Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.     

The hypothesis of sympatric speciation as the dominant generator of endemism in a global hotspot of biodiversity

Allopatric or sympatric speciation influence the degree to which closely related species coexist in different manners, altering the patterns of phylogenetic structure and turnover among and between communities. The objective of this study was to examine whether phylogenetic community structure and turnover in the Brazilian Atlantic Forest permit conclusions about the dominant process for the formation of extant angiosperm richness of tree species. Therefore, we analyzed phylogenetic community structure (MPD, MNTD) as well as taxonomic (Jaccard similarity) and phylogenetic turnover (betaMPD, betaMNTD) among and between 49 tree communities distributed among three different habitat types. Mean annual precipitation and mean annual temperature in each survey area were estimated. Phylogenetic community structure does not differ between habitat types, although MPD reduces with mean annual temperature. Jaccard similarity decreases and betaMNTD increases with spatial distance and environmental differences between study sites. Spatial distance explains the largest portions of variance in the data, indicating dispersal limitation and the spatial aggregation of recently formed taxa, as betaMNTD is related to more recent evolutionary events. betaMPD, that is related to deep evolutionary splits, shows no spatial or environmental pattern, indicating that older clades are equally distributed across the Brazilian Atlantic Forest. While similarity pattern indicates dispersal limitations, the spatial turnover of betaMNTD is consistent with a high degree of sympatric speciation generating extant diversity and endemism in the Brazilian Atlantic Forest. More comprehensive approaches are necessary to reduce spatial sampling bias, uncertainties regarding angiosperm diversification patterns and confirm sympatric speciation as the dominant generator for the formation of extant species diversity in the Brazilian Atlantic Forest.     

Molecular Evolution of Peptide Ligands with Custom-Tailored Characteristics for Targeting of Glycostructures

As an advanced approach to identify suitable targeting molecules required for various diagnostic and therapeutic interventions, we developed a procedure to devise peptides with customizable features by an iterative computer-assisted optimization strategy. An evolutionary algorithm was utilized to breed peptides in silico and the “fitness” of peptides was determined in an appropriate laboratory in vitro assay. The influence of different evolutional parameters and mechanisms such as mutation rate, crossover probability, gaussian variation and fitness value scaling on the course of this artificial evolutional process was investigated. As a proof of concept peptidic ligands for a model target molecule, the cell surface glycolipid ganglioside G_M1, were identified. Consensus sequences describing local fitness optima were reached from diverse sets of L- and proteolytically stable D lead peptides. Ten rounds of evolutional optimization encompassing a total of just 4400 peptides lead to an increase in affinity of the peptides towards fluorescently labeled ganglioside G_M1 by a factor of 100 for L- and 400 for D-peptides.     

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient’s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.     

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody in Escherichia coli

Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.     

Macroarthropod species richness and conservation priorities in Stratiotes aloides (L.) lakes

Species with narrow ranges and specialised traits are most at risk, and the extinction wave is further enhanced by coextinctions. We studied the conservation value and indicator potential of Stratiotes aloides, an aquatic macrophyte that has declined considerably in Europe. Our purpose was to determine whether S. aloides could be used as an indicator of a valuable habitat in terms of macroarthropod diversity and species richness. The potential occurrence of an internationally endangered Stratiotes-habitat specialist, the dragonfly Aeshna viridis, can increase the conservation value of plant colonies. S. aloides beds harboured diverse macroarthropod fauna often containing species of conservation concern, including A. viridis. Stratiotes is a potential indicator of a valuable habitat, and its indicator value is enhanced by the easy identification of the species. However, its use as an indicator of a defined macroarthropod community is limited because no particular community type is connected to it. We suggest that protecting Stratiotes simultaneously conserves valuable arthropod fauna, including A. viridis.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Light Activation of Rhodopsin: Insights from Molecular Dynamics Simulations Guided by Solid-State NMR Distance Restraints

Structural restraints provided by solid-state NMR measurements of the metarhodopsin II intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin II intermediate (> 1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from ¹³C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor.