Prediction of protein mutation effects based on dehydration and hydrogen bonding – A large‐scale study

Reliable computational prediction of protein side chain conformations and the energetic impact of amino acid mutations are the key aspects for the optimization of biotechnologically relevant enzymatic reactions using structure‐based design. By improving the protein stability, higher yields can be achieved. In addition, tuning the substrate selectivity of an enzymatic reaction by directed mutagenesis can lead to higher turnover rates. This work presents a novel approach to predict the conformation of a side chain mutation along with the energetic effect on the protein structure. The HYDE scoring concept applied here describes the molecular interactions primarily by evaluating the effect of dehydration and hydrogen bonding on molecular structures in aqueous solution. Here, we evaluate its capability of side‐chain conformation prediction in classic remutation experiments. Furthermore, we present a new data set for evaluating “cross‐mutations,” a new experiment that resembles real‐world application scenarios more closely. This data set consists of protein pairs with up to five point mutations. Thus, structural changes are attributed to point mutations only. In the cross‐mutation experiment, the original protein structure is mutated with the aim to predict the structure of the side chain as in the paired mutated structure. The comparison of side chain conformation prediction (“remutation”) showed that the performance of HYDE_protein is qualitatively comparable to state‐of‐the art methods. The ability of HYDE_protein to predict the energetic effect of a mutation is evaluated in the third experiment. Herein, the effect on protein stability is predicted correctly in 70% of the evaluated cases. Proteins 2017; 85:1550–1566. © 2017 Wiley Periodicals, Inc.     

Variation in growth,yield and protein concentration in <Emphasis Type="Italic">Saccharina latissima</Emphasis> (Laminariales,Phaeophyceae) cultivated with different wave and current exposures in the Faroe Islands

Ropes seeded directly with Saccharina latissima were deployed at different exposures (sheltered, current exposed and wave exposed) in a Faroese sound and characteristics of growth and quality of the biomass and surroundings were evaluated during the growth season from March to August 2015. Saccharina latissima individuals cultivated at the current exposed location were heavier compared to the individuals cultivated at the other locations; however, the total biomass yield was significantly lower at the current exposed location. The protein and nutritional value of the biomass varied with season but showed no correlation with exposure. The highest protein levels and EAA (essential amino acid) score were measured in the spring (April and May) samples. The amino acid composition was dominated by glutamate followed by aspartic acid; however, this was replaced by methionine in the July samples. Total Kjeldahl nitrogen was significantly higher in May and August compared to July, and the nitrate concentration in the biomass was significantly lower in May and August compared to July. Nitrate was most available in the seawater at the time of deployment (3rd of March) and decreased during spring and summer. Saccharina latissima was cultivated successfully at the sheltered, current exposed and wave exposed locations using a direct seeding method. However, our results indicate that the lower biomass yield at the current exposed cultivation location compared to the sheltered and wave exposed are due to the direct seeding method, and possible limitations using this method need further testing and optimization.     

Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella

Ubiquitylation is one of the cardinal post‐translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image‐based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING‐between‐RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response.     

The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogen Candida glabrata

The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin‐5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up‐regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin‐encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross‐road between multidrug resistance and pathogenesis.     

Isolation and characterization of polymorphic microsatellite markers in Eurasian vulture Gyps fulvus

To obtain polymorphic molecular markers for population genetic and conservation studies in the Eurasian vulture Gyps fulvus populations, we screened a size‐selected partial genomic library enriched for microsatellites with dinucleotide motifs. A total of five polymorphic markers were obtained. The number of alleles ranged from two to nine and the observed and expected heterozygosity were very similar. These markers will be very useful for studying population structure and to evaluate conservation programmes.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

Polar auxin transport is required for the inhibition by blue light of the elongation-related LeEXT tomato gene

Hypocotyl elongation is an early developmental process regulated antagonistically by light and auxin. To highlight the interaction between both signals, we studied the photoregulation of the auxin-induced tomato (Lycopersicon esculentum Mill.) gene LeEXT involved in this process. RNA gel blot analysis indicated that this gene is down-regulated in response to blue light. We demonstrate that this response is principally mediated by the blue light photoreceptor cry1, but an interaction with the red/far-red light photoreceptors phyA, phyB1 and phyB2 has also been established. Furthermore, the polar auxin transport inhibitor NPA reverts the blue light inhibition of Lycopersicon esculentum gene encoding xyloglucan endotransglycosylase (LeEXT) expression, when it has the opposite effect in the dark or under red light. These results provide strong support for a specific interaction between auxin and blue light transduction pathways in the control of LeEXT expression, and therefore, of hypocotyl elongation in tomato.     

Facteurs impliqués dans le remodelage de la chromatine au cours de la spermiogenèse

Round spermatids are post-meiotic cells with a haploid genome contained in a nucleus, with a structure initially similar to that of the somatic cell nucleus. During spermatogenesis, the spermatid nucleus undergoes drastic remodelling during which it first elongates and then condenses into the very specific and tightly packaged structure of the sperm nucleus. During this remodelling dthe histones are replaced by transition proteins, which, in turn, are replaced by protamines, the specific nuclear proteins of the spermatozoa. Immediately prior to their replacement, the histones are hyperacetylated. The first part of our work was to precisely characterise the changes in histone acetylation during murine spermatogenesis. We have shown that the core histones H2A, H2B, H3 and H4 are hyperacetylated in the elongating spermatids. We have also shown that these changes in acetylation are associated with degradation of the enzymes responsible for histone deacetylation, histone deacetylases or HDACs, while histone acetyl transferases are still present in these cells. The histone acetylation pattern was also investigated during human spermatogenesis, revealing that histone hyperacetylation in the nucleus of elongating spermatids, which appears to be conserved during the course of evolution, also occurs during human spermatogenesis. Moreover, our data obtained from the testes of men with severely altered spermatogenesis, including SCO syndromes (Sertoli Cells Only Syndromes), show that a global hyperacetylation of the Sertoli cell nuclei is associated with an absence of meiotic and post-meiotic cells. This suggests that the global histone acetylation variations observed during spermatogenesis are part of a signalling pathway involving germ cell — Sertoli cell communication. Altogether, these data provide a basis for a better understanding of the mechanisms and identification of the factors involved in post-meiotic remodelling of chromatin.     

Novel Derivatives of 9,10-Anthraquinone Are Selective Algicides against the Musty-Odor Cyanobacterium Oscillatoria perornata

Musty “off-flavor” in pond-cultured channel catfish (Ictalurus punctatus) costs the catfish production industry in the United States at least $30 million annually. The cyanobacterium Oscillatoria perornata (Skuja) is credited with being the major cause of musty off-flavor in farm-raised catfish in Mississippi. The herbicides diuron and copper sulfate, currently used by catfish producers as algicides to help mitigate musty off-flavor problems, have several drawbacks, including broad-spectrum toxicity towards the entire phytoplankton community that can lead to water quality deterioration and subsequent fish death. By use of microtiter plate bioassays, a novel group of compounds derived from the natural compound 9,10-anthraquinone have been found to be much more selectively toxic towards O. perornata than diuron and copper sulfate. In efficacy studies using limnocorrals placed in catfish production ponds, application rates of 0.3 μM (125 μg/liter) of the most promising anthraquinone derivative, 2-[methylamino-N-(1′-methylethyl)]-9,10-anthraquinone monophosphate (anthraquinone-59), dramatically reduced the abundance of O. perornata and levels of 2-methylisoborneol, the musty compound produced by O. perornata. The abundance of green algae and diatoms increased dramatically 2 days after application of a 0.3 μM concentration of anthraquinone-59 to pond water within the limnocorrals. The half-life of anthraquinone-59 in pond water was determined to be 19 h, making it much less persistent than diuron. Anthraquinone-59 appears to be promising for use as a selective algicide in catfish aquaculture.     

N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues as vanilloid receptor ligands.

The vanilloid receptor represents a promising target for drug development. Building on our previous strategies which have generated potent agonists for VR1, we now describe a series of novel N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues, several of which are potent VR1 antagonists. We report here the rationale for the design, the synthesis, and the in vitro characterization of activity in assays for [(3)H]resiniferatoxin binding and (45)Ca influx using heterologously expressed rat VR1.