Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N -Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N -glycans and that the ELP fusions had no significant effect on N -glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.     

Distinct isocomplexes of the TRAPP trafficking factor coexist inside human cells

The transport protein particle (TRAPP) complex is required for proper vesicular transport from the ER to the Golgi. The composition of yeast TRAPP is well characterized, but the organization of mammalian TRAPP complex remains elusive. Using a tandem affinity purification (TAP) approach, we provide first experimental proof for the association of NIBP (NIK/IKKβ binding protein) with Bet3 and find two human paralogs of Trs33 (A and B) associated with Bet3. Interaction studies and gel filtration analysis reveal that both proteins are part of human TRAPP and might mark two distinct isocomplexes that exert different functions in the regulation of ER-to-Golgi traffic.

Structured summary

MINT-6784845:

Bet3 (uniprotkb:O43617) physically interacts (MI:0218) with Trs33B (uniprotkb:Q86SZ2) by anti bait coimmunoprecipitation (MI:0006)

MINT-6785053:

Trs33B (uniprotkb:Q86SZ2) physically interacts (MI:0218) with Bet3 (uniprotkb:O43617) and Sedl (uniprotkb:O14582) by anti bait coimmunoprecipitation (MI:0006)

MINT-6784856:

Bet3 (uniprotkb:O43617) physically interacts (MI:0218) with Trs33A2 (uniprotkb:O75865-2) by anti bait coimmunoprecipitation (MI:0006)

MINT-6785038:

Trs33A1 (uniprotkb:O75865-2) physically interacts (MI:0218) with Sedl (uniprotkb:O14582) and Bet3 (uniprotkb:O43617) by anti bait coimmunoprecipitation (MI:0006)

MINT-6784879:

Bet3 (uniprotkb:O43617) physically interacts (MI:0218) with NIBP (uniprotkb:Q96Q05) by tandem affinity purification (MI:0676)

MINT-6785068:

Trs33B (uniprotkb:Q86SZ2), Trs33A2 (uniprotkb:O75865-2) and Bet3 (uniprotkb:O43617) colocalize (MI:0403) by molecular sieving (MI:0071)

MINT-6785415:

Bet3 (uniprotkb:O43617) physically interacts (MI:0218) with Trs33A1 (uniprotkb:O75865) by anti bait coimmunoprecipitation (MI:0006)

     

Identification of androgen-selective androgen-response elements in the human aquaporin-5 and Rad9 genes

The AR (androgen receptor) is known to influence the expression of its target genes by binding to different sets of AREs (androgen-response elements) in the DNA. One set consists of the classical steroid-response elements which are partial palindromic repeats of the 5'-TGTTCT-3' steroid-receptor monomer-binding element. The second set contains motifs that are AR-specific and that are proposed to be partial direct repeats of the same motif. On the basis of this assumption, we used an in silico approach to identify new androgen-selective AREs in the regulatory regions of known androgen-responsive genes. We have used an extension of the NUBIScan algorithm to screen a collection of 85 known human androgen-responsive genes compiled from literature and database searches. We report the evaluation of the most promising hits resulting from this computational search by in vitro DNA-binding assays using full-size ARs and GRs (glucocorticoid receptors) as well as their isolated DBDs (DNA-binding domains). We also describe the ability of some of these motifs to confer androgen-, but not glucocorticoid-, responsiveness to reporter-gene expression. The elements found in the aquaporin-5 and the Rad9 (radiation-sensitive 9) genes showed selective AR versus GR binding in band-shift assays and a strong activity and selectivity in functional assays, both as isolated elements and in their original contexts. Our data indicate the validity of the hypothesis that selective AREs are recognizable as direct 5'-TGTTCT-3' repeats, and extend the list of currently known selective elements.     

Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation

Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted ‘OMIC'' investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH₄NO₃ and KH₂PO₄ and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct ‘presumptive'' degradation networks for complex microbial communities.     

Insertion Mutations in pilE Differentially Alter Gonococcal Pilin Antigenic Variation

Pilus antigenic variation in Neisseria gonorrhoeae occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of the pilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions in pilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilS and recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.     

Differential Expression of CRH,UCN, CRHR1 and CRHR2 in Eutopic and Ectopic Endometrium of Women with Endometriosis

Endometriosis is considered as a benign aseptic inflammatory disease, characterised by the presence of ectopic endometrium-like tissue. Its symptoms (mostly pain and infertility) are reported as constant stressors. Corticotropin releasing hormone (CRH) and urocortin (UCN) are neuropeptides, strongly related to stress and inflammation. The effects of CRH and UCN are mediated through CRHR1 and CRHR2 receptors which are implicated in several reproductive functions acting as inflammatory components. However, the involvement of these molecules to endometriosis remains unknown. The aim of this study was to examine the expression of CRHR1 and CRHR2 in endometriotic sites and to compare the expression of CRHR1 and CRHR2 in eutopic endometrium of endometriotic women to that of healthy women. We further compared the expression of CRH, UCN, CRHR1 and CRHR2 in ectopic endometrium to that in eutopic endometrium of women with endometriosis. Endometrial biopsy specimens were taken from healthy women (10 patients) and endometrial and endometriotic biopsy specimens were taken from women with endometriosis (16 patients). Τhe expression of CRH, UCN, CRHR1, and CRHR2 was tested via RT-PCR, immunohistochemistry and Western blotting. This study shows for the first time that CRH and UCN receptor subtypes CRHR1β and CRHR2α are expressed in endometriotic sites and that they are more strongly expressed (p<0.01) in eutopic endometrium of women with endometriosis compared to healthy women endometrium at the mRNA and protein level. CRH, UCN, CRHR1 and CRHR2 mRNA were also more highly expressed in ectopic rather than eutopic endometrium (CRH, UCN, CRHR2α: p<0.01, CRHR1β: p<0.05) and protein (CRH and UCN: p<0.05, CRHR1 and CRHR2: p<0.01) in women with endometriosis. These data indicate that CRH and UCN might play an immunoregulatory role in endometriotic sites by affecting reproductive functions such as decidualization and implantation of women with endometriosis.     

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift

Firefly bioluminescence reaction in the presence of Mg^2 +, ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350–359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7 Å and 2.2 Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351–359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission.     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Day vs. night predation on artificial caterpillars in primary rainforest habitats – an experimental approach

The influence of natural enemies has led to the evolution of various predator avoidance strategies in herbivorous insects. Many caterpillars are exclusively active at night and rest during the day. It is widely assumed that nocturnal activity in caterpillars reduces their risk of falling prey to their natural enemies. To test this hypothesis, we compared predation pressure between day and night in tree‐fall gaps and closed‐canopy forest sites in an Amazonian primary lowland rainforest. Artificial clay caterpillars, showing camouflaged colouration (green), were exposed as potential prey to a natural predator community. Attacks were significantly more frequent during daytime and were reduced by about a quarter at night in tree‐fall gaps, and by a third in closed‐canopy forest sites. This supports the idea of time‐dependent activity in caterpillars as an antipredatory adaptation. Further, independent of the time of day, predation pressure on caterpillars was significantly higher in tree‐fall gaps compared to closed‐canopy forest habitats. Nearly all predation events were caused by arthropods, whereas birds played a negligible role. Across both habitat types and time scales, ants acted as major predator group, emphasising their important role in population control of herbivorous insects in lowland rainforest ecosystems. This is the first experimental study using artificial caterpillars to examine whether time‐scheduling of exposition might influence predation risk amongst undefended, solitary, free‐living lepidopteran larvae.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.