Aa Polypeptide Research Articles

Identification of Omega-class glutathione transferases in helminths of the Taeniidae family

Glutathione transferase enzymes (GSTs) are believed to be a major detoxification system in helminth parasites and have been associated with immunomodulation of the host response. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least five different GSTs, but no Omega-class enzymes have been reported in this parasite or in any other cestode. Herein we report the identification of a new member of the GST superfamily in E. granulosus s.l., which is phylogenetically related to the Omega-class: EgrGSTO. Through mass spectrometry, we showed that the 237 amino acids protein EgrGSTO is expressed by the parasite. Moreover, we identified homologues of EgrGSTO in other eight members of the Taeniidae family, including E. canadensis, E. multilocularis, E. oligarthrus, Hydatigera taeniaeformis, Taenia asiatica, T. multiceps, T. saginata and T. solium. A manual sequence inspection and rational modification yielded eight Taeniidae’s GSTO sequences, each one encoding for a 237 aa polypeptide showing 80.2% overall identity. To the best of our knowledge, this is the first description of genes encoding for Omega-class GSTs in worms belonging to the Taeniidae family -that at least in E. granulosus s.l. is expressed as a protein- suggesting the gene encodes for a functional protein.

Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

Unveiling the Essential Role of Arkadia's Non-RING Elements in the Ubiquitination Process.

Arkadia is a positive regulator of the TGFβ-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin.

Molecular characterization and expression analysis of double‐stranded RNA‐dependent protein kinase (PKR) in Dabry’s sturgeon ( Acipenser dabryanus )

Protein kinase RNA-activated (PKR) is an important component of the innate immune system induced by interferon. This antiviral protein senses a variety of stresses and pathogen-driven double-stranded RNAs (dsRNAs). In the present study, we cloned and characterized the PKR gene from Dabry's sturgeon (Acipenser dabryanus). Bioinformatics analysis revealed that the AdPKR gene comprises a coding sequence (CDS) of 1848 bp, encoding a 616 amino acids (aa) polypeptide. AdPKR shares high aa identity/similarity with proteins from other fish, ranging from 33.4%/51.8% to 87.6%/91.2%, and relatively low aa identity (13.9%) and similarity (26.8%) with human PKR. The AdPKR polypeptide has two dsRNA-binding motifs at the N-terminus and a conserved catalytic kinase domain at the C-terminus. The sturgeon PKR gene contains 17 exons split by 16 introns. Phylogenetic analysis clustered AdPKR together with its counterparts from other teleost fishes. Real-time fluorescene quantitative PCR (RT-qPCR) transcription profiles revealed highest AdPKR expression levels in brain, followed by middle kidney, skin, muscle and head kidney. AdPKR mRNA transcripts were significantly increased at 6 hr post-stimulation (hps) with polyinosinic:polycytidylic acid (poly I:C), remained high at 24 hps, then decreased to normal levels at 48 hps. These results indicate that AdPKR might play a vital role in antiviral immune responses.

Analysis of TabZIP15 transcription factor from Trichoderma asperellum ACCC30536 and its function under pathogenic toxin stress

The TabZIP15 gene encoding a 396 amino acid (aa) polypeptide in the fungus Trichoderma asperellum ACCC30536 was cloned and characterised. The protein includes a basic region motif (NR-x2-QR-x2-R) and has a pillar-like structure. The 25 basic region/leucine zipper transcription factors (TFs) identified in the T. asperellum genome were divided into YAP (14 TFs), ATF2 (5), GCN4 (2), Zip1 (2), BRLZ (1) and u1 (1) subfamilies based on conserved domains. T. asperellum was cultured in minimal media (MM) control, C-Hungry and N-Hungry medium (to simulate nutrient competition and interaction with pathogens, respectively), and differential expression analysis showed that 14 TabZIP genes (including TabZIP15) were significantly altered under both conditions; TabZIP23 responded strongly to N-Hungry media and TabZIP24 responded strongly to C-Hungry media. However, only YAP genes TabZIP15, TabZIP12 and TabZIP2 were significantly upregulated under both conditions, and expression levels of TabZIP15 were highest. T. asperellum was also cultured in the presence of five fungal pathogenic toxins, and RT-qPCR results showed that TabZIP15 was significantly upregulated in four of the five toxin stress conditions (MM + Rhizoctonia solani, MM + Fusarium oxysporum, MM + Alternaria alternata and MM + Cytospora chrysosperma).

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847.

Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes.

Grouper ubiquitin-specific protease 14 promotes iridovirus replication through negatively regulating interferon response

Ubiquitin-specific protease 14 (USP14), one of the USP family members which belong to deubiquitinating enzymes (DUBs), plays a key role in maintaining cellular protein homeostasis by trimming ubiquitin chains from their substrates. However, the roles of USP14 in response to virus infection still remains largely unknown. In the current study, a USP14 homolog from orange spotted grouper (EcUSP14) was cloned and its roles in innate immune response were investigated. EcUSP14 was composed of 1479 base pairs encoding a 492-amino acid (aa) polypeptide. Sequence analysis indicated that EcUSP14 shared 96.14% and 81.30% identity to USP14 of bicolor damselfish (Stegastes partitus) and humans (homo sapiens), respectively. EcUSP14 contains conserved ubiquitin-like (UBL) domain (aa 3–76) and peptidase-C19A domain (aa 106–481). In response to Singapore grouper iridovirus (SGIV) infection in vitro, EcUSP14 was significantly up-regulated. Subcellular localization showed that EcUSP14 was predominantly localized in the cytoplasm of grouper spleen (GS) cells and mostly co-localized with the viral assembly sites after SGIV infection. The ectopic expression of EcUSP14 significantly promoted the replication of SGIV, as demonstrated by the accelerated progression of severity of cytopathic effect (CPE), the increased viral gene transcription and viral protein synthesis during infection. Consistently, treatment with IU1, a USP14 specific inhibitor, significantly inhibited the replication of SGIV, suggesting that USP14 function as a pro-viral factor during SGIV replication. Further analysis showed that EcUSP14 overexpression decreased the promoter activities of interferon (IFN)-1, IFN-3, IFN-stimulated response element (ISRE), and nuclear factor of kappa B (NF-κB). Furthermore, the ectopic expression of EcUSP14 decreased the activities of IFN-1 promoter evoked by TANK-binding kinase (TBK)-1 and melanoma differentiation-associated protein (MDA)-5, but not stimulator of interferon genes (STING). Thus, we speculated that EcUSP14 facilitated virus replication by negatively regulating the IFN response. Taken together, our results firstly demonstrated that fish USP14 functioned as a pro-viral factor by negatively regulating interferon response against virus infection.

Labeo rohita Mx1 exhibits the critical structural motifs of the family of large GTPases of mammals and is activated by rhabdovirus vaccination and bacterial RNA stimulations

Myxovirus resistance (Mx) proteins belonging to the dynamin superfamily of high molecular weight GTPases exist in various isoforms and play crucial role in innate immunity. In addition to the isoforms, Mx1 also plays important role in exerting its anti-viral actions against a broad range of animal RNA viruses. In rohu (Labeo rohita), mx1 full-length cDNA sequence consists of 2440 nucleotides (nt) encoding 628 amino acids (aa) polypeptide of 71.289 kDa. Structurally, it belongs to the family of large GTPases with one DYNc domain (13-257aa) comprising of dynamin family motifs (LPRGSGIVTR) and the tripartite GTP-binding motifs (GDQSSGKS, DLPG and TKPD) at the N-terminal and one GED domain (537-628aa) at C-terminus. Rohu Mx1 is closely related to zebrafish Mx1 and is widely expressed in gill, liver, kidney, spleen and blood. In response to rhabdovirus vaccinations, poly I:C stimulation and bacterial infections, mx1 gene expression in rohu was significantly (p < 0.05) induced in majority of the tested organs/tissues. Stimulation of rohu gill cell line with bacterial RNA also induced mx1 gene expression. Together these data suggest the important role of Mx1 in innate immunity in rohu against wide spectrum of fish pathogens.

Silencing Br-C impairs larval development and chitin synthesis in Lymantria dispar larvae

In insects, 20-hydroxyecdysone (20E) mediates developmental transitions and regulates molting processes through activation of a series of transcription factors. Broad-Complex (Br-C), a vital gene in the 20E signalling pathway, plays crucial roles during insect growth processes. However, whether Br-C affects chitin synthesis in insects remains unclear. In the present study, the Br-C gene from Lymantria dispar, a notorious defoliator of forestry, was identified based on transcriptome data, and subjected to bioinformatic analysis. The regulatory functions of LdBr-C in chitin synthesis and metabolism in L. dispar larvae were analysed by RNA interference (RNAi). The full-length LdBr-C gene (1431 bp) encodes a 477 amino acid (aa) polypeptide containing a common BRcore region (391 aa) at the N-terminus and a C-terminal Zinc finger domain (56 aa) harbouring two characteristic C2H2 motifs (CXXC and HXXXXH). Phylogenetic analyses showed that LdBr-C shares highest homology and identity with Br-C isoform 7 (83.12%) of Helicoverpa armigera. Expression profiles indicate that LdBr-C was expressed throughout larval and pupal stages, and highly expressed in prepupal and pupal stages. Furthermore, LdBr-C expression was strongly induced by exogenous 20E, and suppressed dramatically after application of dsLdBr-C. Bioassay results showed that knockdown of LdBr-C caused larval developmental deformity, significant weight loss, and a mortality rate of 67.18%. Knockdown of LdBr-C significantly down-regulated transcription levels of eight critical genes (LdTre1, LdTre2, LdG6PI, LdUAP, LdCHS1, LdCHS2, LdTPS and LdCHT) related to chitin synthesis and metabolism, thereby lowering the chitin content in the midgut and epidermis. Our findings demonstrate that Br-C knockdown impairs larval development and chitin synthesis in L. dispar.

LncRNA-encoded polypeptide ASRPS inhibits triple-negative breast cancer angiogenesis.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BC) with the most aggressive phenotype and poor overall survival. Using bioinformatics tools, we identified LINC00908 encoding a 60-aa polypeptide and differentially expressed in TNBC tissues. We named this endogenously expressed polypeptide ASRPS (a small regulatory peptide of STAT3). ASRPS expression was down-regulated in TNBCs and associated with poor overall survival. We showed that LINC00908 was directly regulated by ERα, which was responsible for the differential down-regulation of LINC00908 in TNBCs. ASRPS directly bound to STAT3 through the coiled coil domain (CCD) and down-regulated STAT3 phosphorylation, which led to reduced expression of VEGF. In human endothelial cells, a mouse xenograft breast cancer model, and a mouse spontaneous BC model, ASRPS expression reduced angiogenesis. In a mouse xenograft breast cancer model, down-regulation of ASRPS promoted tumor growth, and ASRPS acted as an antitumor peptide. We presented strong evidence that LINC00908-encoded polypeptide ASRPS represented a TNBC-specific target for treatment.

Genome-Wide Identification, Expression Profiling, and Functional Validation of Oleosin Gene Family in Carthamus tinctorius L.

Carthamus tinctorius L., commonly known as safflower, is an important oilseed crop containing oil bodies. Oil bodies are intracellular organelles in plant cells for storing triacylglycerols (TAGs) and sterol esters. Oleosins are the most important surface proteins of the oil bodies. We predicted and retrieved the sequences of eight putative C. tinctorius oleosin (Ctoleosin) genes from the genome database of safflower. The bioinformatics analyses revealed the size of their open reading frames ranging from 414 to 675 bp, encoding 137 to 224 aa polypeptides with predicted molecular weights of 14.812 to 22.155 kDa, all containing the typical “proline knot” motif. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) determined the spatiotemporal expression pattern of Ctoleosin genes, which gradually increased and peaked during flowering and seed ripening, and decreased thereafter. To validate their role in plant development, we transformed and overexpressed these eight putative Ctoleosin genes in Arabidopsis. Overexpressing Ctoleosins did not affect leaf size, although silique length was altered. Arabidopsis transformed with Ctoleosin3, 4, and 5 grew longer siliques than did the wild-type plants, without altering seed quantity. The 100-grain weight of the transgenic Arabidopsis seeds was slightly more than that of the wild-type seeds. The seed germination rates of the plants overexpressing Ctoleosin4 and 6 were slightly lower as compared with that of the wild-type Arabidopsis, whereas that in the other transgenic lines were higher than that in the wild-type plants. The overexpression of Ctoleosin genes elevated the oil content in the seeds of transgenic Arabidopsis. Our findings not only provide an approach for increasing the oil content, but also for elucidating the intricate mechanisms of oil body synthesis.

Characterization of a novel secretory spherical body protein in Babesia orientalis and Babesia orientalis-infected erythrocytes

BackgroundThe spherical body, a membrane bound organelle localized in the apical organelle complex, is unique to Babesia and Theileria spp. The spherical body proteins (SBPs) secreted by spherical bodies include SBP1, SBP2, SBP3 and SBP4. Up to now, only SBP3 has been characterized in Babesia orientalis.MethodsThe BoSBP4 gene was amplified from cDNA and gDNA and cloned into the pGEX-6P-1 vector by homologous recombination, sequenced and analyzed by bioinformatics tools. The amino acid (aa) sequence of BoSBP4 was compared with that of Babesia bovis and Babesia bigemina as well as SBP3 of B. orientalis. The immunoreactivity was evaluated by incubating recombinant BoSBP4 (rBoSBP4) with the serum of B. orientalis-infected water buffalo. The native form of BoSBP4 was identified by incubating lysate of B. orientalis-infected water buffalo erythrocytes with the anti-rBoSBP4 mouse serum. The cellular localization of BoSBP4 was determined by indirect immunofluorescence assay.ResultsThe full length of the BoSBP4 gene was estimated to be 945 bp without introns, encoding a 314 aa polypeptide with a predicted molecular weight of 37 kDa. The truncated recombinant protein was expressed from 70 to 945 bp as a GST fusion protein with a practical molecular weight of 70 kDa. BoSBP4 shared a 40% and 30% identity with B. bovis and B. bigemina, respectively. Furthermore, it was 31% identical to SBP3 of B. orientalis. BoSBP4 was identified in the lysate of B. orientalis-infected water buffalo erythrocytes with a molecular weight of 37 kDa, corresponding to the expected molecular mass of BoSBP4. The result of rBoSBP4 with positive serum revealed that BoSBP4 can elicit an immune response to B. orientalis-infected water buffalo. The cellular localization of BoSBP4 was detected to be adjacent to the merozoite nucleus in the intracellular phase, followed by the diffusion of the fluorescence of BoSBP4 into the cytoplasm of B. orientalis-infected erythrocytes as puncta-like specks and a gradual increase of the fluorescence.ConclusionsIn this study, SBP4 in B. orientalis was characterized for the first time. It may play a key role in interaction with the host cell by being secreted into the cytoplasm of the B. orientalis-infected erythrocytes to facilitate parasite growth and reproduction.

Role Of Insulin Like Growth Factor in Polycystic Ovary Syndrome

Objectives: The polycystic ovary syndrome (PCOS) is conventionally defined as a combination of hyperandrogenism and anovulation with polycystic ovaries. Insulin-like growth factor I (IGF-I) is a 70 aa polypeptide hormone with endocrine, paracrine, and autocrine effects. it is now established that the ovary is a site of IGF-I gene expression and reception. However, this huge amount of data could be somehow distractive since different species may produce distinct IGFs (or their relative binding proteins and receptors) at different stages of follicular development. Objective: The aim of this work was to determine the serum level of IGF-I to evaluate its role in PCOS. Subjects and Methods: Twenty-five infertile women with PCOS diagnosed by ultrasound examination and a history of oligomenorrhea, hirsutism and obesity were studied. Serum levels of insulin-like growth factor-1 (IGF-1) and hormonal profile were measured. Fifteen healthy and fertile women with regular menstrual cycles served as a comparison group. Results: The serum levels of IGF-I were significantly elevated in women with PCOS compared with control and there was positive correlation between IGF-I from one side and each of BMI, W/H ratio, LH, LH/FSH ratio, TSN, T/SHBG ratio, right and left ovarian volumes. On the other side, IGF-I was inversely correlated with SHBG, right and left uterine artery PI and RI. Conclusion: The significant finding of this study is that PCOS patients have increased circulating IGF-I levels than healthy controls and it can be used as a biochemical marker for PCOS.

LrHMGB1 Shares Structural Similarities with Human HMGB1, and Its Expression Is Induced in Bacterial Infection, Antiviral Vaccination, and Pathogen-Associated Molecular Patterns Stimulation.

The high-mobility group box 1 (HMGB1) protein is a highly conserved nonhistone chromosomal protein ubiquitously present in almost all cell types. In the nucleus, it facilitates DNA repair and replication, V(D)J recombination, stabilization of nucleosome, and in the cytoplasm, it regulates autophagy and apoptosis. In addition to these intracellular functions, HMGB1 also facilitates activation of innate immune responses and plays key roles in host defense. To investigate its role in fish, we cloned and characterized HMGB1 in Labeo rohita (LrHMGB1), the most important freshwater fish species in the Indian subcontinent. The full-length cDNA sequence of LrHMGB1 consisted of 787 bp having an open reading frame of 618 bp encoding 205 amino acids (aa) polypeptide, with an estimated molecular mass of 23.61 kDa and isoelectric point (pI) of 5.96. Motif search of LrHMGB1 revealed two homologous DNA-binding domains, the A-box and B-box comprising 8-78 aa and 94-162 aa, respectively, and a negatively charged acidic C-terminal tail (179-204) that consisted of 26 consecutive aspartic and glutamic acid residues. The amino acids sequence of LrHMGB1 protein and the secondary structure having helix (H) and coils (C) in tandem in the A- and B-box region and only coils in the acidic tail region shared significant similarity with mouse and human HMGB1. In addition to the three prominent motifs (A-box, B-box, and the acidic tail), the site of acetylation (lys27-29), phosphorylation (serine38,41,45,52), methylation (lys43), and oxidation (cysteine44,105) in LrHMGB1 was also conserved across the fish species, mouse, and human. LrHMGB1 was expressed during embryogenesis and was widely expressed in various organs/tissues having highest expression in blood. In response to bacterial infection, antiviral vaccination, and pathogen-associated molecular patterns stimulations, LrHMGB1 gene expression was significantly (p < 0.05) induced in various organs and tissues. These results together suggest an evolutionary conserved structure and function of HMGB1 from fish to human.

Effect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90β1 in the river pufferfish (Takifugu obscurus): Evidences for its immunologic function involving in exploring process

HSP90β1 (known as glyco-protein 96, GP96) is a vital endoplasmic reticulum (ER) depended chaperonin among the HSPs (heat shock proteins) family. Furthermore, it always processes and presents antigen of the tumor and keeps balance for the intracellular environment. In the present study, we explored the effect of tributyltin chloride (TBT-Cl) exposure on HSP90β1 expression in river pufferfish, Takifugu obscurus. The full length of To-HSP90β1 was gained with 2775 bp in length, with an ORF (open reading frame) encoding an 803 aa polypeptide. A phylogenetic tree was constructed and showed the close relationship to other fish species. The HSP90β1 mRNA transcript was expressed in all tissues investigated with higher level in the gill and liver. After the acute and chronic exposure of TBT-Cl, the To-HSP90β1 mRNA transcript significantly was up-regulated in gills. Moreover, the histology study indicated the different injury degree of TBT-Cl in liver and gill. Immunohistochemistry (IHC) staining results implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-HSP90β1 to TBT-Cl exposure. All the results indicated that HSP90β1 may be involved in the resistance to the invasion of TBT-Cl for keeping autoimmune homeostasis.

Molecular characterization and functional analysis of two phospholipid hydroperoxide isoforms from Larimichthys crocea under Vibrio parahaemolyticus challenge

Glutathione peroxidases family is a key role in the antioxidant system in oxybiotic organisms for cell redox homeostasis. One of their members, phospholipid hydroperoxide glutathione peroxidase (GPx4) have unique monomeric structure and can directly react with complex lipid and membrane-bound peroxides under the presence of glutathione(GSH). In this paper, two complete GPx4 cDNAs (designated as LcGPx4a and LcGPx4b) from Larimichthys crocea are identified by rapid amplification of cDNA ends. The cDNA of LcGPx4a was consisted of a 5′-untranslated region (UTR) of 258 bp, a 3′-UTR of 330 bp, and an open reading frame (ORF) of 561 bp encoding 186 amino acid (aa) polypeptides. And the full-length sequence of LcGPx4b was 1164 bp with a 5′-UTR of 34 bp, a 3′-UTR of 551 bp and an ORF of 576 bp encoding a polypeptide of 191 aa residues with a predicted signal peptide of 15 aa. The characteristic selenocysteine insertion (SECIS) sequence was detected in the 3′UTR of the two sequences with 78 bp in length. The conserved active site of selenocysteine (Sec) encoded by TGA was also identified and formed a tetrad functional structure with glutamine, tryptophan, and asparagine in LcGPx4a and LcGPx4b. Two signature site motifs (“LRILAFPSNQFGNQEPG” and “LRILGFPCNQFGGQEPG”) were both conserved in the deduced amino acid of LcGPx4a and LcGPx4b. The genomic structure analysis revealed that the two sequences both had 7 exons and 6 introns, and the Sec opal codon and SECIS element were located at the third and seventh exons, respectively. LcGPx4a and LcGPx4b both have a wide distribution in 9 tissues with various relative expression levels and a highest expression pattern in the liver. Under Vibrio parahaemolyticus challenge, their relative expression levels were altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx4a and LcGPx4b showed a significantly up-regulated trend in the spleen during experimental period. Above results suggested that LcGPx4a and LcGPx4b were two conserved immune molecules and might play a role in the immune response of fish with a tissue-depemdent manners.

Tissue expression pattern of ABCG transporter indicates functional roles in reproduction of Toxocara canis.

Toxocara canis is a zoonotic parasite with worldwide distribution. ATP-binding cassette (ABC) transporters are integral membrane proteins which involve in a range of biological processes in various organisms. In present study, the full-length coding sequence of abcg-5 gene of T. canis (Tc-abcg-5) was cloned and characterized. A 633 aa polypeptide containing two conserved Walker A and Walker B motifs was predicted from a continuous 1902nt open reading frame. Quantitative real-time PCR was employed to determine the transcriptional levels of Tc-abcg-5 gene in adult male and female worms, which indicated high mRNA level of Tc-abcg-5 in the reproductive tract of adult female T. canis. Tc-abcg-5 was expressed to produce rabbit polyclonal antiserum against recombinant TcABCG5. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of TcABCG5, which showed predominant distribution of TcABCG5 in the uterus (especially in the germ cells) of adult female T. canis. Tissue transcription and expression pattern of Tc-abcg-5 indicated that Tc-abcg-5 might play essential roles in the reproduction of this parasitic nematode.

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene.

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is “matryoshka,” containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5′-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.

Identification and characterization of two selenium-dependent glutathione peroxidase 1 isoforms from Larimichthys crocea

Glutathione peroxidases, a vital family of antioxidant enzymes in oxybiotic organisms, are involved in anti-pathogen immune response. In this study, two complete selenium-dependent glutathione peroxidase 1 cDNAs (designated as LcGPx1a and LcGPx1b) were obtained from the large yellow croaker Larimichthys crocea by rapid amplification of cDNA ends. The full-length sequence of LcGPx1a was 917 bp with a 5′-untranslated region (UTR) of 52 bp, a 3′-UTR of 289 bp, and an open reading frame of 576 bp encoding 191 amino acid (aa) polypeptides. The cDNA of LcGPx1b was composed of 884 bp with a 5′-UTR of 59 bp, a 3′-UTR of 258 bp, and an open reading frame of 567 bp encoding 188 aa polypeptides. The conserved selenocysteine insertion sequence was detected in the 3′-UTR of both isoforms, which can classify types I and II. Protein sequence analysis revealed that both isoforms included a selenocysteine encoded by an opal codon (TGA) and formed the functioning tetrad site with glutamine, tryptophan, and asparagine. Three conservative motifs, including one active site motif (“GKVVLIENVASLUGTT”) and two signature site motifs (“LVILGVPCNQFGHQENC” and “V(A/S)WNFEKFLI”), were conserved both in sequence and location. Multiple alignments revealed that they exhibited a high level of identities with GPx1 from other organisms, especially in the abovementioned conserved amino acid sequence motifs. Tissue expression analysis indicated that LcGPx1a and LcGPx1b had a wide distribution in nine tissues with various abundances. The transcript level of LcGPx1a was not significantly different among the nine tissues, whereas that of LcGPx1b was higher in the kidney and head kidney than in the other tissues. After Vibrio parahaemolyticus stimulation, the expression levels of LcGPx1a and LcGPx1b were unanimously altered in the liver, spleen, kidney, and head kidney but with different magnitudes and response time. LcGPx1a and LcGPx1b showed distinct expression trends in the liver, where LcGPx1b was induced and LcGPx1a was depressed in response to pathogen infection. These results indicate that LcGPx1a and LcGPx1b display functional diversities and play crucial roles in mediating the immune response of fish.

Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code

BackgroundMiddle-down mass spectrometry (MS), i.e., analysis of long (~50–60 aa) polypeptides, has become the method with the highest throughput and accuracy for the characterization of combinatorial histone posttranslational modifications (PTMs). The discovery of histone readers with multiple domains, and overall the cross talk of PTMs that decorate histone proteins, has revealed that histone marks have synergistic roles in modulating enzyme recruitment and subsequent chromatin activities. Here, we demonstrate that the middle-down MS strategy can be combined with metabolic labeling for enhanced quantification of histone proteins and their combinatorial PTMs in a dynamic manner.MethodsWe used a nanoHPLC-MS/MS system consisting of hybrid weak cation exchange–hydrophilic interaction chromatography combined with high resolution MS and MS/MS with ETD fragmentation. After spectra identification, we filtered confident hits and quantified polypeptides using our in-house software isoScale.ResultsWe first verified that middle-down MS can discriminate and differentially quantify unlabeled from heavy labeled histone N-terminal tails (heavy lysine and arginine residues). Results revealed no bias toward identifying and quantifying unlabeled versus heavy labeled tails, even if the heavy labeled peptides presented the typical skewed isotopic pattern typical of long protein sequences that hardly get 100% labeling. Next, we plated epithelial cells into a media with heavy methionine-(methyl-13CD3), the precursor of the methyl donor S-adenosylmethionine and stimulated epithelial to mesenchymal transition (EMT). We assessed that results were reproducible across biological replicates and with data obtained using the more widely adopted bottom-up MS strategy, i.e., analysis of short tryptic peptides. We found remarkable differences in the incorporation rate of methylations in non-confluent cells versus confluent cells. Moreover, we showed that H3K27me3 was a critical player during the EMT process, as a consistent portion of histones modified as H3K27me2K36me2 in epithelial cells were converted into H3K27me3K36me2 in mesenchymal cells.ConclusionsWe demonstrate that middle-down MS, despite being a more scarcely exploited MS technique than bottom-up, is a robust quantitative method for histone PTM characterization. In particular, middle-down MS combined with metabolic labeling is currently the only methodology available for investigating turnover of combinatorial histone PTMs in dynamic systems.