localized either uniformly and/or discretely to the cytoplasm and/or to the nucleus. Oryctolagus cuniculus (oc)GBP5L1 and rarely ocGBP5L2 were an exception, colocalizing with the trans-Golgi network (TGN). In addition, four ocGBPs were IFN-inducible and only ocGBP5L2 inhibited furin activity. In conclusion, from an evolutionary perspective, lagomorph GBPs experienced multiple gain and loss events, and the molecular and functional characteristics of ocGBP suggest a role in innate immunity.

Evolution of primate interferon-induced transmembrane proteins (IFITMs): a story of gain and loss with a differentiation into a canonical cluster and IFITM retrogenes.

Interferon-inducible transmembrane proteins (IFITMs) are a family of transmembrane proteins. The subgroup of immunity-related (IR-)IFITMs is involved in adaptive and innate immune responses, being especially active against viruses. Here, we suggest that IFITMs should be classified as (1) a canonical IFITM gene cluster, which is located on the same chromosome, and (2) IFITM retrogenes, with a random and unique location at different positions within the genome. Phylogenetic analyses of the canonical cluster revealed the existence of three novel groups of primate IFITMs (pIFITM) in the IR-IFITM clade: the prosimian pIFITMs(pro), the new world monkey pIFITMs(nwm) and the old world monkey pIFITMs(owm). Therefore, we propose a new nomenclature: IR-pIFITM1, IR-pIFITM2, IR-pIFITM3, IR-pIFITMnwm, IR-pIFITMowm, and IR-pIFITMpro. We observed divergent evolution for pIFITM5 and pIFITM10, and evidence for concerted evolution and a mechanism of birth-and-death evolution model for the IR-pIFITMs. In contrast, the IFITMs scattered throughout the genomes possessed features of retrogenes retrotransposed by class 1 transposable elements. The origin of the IFITM retrogenes correspond to more recent events. We hypothesize that the transcript of a canonical IFITM3 has been constantly retrotransposed using class 1 transposable elements resulting in the IFITM retro(pseudo)genes. The unique pattern of each species has most likely been caused by constant pseudogenization and loss of the retro(pseudo)genes. This suggests a third mechanism of evolution for the IR-IFITMs in primates, similar to the birth-and-death model of evolution, but via a transposable element mechanism, which resulted in retro(pseudo)genes.

Functional cross-species conservation of guanylate-binding proteins in innate immunity.

Guanylate binding proteins (GBPs) represent an evolutionary ancient protein family widely distributed among eukaryotes. They are interferon (IFN)-inducible guanosine triphosphatases that belong to the dynamin superfamily. GBPs are known to have a major role in the cell-autonomous innate immune response against bacterial, parasitic and viral infections and are also involved in inflammasome activation. Evolutionary studies depicted that GBPs present a pattern of gain and loss of genes in each family with several genes pseudogenized and some genes more divergent, indicative for the birth-and-death evolution process. Most species harbor large GBP gene clusters encoding multiple paralogs. Previous functional studies mainly focused on mouse and human GBPs, but more data are becoming available, broadening the understanding of this multifunctional protein family. In this review, we will provide new insights and give a broad overview about GBP evolution, conservation and their roles in all studied species, including plants, invertebrates and vertebrates, revealing how far the described features of GBPs can be transferred to other species.

Engineering of Thermostable ß-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

The ß-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-ßHAD), which catalyzes the NADP+ -dependent oxidation of ß-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-ßHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.

Same same but different - Antiviral factors interfering with the infectivity of HIV particles.

Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Novel strategies to combat this pandemic include the discovery of cellular proteins targeting distinct steps of the HIV replication cycle. Here, we summarize our current knowledge on antiviral proteins interfering with the infectivity of released HIV particles.

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.