High Amino Acid Sequence Identity Research Articles

Structure, catalytic mechanism, posttranslational lysine carbamylation, and inhibition of dihydropyrimidinases.

Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-β-alanine and N-carbamyl-β-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase.

Genetic diversity of limonene synthase genes in Rongan kumquat (Fortunella crassifolia).

D-limonene is the main component of citrus essential oils. In the monoterpene biosynthetic pathway, geranyl diphosphate reacts with monoterpenes to form the prenyl-carbocation intermediate to produce d-limonene. In this study, d-limonene synthase (FcLS) genes were first isolated from Rongan kumquat (Fortunella crassifolia Swingle). Sequencing analysis revealed that the open reading frames of 18 FcLS genes contain 12 single nucleotide polymorphisms, which resulted in the variation of FcLS proteins, indicating that the limonene synthase genes are a large family in F. crassifolia. This phenomenon has not been reported in Citrus. The predicted FcLS proteins showed a high amino acid sequence identity with other Citrus limonene synthases and also had the typical structures of limonene synthase protein. FcLS1 was validated to be a functional d-limonene synthase by prokaryotic expression.

A putative tomato inositol polyphosphate 5-phosphatase, Le5PT1, is involved in plant growth and abiotic stress responses.

Based on sequence similarity to Arabidopsis inositol polyphosphate 5-phosphatases (5PTases) involved in abiotic stress responses and development, four tomato cDNAs (Le5PT1-4) encoding putative 5PTase proteins were identified. The predicted protein sequences of the Le5PTs include conserved catalytic domains required for 5PTase enzyme activity. Le5PT1, 2, and 3 showed high amino acid sequence identity with At5PTase2, At5PTase1 and At5PTase3, and At5PTase5 and At5PTase6, respectively. The expression of Le5PT1 was downregulated soon after initiation of dehydration and salt stress as well as exposure to polyethylene glycol (PEG) and NaCl, but not by exogenous ABA treatment. On the other hand, the expression of Le5PT2 gradually increased with time in all treatments. Transgenic tobacco plants overexpressing Le5PT1 exhibited reduced growth in height, leaf area, and dry weight compared to wild type plants. Transgenic plants also had lower water use efficiency (WUE) than wild type and the downregulation of the drought-responsive gene, NtERD10B. Together these results suggest that Le5PT1 may have a negative role in response to water deficit through the repression of drought-inducible genes that in turn affects plant growth and development.

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation.

In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities.IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

Molecular cloning and functional characterization of a glyceraldehyde-3-phosphate dehydrogenase gene from Spartina alterniflora reveals its involvement in salt stress response

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been emerging to be a multifunctional protein involved in various cellular processes, in addition to its role in energy metabolism. In this study, the SaGAPDH gene was cloned from Spartina alterniflora based on the full-length cDNA library. The open reading frame of SaGAPDH was 1014 bp, and it was encoding 337 amino acids with a calculated molecular mass of 36.40 kDa. Multiple sequence alignment showed that SaGAPDH had high amino acid sequence identity with other plant GAPDHs, and phylogenetic analysis demonstrated that SaGAPDH had a closer affinity to GAPDH in Aeluropus lagopoides (AlGAPDH). Subcellular localization suggested that SaGAPDH was located in cytosol. The recombinant SaGAPDH protein was expressed in Escherichia coli cells to characterize its catalytic activity. And E. coli carrying SaGAPDH gene showed an increased salt stress resistance. SaGAPDH gene was induced by salt stress, and to further investigate its function, transgenic Arabidopsis plants ectopically antisense-overexpressing SaGAPDH was generated. The transgenic Arabidopsis plants showed a specific down-regulation of AtGAPC1 transcript and the GAPDH enzyme activity. They also showed decreased tolerance to salt stress and down-regulation of antioxidant enzymes including catalase, ascorbate peroxidase, superoxide dismutase, and peroxidase, as well as their transcripts. Above results were further confirmed by the aggravation of oxidative damage in SaGAPDH antisense-overexpressing transgenic Arabidopsis lines, which accumulated more reactive oxygen species (ROS) such as superoxide anion (O 2 ·− ) and hydrogen peroxide (H2O2) under salt stress. This study indicated that SaGAPDH may play an important role in response to salt stress by the regulation of redox homeostasis.

Structure and tailspike glycosidase machinery of ORF212 from E. coli O157:H7 phage CBA120 (TSP3)

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a β-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.

Subversion of natural killer cell responses by a cytomegalovirus-encoded soluble CD48 decoy receptor.

Throughout evolution, cytomegaloviruses (CMVs) have been capturing genes from their hosts, employing the derived proteins to evade host immune defenses. We have recently reported the presence of a number of CD48 homologs (vCD48s) encoded by different pathogenic viruses, including several CMVs. However, their properties and biological relevance remain as yet unexplored. CD48, a cosignaling molecule expressed on the surface of most hematopoietic cells, modulates the function of natural killer (NK) and other cytotoxic cells by binding to its natural ligand 2B4 (CD244). Here, we have characterized A43, the vCD48 exhibiting the highest amino acid sequence identity with host CD48. A43, which is encoded by owl monkey CMV, is a soluble molecule released from the cell after being proteolytically processed through its membrane proximal region. A43 is expressed with immediate-early kinetics, yielding a protein that is rapidly detected in the supernatant of infected cells. Remarkably, surface plasmon resonance assays revealed that this viral protein binds to host 2B4 with high affinity and slow dissociation rates. We demonstrate that soluble A43 is capable to abrogate host CD48:2B4 interactions. Moreover, A43 strongly binds to human 2B4 and prevents 2B4-mediated NK-cell adhesion to target cells, therefore reducing the formation of conjugates and the establishment of immunological synapses between human NK cells and CD48-expressing target cells. Furthermore, in the presence of this viral protein, 2B4-mediated cytotoxicity and IFN-γ production by NK cells are severely impaired. In summary, we propose that A43 may serve as a functional soluble CD48 decoy receptor by binding and masking 2B4, thereby impeding effective NK cell immune control during viral infections. Thus, our findings provide a novel example of the immune evasion strategies developed by viruses.

A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis

BackgroundCellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals.ResultsA novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65 °C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0–11.0) after being incubated at 25 °C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60 °C for 60 h, and its half-lives at 65 °C and 70 °C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-d-cellobioside (pNPC), and p-nitrophenyl-β-d-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%.ConclusionsXynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure–function relationship of bifunctional xylanase/cellulase.

Cloning and functional characterization of rockfish peroxiredoxin 4 homolog with its innate immune responses

The fourth member of the typical 2-cysteine peroxiredoxin (Prx4) is a well-known antioxidant enzyme, which reduces different peroxides in their catalytic process. The present study reports the identification of the rockfish Sebastes schlegelii Prx4 (SsPrx4) at a genomic level, as well as the characterization of its structural and functional features. SsPrx4 harbors a complete ORF of 786 bp encoding a polypeptide (29 kDa) of 262 amino acids (aa) with an isoelectric point of 6.2. Thioredoxin 2 domain was prominent in the SsPrx4 sequence, which has a signal peptide (31 bp) at the N-terminus. Hence, the SsPrx4 may be functionally active in the cytoplasm of rockfish cells. Moreover, two VCP motifs and three catalytic triad residues (112T, 115C, 191R) were identified in the SsPrx4 protein sequence. A peroxidatic cysteine (115CP) and resolving cysteines (236CR) were detected at the VCP motifs. The rockfish Prx4 genome consists of seven exons, which are similar to the architecture of other Prx4 orthologs. The deduced amino acid sequence of SsPrx4 shares a relatively high amino acid sequence identity (91.6%) and close evolutionary relationship with Miichthys miiuy and Stegastes partitus Prx4. The potential for scavenging extracellular H2O2 was evidenced by the purified recombinant SsPrx4 protein (rSsPrx4) in vitro system. Moreover, rSsPrx4 may protect the plasmid DNA in a metal-catalyzed oxidation system and catalyze the reduction of an insulin disulfide bond. Quantitative real-time PCR revealed that SsPrx4 mRNA was ubiquitously expressed in fourteen different tissues, with the highest expression observed in the liver followed by the ovary, and kidney tissues. Transcriptional modulations were observed in liver and spleen tissues of rockfish after injecting them with bacterial stimuli, including Streptococcus iniae, LPS, and a viral mimic of poly I:C. Together, the results suggest that SsPrx4 may play an important role in both the antioxidant and innate immune defense of black rockfish. These findings provide structural and functional insights into the SsPrx4 of the teleost.

Microbial Degradation of Nicotinamide by a Strain Alcaligenes sp. P156

A novel Alcaligenes sp. strain P156, which can utilize nicotinamide as its sole source of carbon, nitrogen and energy, was enriched and isolated from soil in a solid waste treatment plant. Aerobic growth and degradation with nicotinamide were characterized. Seven nicotinamide degradation-related genes were obtained by sequence alignment from the genome sequence of strain P156. Four genes, designated naaA, naaD, naaE and naaF, were cloned and heterologously expressed. Nicotinamide degradation is initiated by deamination to form nicotinic acid catalyzed by the nicotinamidase NaaA, which shares highest amino acid sequence identity (27.2%) with nicotinamidase from Arabidopsis thaliana. Nicotinic acid is converted to 6-hydroxynicotinic acid, which is further oxidized to 2,5-dihydroxypyridine (2,5-DHP). 2,5-DHP is then transformed to a ring-cleavage product, N-formylmaleamic acid, by an Fe2+ dependent dioxygenase NaaD. N-formylmaleamic acid is transformed to fumaric acid through maleamic acid and maleic acid by NaaE and NaaF, respectively. To our knowledge, this is the first report of the complete microbial degradation of nicotinamide in bacteria. Nicotinamide is considered as a model compound for the study of microbial degradation of pyridinic compounds, and the nicotinamide degrading related genes in strain P156 were distributed differently from the reported similar gene clusters. Therefore, this study contribute to the knowledge on the degradation of pyridinic compounds.

Molecular cloning, characterization, and expression analysis of Megalobrama amblycephala melanocortin receptor 3 during fasting

PDF HTML阅读 XML下载 导出引用 引用提醒 团头鲂黑素皮质素3型受体基因克隆及表达分析 DOI: 作者: 作者单位: 1. 南京农业大学无锡渔业学院, 江苏 无锡 214081;2. 中国水产科学研究院淡水渔业研究中心, 农业农村部淡水渔业和种质资源利用重点实验室, 江苏 无锡 214081 作者简介: 廖盛臣(1992-),男,硕士研究生,主要从事水产病害学研究.E-mail:602468594@qq.com 通讯作者: 中图分类号: S917 基金项目: 国家自然科学基金项目（31572662）；现代农业产业技术体系建设专项（CARS-45）. Molecular cloning, characterization, and expression analysis of Megalobrama amblycephala melanocortin receptor 3 during fasting Author: Affiliation: 1. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China;2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural affairs;Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:黑素皮质素3型受体（MC3R）系统与动物摄食行为以及能量调控密切相关。为丰富鱼类MC3R相关基础研究，探索鱼类摄食行为与能量调控机制，本研究克隆了团头鲂（基因，采用分子生物信息学和相对荧光定量PCR方法分别对氨基酸序列保守结构域、组织表达分布和禁食条件下的表达变化等开展分析研究。结果显示，团头鲂mc3r mRNA在下丘脑、垂体、肝脏和卵巢有相对较高的表达。禁食试验结果显示，下丘脑和垂体中的转录变化与周期性摄食信号相关，其中下丘脑 mRNA在禁食72 h和禁食72 h后恢复投喂6 h表达量显著升高（<0.05）。此外，对血液皮质醇和血糖的监测结果显示，两者均在禁食14 d有显著变化，其中皮质醇水平显著高于对照组（<0.05）。综合本研究结果表明，MC3R在鲤科鱼类中具有高度相似的氨基酸序列和结构域；在组织中的转录表达变化与食物摄取有明显相关性，对调控鱼体摄食行为和能量代谢具有重要作用。 Abstract:The melanocortin system consists of melanocortin peptides derived from the proopiomelanocortin gene, five melanocortin receptors, two endogenous antagonists, and two ancillary proteins. It plays a role in many important physiological functions of terrestrial animals. Studies on rodents have affirmed the role of melanocortin receptor 3 () in feeding behavior and energy regulation. However, the function of in fish, including basic information, is not well understood. Thus, to determine the initial value of fish and assess the role of MC3R in fish feeding behavior and energy regulation, the full-length cDNA of was cloned. The nucleotide sequence and deduced amino acid sequence were analyzed using bioinformatics methods. Blood glucose and cortisol levels were detected, and real-time quantitative PCR was employed to characterize the tissue expression profile and expression during fasting and refeeding. The results showed that cDNA encodes a protein of 327 amino acids, which shares a high amino acid sequence identity with that of other animals and is characteristic with the typical seven transmembrane domain. Phylogenetic analyses showed that the cluster was within the MC3R clade of the Cyprinidae MC3Rs. The tissue expression analysis revealed that was highly expressed in the hypothalamus, pituitary, liver, and ovary, whereas its expression was much lower in the other detected tissues. During fasting and refeeding, mRNA expression in the hypothalamus and pituitary showed a periodic variation with higher expression near the first feeding time every day both in the experimental and control groups. Furthermore, the expression of mRNA was significantly increased and was higher than that in the control group after 72 h and 14 d fasting in the hypothalamus ( < 0.05), whereas similar results occurred in the pituitary at 72 h fasting and 6 h after refeeding ( < 0.05). In addition, an obvious change in blood glucose and cortisol was only observed with long term fasting ( is not only highly conservative in genetic information, but also in its tissue expression profile and physiological function during the evolutionary process, and may play an important role in feeding behavior and energy regulation. 参考文献 相似文献 引证文献

Structure of human cortisol-producing cytochrome P450 11B1 bound to the breast cancer drug fadrozole provides insights for drug design

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing's disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active-site residues, the overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. Whereas fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, and CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of each human CYP11B enzyme.

First natural crossover recombination between two distinct species of the family Closteroviridae leads to the emergence of a new disease.

Lettuce chlorosis virus-SP (LCV-SP) (family Closteroviridae, genus Crinivirus), is a new strain of LCV which is able to infect green bean plants but not lettuce. In the present study, high-throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt long RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 3´ end encodes proteins which share high amino acid sequence identity with RNA1 of Bean yellow disorder virus (BnYDV; EU191904). The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA (virus-derived small interfering RNA) along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus.

Crystal structure determination of Scylla paramamosain arginine kinase, an allergen that may cause cross-reactivity among invertebrates

Shellfish are one of the most common causes of food allergy. Arginine kinase (AK) is known as an important allergen in shellfish. In the present study, AK from crab (Scylla paramamosain) was purified and its crystal structure was determined. A comparison of AK from S. paramamosain to AKs of other species showed high amino acid sequence and secondary structure identity, while the superposition of crystal structures of AKs from different species revealed only slight differences. Similarity of the linear epitope regions among species was observed in the epitope alignment of AKs; conformational epitopes were located in the regions where secondary structure was conserved. The structure of S. paramamosain AK is an accurate template for the analysis of the IgE binding pattern, and the structure conservation and epitope similarity of AKs among species could help to inform our understanding of the cross-reactivity and contribute to the prediction of cross-reactivity related epitopes.

Genomic characterization of circoviruses associated with acute gastroenteritis in minks in northeastern China.

Mink circovirus (MiCV), a virus that was newly discovered in 2013, has been associated with enteric disease. However, its etiological role in acute gastroenteritis is unclear, and its genetic characteristics are poorly described. In this study, the role of circoviruses (CVs) in mink acute gastroenteritis was investigated, and the MiCV genome was molecularly characterized through sequence analysis. Detection results demonstrated that MiCV was the only pathogen found in this infection. MiCVs and previously characterized CVs shared genome organizational features, including the presence of (i) a potential stem-loop/nonanucleotide motif that is considered to be the origin of virus DNA replication; (ii) two major inversely arranged open reading frames encoding putative replication-associated proteins (Rep) and a capsid protein; (iii) direct and inverse repeated sequences within the putative 5ʹ region; and (iv) motifs in Rep. Pairwise comparisons showed that the capsid proteins of MiCV shared the highest amino acid sequence identity with those of porcine CV (PCV) 2 (45.4%) and bat CV (BatCV) 1 (45.4%). The amino acid sequence identity levels of Rep shared by MiCV with BatCV 1 (79.7%) and dog CV (dogCV) (54.5%) were broadly similar to those with starling CV (51.1%) and PCVs (46.5%). Phylogenetic analysis indicated that MiCVs were more closely related to mammalian CVs, such as BatCV, PCV, and dogCV, than to other animal CVs. Among mammalian CVs, MiCV and BatCV 1 were the most closely related. This study could contribute to understanding the potential pathogenicity of MiCV and the evolutionary and pathogenic characteristics of mammalian CVs.

TbLpn, a key enzyme in lipid droplet formation and phospholipid metabolism, is essential for mitochondrial integrity and growth of Trypanosoma brucei.

Mammalian phosphatidic acid phosphatases, also called lipins, show high amino acid sequence identity to Saccharomyces cerevisiae Pah1p and catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol. Both the substrate and product of the reaction are key precursors for the synthesis of phospholipids and triacylglycerol (TAG). We now show that expression of the Trypanosoma brucei lipin homolog TbLpn is essential for parasite survival in culture. Inducible down-regulation of TbLpn in T. brucei procyclic forms increased cellular PA content, decreased the numbers of lipid droplets, reduced TAG steady-state levels and inhibited in vivo [3 H]TAG formation after labeling trypanosomes with [3 H]glycerol. In addition, fluorescence and transmission electron microscopy revealed that depletion of TbLpn caused major alterations in mitochondrial morphology and function, i.e., the appearance of distorted mitochondrial matrix, and reduced ATP production via oxidative phosphorylation. Effects of lipin depletion on mitochondrial integrity have previously not been reported. N- and C-terminally tagged forms of TbLpn were localized in the cytosol.

Understanding How Two Similar RNA Binding Domains of Paralogous Proteins Mediate Different Protein‐Protein Interactions

Polypyrimidine Tract Binding Protein (PTBP) is an RNA binding protein that has a high affinity for pyrimidine rich regions of RNA sequences. Studies have shown that it acts primarily as a negative regulator of alternative pre‐mRNA splicing events (ASEs); however, it has been shown in some cases to enhance ASEs. Improper regulation of ASEs is linked to neurodegenerative diseases and cancer. Two well studied paralogs of PTBP are PTBP1 and PTBP2. The two proteins demonstrate different levels of splicing repression activity despite sharing high amino acid sequence identity (74%) and similar domain arrangement consisting of four RNA recognition motifs (RRMs) connected by three linker sequences and an N‐terminal region. Recent studies generating chimeras of the two proteins demonstrated that multiple regions of PTBP1 can confer splicing repression activity when integrated in the PTBP2 backbone. These studies also highlighted that despite the high amino acid sequence identity, the RRM 2 region of the two proteins demonstrate different affinities for a partner protein, Raver 1. This protein is required for complete PTB‐mediated splicing repression of the α‐tropomyosin exon 3 sequence. The PTBP‐Raver1 interaction motif has been identified, and PTBP1 and PTBP2 share 87% amino acid sequence identity within their respective Raver1 interaction motifs. These results suggest that additional factors such as post‐translational modifications (PTMs) might influence the PTBP‐Raver1 interaction and in turn, splicing repression activity. To test this, we assayed PTBP1 RRM2 for PTMs via immunoprecipitation followed by mass spectrometry and have identified several ubiquitin modifications. Next, we plan to examine whether the ubiquitin modifications in PTBP1 RRM2 contributes to the Raver1 interaction specifically, as well as to other protein‐protein interactions required for splicing repression activity.Support or Funding InformationThis work was supported by a Maximizing Access to Research Careers grant to CSUF from the National Institutes of Health [5T34GM008612‐21]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification and Characterization of Wheat Yellow Striate Virus, a Novel Leafhopper-Transmitted Nucleorhabdovirus Infecting Wheat.

A new wheat viral disease was found in China. Bullet-shaped viral particles within the nucleus of the infected wheat leave cells, which possessed 180–210 nm length and 35–40 nm width, were observed under transmission electron microscopy. A putative wheat-infecting rhabdovirus vectored by the leafhopper Psammotettix alienus was identified and tentatively named wheat yellow striate virus (WYSV). The full-length nucleotide sequence of WYSV was determined using transcriptome sequencing and RACE analysis of both wheat samples and leafhoppers P. alienus. The negative-sense RNA genome of WYSV contains 14,486 nucleotides (nt) and seven open reading frames (ORFs) encode deduced proteins in the order N-P-P3-M-P6-G-L on the antisense strand. In addition, WYSV genome has a 76-nt 3′ leader RNA and a 258-nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. The entire genome sequence shares 58.1 and 57.7% nucleotide sequence identity with two strains of rice yellow stunt virus (RYSV-A and RYSV-B) genomes, respectively. The highest amino acid sequence identity was 63.8% between the L proteins of the WYSV and RYSV-B, but the lowest was 29.5% between the P6 proteins of these viruses. Phylogenetic analysis firmly established WYSV as a new member of the genus Nucleorhabdovirus. Collectively, this study provided evidence that WYSV is likely the first nucleorhabdovirus described infecting wheat via leafhopper P. alienus transmission.

Expression, purification and crystallization of phosphoribosyl transferase from a mycobacteriophage.

Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transferase from phage BTCU-1, is reported. The crystals belonged to space group C2221, with unit-cell parameters a = 59.71, b = 64.42, c=65.32 Å, α = β = γ = 90°. The crystals diffracted X-rays to 2.2 Å resolution.

New cytokines and TLR pathway signaling molecules in Chinese rare minnow (Gobiocypris rarus): Molecular characterization, basal expression, and their response to chlorpyrifos

In this study, the cDNA fragments of cytokines (il-8) and toll-like receptor (TLR) pathway signaling molecules (myd88, irak-1, irf5, and irf7) in the Chinese rare minnow were cloned and exhibited a high amino-acid sequence identity compared to other cyprinid fish orthologs. The mRNA expressions of these genes in the different tissues (liver, brain, spleen, kidney, and skin) were observed. The highest expression levels of myd88, irak-1, and irf5 were detected in the spleen, whereas il-8 and irf7 were detected in the kidney and liver respectively. The mRNA expression of irak-1, irf5, and irf7 in the liver from 0.1 μg/L and 0.5 μg/L CPF treatments were significantly increased on day 7 (p < 0.05), whereas the levels of only irak-1 and irf7 were markedly increased on day 28 (p < 0.05). Moreover, the mRNA expression of il-8 in the spleen following 0.5 μg/L CPF treatments was significantly decreased on day 7 (p < 0.05), whereas significantly decrease were observed in the levels of irf7 in the spleen at 2.5 μg/L CPF on days 7 and 28 (p < 0.05). The 0.1 μg/L and 0.5 μg/L of CPF significantly induced the levels of irak-1 and myd88 in the spleen after 28 d exposure (p < 0.05). Therefore, the high induction of cytokines and TLR pathway signaling molecules demonstrated that Chinese rare minnow was immune-compromised exposed to CPF. Moreover, our finding indicated that these immune-related genes could be feasible to screen for substances hazardous to the immune system of fish.