Protein engineering for covalent immobilization and enhanced stability through incorporation of multiple noncanonical amino acids

In this study, we demonstrate the application of multiple functional properties of proteins generated through coupling of residue-specific and site-specific incorporation method. With green fluorescent protein (GFP) as a model protein, we constructed multifunctional GFP through sitespecific incorporation of L-3,4-dihydroxyphenylalanine (DOPA) and residue-specific incorporation of (2S, 4S)-4- fluoroproline (4S-FP) or L-homopropargylglycine (hpg). Fluorescence analysis revealed a conjugation efficiency of approximately 20% for conjugation of DOPA-containing variants GFPdopa, GFPdp[4S-FP], and GFPdphpg onto chitosan. While incorporation of 4S-FP improved protein folding and stability, hpg incorporation into GFP allowed conjugation with fluorescent dye/polyethylene glycol (PEG). In addition, the modification of GFPhpg and GFPdphpg with PEG through Cu(I)-catalyzed click reaction increased protein thermal stability by about two-fold of the wild-type GFP.     

Parasitism may alter functional response comparisons: a case study on the killer shrimp <Emphasis Type="Italic">Dikerogammarus villosus</Emphasis> and two non-invasive gammarids

The Ponto-Caspian freshwater amphipod Dikerogammarus villosus has colonized most of the water bodies of continental Europe where it causes strong structural alterations in recipient communities that can lead to changes in ecosystem-level processes, mainly because of a strong predatory behaviour. Most of the D. villosus populations from the invaded range have been found infected with the co-introduced microsporidian parasite Cucumispora dikerogammari, known to decrease the predation rate of its host. Infection might thus mitigate the ecological impact of D. villosus and we wanted to test this assumption using the comparative functional response approach. We compared the relationship between resource use and resource availability (i.e. the functional response, FR) of D. villosus, either with infected individuals or not, to that of two non-invasive gammarids: Gammarus pulex and Echinogammarus berilloni. With infected individuals included, D. villosus displayed a higher FR than the two non-invasive gammarids. Although this effect was not significant, C. dikerogammari infection tended to alter the FR of D. villosus with a slight decrease in attack rate and handling time, resulting in a less steep initial slope and a higher asymptote, respectively. Removing infected D. villosus from the dataset did not affect the FR comparison with G. pulex but suppressed the difference in FR with E. berilloni. Although we cannot exclude the role of sample size reduction in this effect, this suggests that C. dikerogammari infection might increase the predation pressure on local prey populations in case of species replacement between D. villosus and E. berilloni. From a more general perspective, our study illustrates how parasites may alter our capacity to predict invasive species impacts from FR comparisons.     

Phosphorylation regulates interaction of 210-kDa myosin light chain kinase <Emphasis Type="Italic">N</Emphasis>-terminal domain with actin cytoskeleton

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303–314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus <Emphasis Type="Italic">Anguilla</Emphasis>)

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4–100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490–496 and 527–530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.     

The Sonogashira reaction as a new method for the modification of borated analogues of the green fluorescence protein chromophore

The Sonogashira reaction was used for modifications of borated green fluorescence protein chromophore derivatives, 4-(2-(difluoroboryl)benzylidene)-1H-imidazol-5(4H)-ones, for the development of new fluorescent dyes. The derivatives bearing an acetylene fragment and a difluoroboryl group were obtained in high yields. The modification resulted in a significant bathochromic shift of the absorption and emission maxima and is a promising method for the development of new fluorescent dyes.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

     

Contribution of <Emphasis Type="Italic">Eutrema salsugineum</Emphasis> Cold Shock Domain Structure to the Interaction with RNA

Plant cold shock domain proteins (CSDPs) are DNA/RNA-binding proteins. CSDPs contain the conserved cold shock domain (CSD) in the N-terminal part and a varying number of the CCHC-type zinc finger (ZnF) motifs alternating with glycine-rich regions in the C-terminus. CSDPs exhibit RNA chaperone and RNA-melting activities due to their non-specific interaction with RNA. At the same time, there are reasons to believe that CSDPs also interact with specific RNA targets. In the present study, we used three recombinant CSDPs from the saltwater cress plant (Eutrema salsugineum)-EsCSDP1, EsCSDP2, EsCSDP3 with 6, 2, and 7 ZnF motifs, respectively, and showed that their nonspecific interaction with RNA is determined by their C-terminal fragments. All three proteins exhibited high affinity to the single-stranded regions over four nucleotides long within RNA oligonucleotides. The presence of guanine in the single-or double-stranded regions was crucial for the interaction with CSDPs. Complementation test using E. coli BX04 cells lacking four cold shock protein genes (ΔcspA, ΔcspB, ΔcspE, ΔcspG) revealed that the specific binding of plant CSDPs with RNA is determined by CSD.     

The structure of a human voltage-gated potassium Kv10.2 channel which lacks a cytoplasmic PAS domain

The three-dimensional structure of a human voltage-gated potassium Kv10.2 channel which lacks a cytoplasmic N-terminal PAS-domain was determined, and its distribution in eukaryotic cells was investigated. The channel protein was expressed in the COS7 cell line and purified by affinity chromatography. The channel distribution on the cell surface was determined by the immunofluorescence method using the antibodies against its C-terminus. PAS-domain truncation was shown to cause a decrease the expression of the channels on the cell surface. In order to reveal the positions of the channel cytoplasmic domains, the threedimensional structure of the protein lacking the cytoplasmic PAS-domain was compared to the previously obtained full-length structure. We demonstrated that the C-terminal CNBD-domain of the Kv10.2 channel undergoes conformational rearrangements in the absence of its N-terminal PAS-domain.     

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

Opportunistic omnivory impairs our ability to predict invasive species impacts from functional response comparisons

Comparing the relationship between resource use and resource availability (i.e. the functional response, FR) between two predators can provide useful insights on their relative predatory impacts. For instance in invasion ecology, an increase in the predation pressure on local prey populations can be predicted from a significant difference in FR revealing a higher FR for the invasive predator compared to the native trophic analogue it may replace. In traditional FR experiments, the focal prey species is the only source of food. This may lead to misinterpretations with opportunistic omnivores that are able to cope with different resource availabilities in their natural environment, and whose predation rate may therefore be modulated by the presence of alternative resources. To address this question, we compared the FR of two freshwater gammarid species known to behave as opportunistic omnivores: the invasive “killer shrimp” Dikerogammarus villosus and the native Gammarus pulex, in a treatment with a focal prey species as the only food source (the water flea Daphnia magna) and in a treatment with the focal prey and an alternative food source (Carpinus betulus leaves). D. villosus showed a significantly higher FR than G. pulex with water fleas only and providing leaf litter suppressed this difference. The predatory impact of D. villosus might therefore be modulated by the relative availability of live prey compared to the alternative food sources. Increasing the realism of FR experiments through the inclusion of abundant and easily accessible alternative resources, like leaf litter for benthic invertebrates, should refine the predictions made from FR comparisons.     

Towards map-based cloning of <Emphasis Type="Italic">FB_Mfu10</Emphasis>: identification of a receptor-like kinase candidate gene underlying the <Emphasis Type="Italic">Malus fusca</Emphasis> fire blight resistance locus on linkage group 10

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain—Ea3049, and the avrRpt2_EA mutant—ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains.     

Association of <Emphasis Type="Italic">PALB2</Emphasis> sequence variants with the risk of early-onset breast cancer in patients from Turkey

The PALB2 gene, has been accepted as a moderate-penetrance gene associated with breast cancer susceptibility and this gene product is involved in the DNA damage repair pathway via co-localization with BRCA2. Germline PALB2 mutations are associated with an increased breast cancer risk. However, the prevalence of the diverse types of PALB2 variants depend on the population. Thus, the aim of the present study was to determine, for the first time, the prevalence of PALB2 variants in a Turkish population of BRCA1/BRCA2-negative early-onset patients with breast cancer. In total, 223 Turkish patients with BRCA1/BRCA2 negative early-onset breast cancer and 60 unaffected women were included in the study. All the coding exons and intron/exon boundaries of PALB2 were subjected to mutational analysis by heteroduplex analysis (HDA)and DNA sequencing. Eighteen PALB2 variants were found in breast cancer patients within the Turkish population. Three variants (c.271G>A, c.404C>A and c.2981T>A) have not been previously reported. In addition, nine intronic variants were described, and this study is the first to describe the c.1685-44T>A intronic variant. The prevalence of possible pathogenic PALB2 variants was found to be 4.03 % in BRCA1/2-negative Turkish patients with early-onset breast cancer. Different variants of PALB2 have been reported in the literature, and the prevalence of these variants could different for each population. This is the first study to investigate the prevalence of PALB2 variants in Turkish patients with early-onset breast cancer.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Background

Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications.Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia.The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation.

Methods

Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered.

Results

Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p =?0.22), erythrocyte GALT activity (p =?0.87), urinary galactose (p =?0.52) and urinary galactitol levels (p =?0.41). Patients’ fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect.

Conclusions

This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

     

Recombinant production and structural studies of the human Lypd6 and Lypd6b proteins

Lypd6 and Lypd6b are human three-finger proteins expressed in various tissues and sharing a high degree of sequence homology (~54%). Unlike other members of the Ly6/uPAR family, both proteins additionally bear extended N- and С-terminal sequences flanking the LU-domain. The role of these sequences is unclear. It is known that Lypd6 can increase the amplitude of nicotine-induced calcium currents in mouse trigeminal ganglion neurons. The Danio rerio fish Lypd6 is involved in the regulation of the Wnt/β-cathenin signaling pathway and the knockdown of the lypd6 expression leads to the impairment of embryonic development. The Lypd6b expression in X. laevis oocytes increased the sensitivity of nicotinic acetylcholine receptors to acetylcholine and increased their desensitization rate. Molecular mechanisms of Lypd6 and Lypd6b action as well as their spatial structures remain unknown. In this work, the genes encoding water-soluble variants of human Lypd6 and Lypd6b lacking N-terminal sequences (rLypd6 and rLypd6b) and Lypd6 bearing the N-terminal sequence (N-rLypd6) were obtained and expressed. The proteins were expressed in cytoplasmic inclusion bodies followed by solubilization under denaturing conditions and renaturation. The renaturation conditions were screened to optimize recombinant protein yields. The analysis of NMR spectra of recombinant N-rLypd6, rLypd6, and rLypd6b demonstrated that N-rLypd6 may not have a structured form. The production of milligram quantities of isotope-labeled rLypd6 and rLypd6b analogues allowed characterization of the secondary structure of these proteins and study of their intramolecular dynamics. It was found that rLypd6 and rLypd6b have structural elements typical for the Ly6/uPAR family, although with some unique features, particularly, an additional disulfide bond in the third loop and helical regions in the first and third loops.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.