Molecular and bioinformatics characterization of fruit bromelain from ananas comosus

Pineapple scientifically known as Ananas comosus, has several available cultivars in Malaysia, including Moris cv, N36 cv, and Sarawak cv. Bromelain has been identified as an active component and a major protease of A. comosus and has gained wide acceptance and compliance as a phototherapeutic drug. Although a considerable level of research has been devoted to bromelain from A. comosus, less attention has been paid to the fruit bromelain compared to the stem bromelain. Therefore, the purpose of this research is to reveal an in-depth information regarding fruit bromelain from A. comosus. Until recently, the three-dimensional (3D) structure of bromelain remained to be elucidated. A comprehensive information on the thorough structural organisation of bromelain is vital for therapeutic application and in the understanding of their role in cells and in other related molecular mechanisms. In this study, the screening of fruit bromelain from the local pineapple cultivars (Morris cv, N36 cv. and Sarawak cv) was implemented, followed by the isolation and cloning of the fruit bromelain from the best cultivar with the highest proteolytic activity for sequence analysis. Additionally, a comparison of the fruit and stem bromelain was performed using bioinformatics tools, including both amino acids and structural comparisons. From the screening results, the highest proteolytic activity (0.8220 U/mL) was observed from the fruit bromelain of Morris cv, followed by N36 cv (0.7695 U/mL) and Sarawak cv (0.6942 U/mL). A gene encoding for pineapple fruit bromelain was successfully isolated from Morris cv. using Reverse Transcription -Polymerase Chain Reaction (RT-PCR) techniques. The amino acid sequence and domain analysis of the fruit and stem bromelains demonstrated several differences and similarities of the cysteine protease family members. Additionally, an analysis of the modelled fruit (BAA21848) and stem (CAA08861) bromelains revealed the presence of unique properties of the predicted structures Cys-148, His-281, Gln-174 and Asn-275 are the catalytic residues of fruit bromelain whereas stem bromelain Cys-147, His-281, His-141 and Asn-302. This play crucial roles in chemical catalysis as general acid/base catalysts. The sequence analysis and structural prediction of the stem and fruit bromelain from A. comosus, along with the comparison of both structures provided a new insight on their distinct properties for industrial application. From the analysis, stem bromelain is more hydrophobic than fruit bromelain. The knowledge of the structure of these proteolytic enzymes from A. comosus is expected to increase the understanding of their functions and mechanism

Diversidade de fitocistatinas em arroz e suas proteinases cisteínicas alvo, com enfoque em fitocistatinas carboxiéstendidas e inibição de legumaínas

Fitocistatinas são inibidores competitivos de proteases cisteínicas em plantas que atuam principalmente na inibição de papaínas. Entretanto, há uma demonstração in vitro de uma fitocistatina carboxi-estendida com capacidade de inibir também uma protease do tipo legumaína. Em arroz existem 12 genes de fitocistatinas, mas apenas um deles (OcXII), possui a extensão C-terminal. Desta forma, o objetivo geral deste trabalho é compreender a diversidade e os mecanismos de interação entre as fitocistatinas e suas proteases cisteínicas alvo em arroz, com ênfase nas implicações funcionais da OcXII. Neste trabalho, nós demonstramos que, em arroz, os 12 genes de fitocistatinas têm um perfil de expressão gênica diferenciado na germinação e em respostas ambientais, sendo OcI, OcIII e OcXII os genes mais expressos. O peptídeo recombinante, correspondente à extremidade C-terminal da OcXII possui capacidade inibitória de legumaínas e não afeta a atividade das papaínas. Através do silenciamento transcricional da OcXII, via RNAi, foram obtidas plantas com atividades proteolíticas aumentadas tanto de papaínas quanto legumaínas, em adição a um fenótipo de crescimento inicial acelerado. O fenótipo oposto é observado em plântulas crescendo em condições alcalinas. Plantas expressando o promotor OcXII fusionado ao gene repórter gus demonstraram um perfil de ativação de OcXII durante a germinação, principalmente na região do escutelo das sementes. Esta ativação de OcXII permanece alta quando as sementes são mantidas com níveis elevados de ABA e sob estresse alcalino. Como alvos específicos de OcXII, as legumaínas estão relacionadas com a biossíntese de componentes vacuolares e degradação de proteínas de armazenamento. Em arroz, nós encontramos cinco diferentes loci para as legumaínas. Análises filogenéticas e estudos de expressão gênica demonstraram uma maior associação de OsaLeg2 e OsaLeg3 com tecidos de semente, e OsaLeg1, 4 e 5 com tecidos vegetativos. Também foram observadas formas de splicing alternativo, com potencial de originar diferentes isoformas ativas de legumaínas. Em geral, nossos dados demonstram o envolvimento bifuncional da OcXII nos processos de germinação e defesa interagindo e inibindo a atividade de proteases cisteínicas específicas, onde a região N-term de OcXII inibe papaínas, enquanto a C-term inibe legumaínas.Phytocystatins are competitive inhibitors of cysteine proteases in plants, principally inhibiting papain-like proteases activity. However, there is one in vitro demonstration that a carboxy-extended phytocystatin can inhibit legumain-like proteases either. In rice there are 12 phytocystatins genes, but only one (OcXII) has the C-terminal extension. Thus, the aim of this study is to understand the diversity and interaction mechanisms among phytocystatins and its cysteine protease targets in rice, focusing on OcXII functional implications. In this work we demonstrated that in rice, the 12 phytocystatins genes have a different gene expression profile during the germination and environmental responses, while OcI OcIII and OcXII were the most expressed genes. A recombinant peptide corresponding to the different C-terminus of OcXII was produced and its inhibitory capacity was confirmed against legumains without affecting the activity of papains. OcXII transcriptional silencing, via RNAi, resulted in plants with increased proteolytic activity for both papain and legumains, in addition to an accelerated initial growth phenotype. The opposite phenotype is observed in seedlings growing under alkaline conditions. Plants expressing the OcXII promoter fused to gus reporter gene demonstrated an OcXII activation profile during germination, especially in the seed scutellum region. This OcXII activation remains high when the seeds are kept at high ABA levels or under alkaline conditions. As specific OcXII targets, legumains are related to the biosynthesis of vacuolar storage components and protein degradation. In rice, we found 5 different loci for legumains. Phylogenetic analyses and gene expression studies demonstrated a greater association of OsaLeg3 and OsaLeg2 with seed tissues, while OsaLeg1, 4 and 5 were more abundant in vegetative tissues. Also alternatively spliced forms were observed, with the potential to produce different isoforms of active legumains. Overall, our data demonstrate the involvement of the bifunctional OcXII in germination processes and defense, interacting and inhibiting the activity of specific cysteine proteases, where its N-term region inhibits papain, while the C-term inhibits legumains

Isolamento e cristalização de estados oligoméricos de cistatinas de plantas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2020.As cistatinas das plantas representam uma subfamília das cistatinas (Cys), também conhecidas como Fitocistatinas (PhyCys), sendo caracterizadas como proteínas inibidoras das enzimas cisteíno–proteases (Cys–Prot). A atividade proteica das PhyCys pode ser controlada por meio da oligomerização do tipo troca de domínios (domain swap), um evento de dimerização em que as proteínas trocam elementos de estrutura secundária, formando um dímero sem atividade inibitória. As PhyCys são inibidoras de proteases exógenas e endógenas, regulando vários processos fisiológicos como: tolerância a diferentes tipos de estresses bióticos (produzidos por fungos, bactérias e insetos) e abióticos (frio, calor e sal) tornando–as interessantes alvos de pesquisa. Os estudos das PhyCys potencializam que estas sejam ferramentas biotecnológicas aplicadas na agricultura. A presente pesquisa visou selecionar, clonar, expressar, purificar e caracterizar PhyCys. Por meio de análises, de bioinformática, em banco de dados, foram avaliadas várias PhyCys de relevância econômica, resultando na escolha de duas PhyCys, uma da Junglas regia (NOG1) e outra do Coffea arabica (CAF1). As PhyCys utilizadas nesta pesquisa foram obtidas por expressão heteróloga, utilizando as cepas de Escherichia coli (E. coli) Lemo21 (DE3) cultivadas em meio auto–indutor ZYM–5052, pois essas condições apresentaram melhor expressão solúvel de NOG1 e CAF1. As proteínas foram purificadas por cromatografia de afinidade a metal e cromatografia de exclusão molecular (SEC). As purificações de NOG1 resultaram em maior quantidade de dímeros, contudo foram observados também monômeros, e para a CAF1 foram observados principalmente monômeros. A SEC foi utilizada em ensaios de estabilidade térmica e nos ensaios de oligomerização das PhyCys. As PhyCys foram incubadas em diferentes temperaturas (T) e as duas PhyCys se mostraram estáveis em gelo. Após incubação por diferentes períodos em intervalos discretos de 20 °C a 70 °C foi possível monitorar a conversão da forma monomérica na forma dimérica, e vice–versa. Os resultados demonstram que as mudanças no estado oligomérico das PhyCys ocorreram em função da temperatura, do tempo de incubação e da concentração. As triagens para obtenção de cristais foram realizadas com o auxilio do robô Mosquito® em diferentes condições, depois refinadas manualmente, e os cristais obtidos foram submetidos à coleta de dados de difração de raios–X in–house em um difratômetro D8 VENTURE (Bruker). Dados obtidos a 3,1 Å de resolução provenientes de cristais monoclínicos de grupo espacial C 1 2 1 da NOG1 foram resolvidos por substituição molecular. Foram observados dois dímeros por troca de domínios na unidade assimétrica.Fundação de Apoio a Pesquisa do Distrito Federal (FAP/DF) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Plant cystatins, also known as phytocystatins (PhyCys) represent a subfamily of cystatins (Cys), and are proteins that inhibit the enzymes cysteine–proteases (CysProt). The protein activity of PhyCys can be controlled through domain swap oligomerization. In the dimerization process, proteins exchange elements of secondary structure, forming a dimer without inhibitory activity. PhyCys are inhibitors of exogenous and endogenous proteases, acting in tolerance to different types of biotic stresses (produced by fungi, bacteria and insects) and abiotic (cold, heat and salt) making them interesting targets for research, and as possible biotechnological tools to be employed in agriculture. The present research aimed to select, clone, express, purify and characterize PhyCys. Several PhyCys of economic relevance were evaluated, resulting in one from Junglas regia (NOG1) and another from Coffea arabica (CAF1). In order to obtain the PhyCys used in this research, the heterologous expression process was carried out in a prokaryotic system using the pET24a expression vector. In the expression tests, the strains of Escherichia coli (E. coli) Lemo21 (DE3), grown in a self–inducing medium ZYM–5052, were more efficient, both in the production of NOG1 as well as CAF1. The proteins were purified by metal affinity chromatography and molecular exclusion chromatography (SEC). The purifications of NOG1 resulted in a greater amount of dimers, however, monomers and tetramers were also observed, and for CAF1, mainly monomers were observed. SEC was used in thermal stability tests and in PhyCys oligomerization tests. The PhyCys were incubated at different temperatures (T) and the two PhyCys were stable in ice. After incubation for different periods at discrete temperature intervals it was possible to monitor the conversion from monomer to dimer, and vice–versa. The results demonstrate that the changes in the PhyCys oligomeric state occurred as a function of temperature, incubation time and concentration. The screenings for obtaining crystals were performed with the help of the Mosquito® robot in different conditions, and then manually refined. The obtained crystals were subjected to in– house X–ray diffraction on a D8 VENTURE diffractometer (Bruker). Data obtained at 3.1 Å resolution from monoclinic crystals of space group C 1 2 1 of NOG1 were resolved by molecular substitution. Two domain–swapped dimers were observed in the asymmetric unit

Identification and Characterization of Stress Responsive Genes in Soybean and Sunflower

Stress responsive genes encode proteins involved in plants’ response to abiotic and biotic stresses. Among such stress responsive proteins, proteins encoded by resistance genes (R genes) or nucleotide binding site-leucine-rich repeats (NBS-LRRs) and mitogen-activated protein kinases (MAPKs) are the major groups of proteins regulating biotic and abiotic stresses, respectively. Previous studies in Nepal’s lab at SDSU identified and characterized coiled coil (CC)-NBS-LRRs (CNLs), resistance to powdery mildew8 (RPW8)-NBS-LRRs (RNLs), NBS-LRR (NLs), and MAPK proteins in soybean. This study focuses on R and MAPK genes in the recently sequenced genome of sunflower as well as the toll-interleukin-1 receptor-like nucleotide-binding site leucine-rich repeat (TNL) R genes of soybean. This study also uses greenhouse experiments and RNA sequencing (RNA-seq) data to characterize stress responsive genes involved in interaction effects of soybean aphid (SBA) and soybean cyst nematode (SCN) interactions on soybean. Thus the major objectives of this dissertation work were to 1) explore the TNL genes in soybean and R (CNL, TNL, RNL) genes in sunflower genomes to assess how they may have evolved and their possible role in resistance against pathogens using available transcriptomic data, 2) identify and characterize MAPK genes in sunflower, and 3) characterize induced susceptibility effects of soybean-soybean aphid and interaction effects of soybean soybean aphid-soybean cyst nematode on soybean. In this dissertation, we used in silico approaches to report genome-wide identification and characterization of soybean TNL proteins as well as sunflower R and MAPK proteins. In order to achieve these objectives, numerous bioinformatics tools were utilized: hidden markov model (HMM) profilings were performed, and annotation of protein domains were conducted. Maximum Likelihood phylogenetic trees were constructed, and nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site ratios (Ka/Ks) as a proxy for selection pressure of R genes were calculated. In addition, chromosomal distribution, intron-exon architecture; synteny as well as gene expression patterns were assessed. In order to characterize stress responsive genes involved in defense responses, we used soybean aphid (Aphis glycines; SBA) and soybean cyst nematode (Heterodera glycines; SCN) to infest soybean cultivars. We conducted greenhouse experiments to characterize induced susceptibility effects of soybean-SBA interaction, and three-way interactions among soybean, SBA, and SCN. We utilized both demographic and genetic (RNA-seq) datasets to characterize the genes involved in such interactions using biotype 1, biotype 2 soybean aphids and HG type 0 SCN on soybean. FastQC, Btrim, Trimmomatic, Salmon, iDEP, MapMan tools were used to assess the quality, trim, map, assemble, visualize, pathway analysis and biological significance of RNA sequencing data to host genome. We identified an inventory of 117 of 153 regular TNL genes in soybean, and 352 NBS-encoding genes (100 CNLs, 77 TNLs, 13 RNLs, and 162 NLs), 28 MPKs and eight MKKs in sunflower through in silico analyses. R genes in soybean and sunflower formed several gene clusters suggesting their origin by tandem duplications. The selection pressure analysis revealed R genes experiencing purifying selection (Ka/Ks \u3c 1) in both soybean and sunflower. Sunflower MAP Kinases revealed within and between clade functional divergence, and MKK3 orthologues were highly conserved across the species representing diverse taxonomic groups of the plant kingdom. Demographic data obtained from greenhouse experiments showed that induced susceptibility as initial feeding with virulent SBA (biotype 2) increased the population of subsequent avirulent SBA (biotype 1) in both susceptible and resistant cultivars. In the three-way interaction among soybean, SBA, and SCN, the number of SCN eggs was significantly greater on the susceptible cultivar and there was no effect in the resistant cultivar in the presence of SBA. The SBA population density was negatively affected by SCN populations. RNA-seq analysis in both studies have revealed differentially expressed genes (DEGs) and transcription factor (TF) binding motifs, which were enriched for various biological processes and pathways at different time points. The DEGs were common and unique in susceptible and resistant cultivars and treatments that were enriched for various biological processes and pathways. These DEGs were also functionally related to known defense mechanisms previously reported in various hostaphid and host-nematode systems. The responses to aphid biotype 1 infestation in the presence or absence of inducer population (biotype 2) at two time points (day1 and 11 post inducer infestation) revealed significant differences on the gene enrichment and regulation in SBA resistant and susceptible cultivars. For instance, enrichment analysis showed ‘response to chitin’, ‘lignin catabolic and metabolic process’, ‘asparagine metabolic process’, ‘response to chemical’ unique to treatment with no inducer population, whereas, ‘response to reactive oxygen species’, ‘photosynthesis’, ‘regulation of endopeptidase activity’ unique to treatment with inducer population. Likewise, Soybean-SBA-SCN interaction study showed enrichment of genes in ‘Plant Pathogen Interaction’ and ‘cutin, suberine, and wax biosynthesis’ pathways at 5 (days post SBA infestation) dpi; ‘isoflavonoid biosynthesis’ and ‘one carbon pool by folate’ pathways enriched at 30 dpi in SCN resistant and susceptible cultivars. Overall, the results from this study have improved the current understanding of diversity and evolution of MAPK and R genes in sunflower and soybean, as well as have first time reported a molecular characterization of induced susceptibility effects due to SBA on soybean, and soybean- SBA-SCN interactions, which has a direct implication in disease and pest management

Characterization of P1 leader proteases of the Potyviridae family and identification of the host factors involved in their proteolytic activity during viral infection

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-05-2018Esta tesis tiene embargado el acceso al texto completo hasta el 25-11-201

Allergic Diseases

The present Edition "Allergic diseases - highlights in the clinic, mechanisms and treatment" aims to present some recent aspects related to one of the most prevalent daily clinical expression disease. The effort of a group of outstanding experts from many countries reflects a set of scientific studies very promising for a better clinical care and also to the treatment and control of the allergy. This book provides a valuable reference text in several topics of the clinical allergy and basic issues related to the immune system response. The inflammatory reaction understanding in allergic disease is clearly evidenced, as well as new strategies for further researches

Molecular and immunological characterisation of proteins from Anisakis pegreffii and their immune stimulatory effect on the human health system

With the fast growth of seafood trading worldwide, the potential health risks of eating contaminated seafood have greatly increased. Biological effects of contaminated seafood are associated and caused by a variety of bacteria, viruses, and parasites; this various groups of pathogens results in an extensive diversity of clinical syndromes, each with its own epidemiological characteristics. According to a World Health Organization investigation, more than 1 billion people worldwide are infected with different species of the Ascaris lumbricoides worm parasite (282), which causes serious conditions ranging from mild to lethal. Anisakis parasites in marine fish have imposed a significant economic burden, reducing productivity and requiring elaborate and expensive control methods. Nematodes of the family Anisakidae is a major group parasitise fish, mammals, birds and reptiles, with the larval stages of some species. Several species of Anisakis have been found to be parasitic in marine mammals such as whales and dolphins in their adult stages, and their larvae are found in a variety of fish species. Anisakid nematodes have complex life cycles that include invertebrate and vertebrate hosts at various developmental stages during their life. Most reports of anisakiasis infections are associated with eating raw or undercooked fish that contain larval Anisakis. Humans can also be accidental hosts for larval Anisakis, however they cannot progress their life cycles, but they can frequently cause hypersensitivity IgE-mediated reactions with or without several gastrointestinal manifestations ranging from urticaria to angioedema, called anisakiasis diseases. The development of an allergic reaction mediated by IgE may be against ES and somatic allergens of larval Anisakis. Nuclear ribosomal DNA (rDNA) provides suitable genetic markers for the identification of larval Anisakis. Therefore, the sequences of internal transcribed spacers (ITS-1 and ITS-2) are a powerful approach to identify and distinguish anisakid nematodes (at any developmental stage) for diagnostic or taxonomic purposes, for exploring the genetic composition of larval anisakid populations and for investigating their ecology. Although several allergens have been identified in Anisakis simplex, little information is available for other parasite species infecting fish. Therefore, this project aims to investigate the allergenicity of tropomyosin (Ani p 3) and a cysteine protease inhibitor (Ani p 4) from the sibling species, Anisakis pegreffii, using biochemical, genetic and immunological approaches. Larvae (L3) were harvested manually from tiger flathead fish and identified morphologically using a light microscope. For molecular characterisation and genetic diversity, total genomic DNA was extracted from individual samples and verified by species-specific PCR amplification using the sequences of the first and/or second internal transcribed spacers (ITS-1 and/or ITS-2) of ribosomal DNA (rDNA) as the species-specific genetic markers. For molecular characterisation of Anisakis allergens, RNA of Anisakis pegreffii was extracted, then cDNA synthesised and amplified using designed primers and used to amplify tropomyosin and cysteine protease inhibitor genes from Anisakis pegreffii. Whole muscle proteins from Anisakis larvae were extracted directly with extraction solution and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by immunoblotting assays. Tropomyosin protein was detected using anti-tropomyosin and anti-crustacean polyclonal antibodies raised in rabbits while a cysteine protease inhibitor was detected using anti-cysteine protease inhibitor polyclonal antibodies raised in rabbit. The allergenicity of the nematode tropomyosin and cysteine protease inhibitor was evaluated using human serum of atopic patient with a shellfish allergy. The result of SDS-PAGE and immunoblotting assays of Anisakis pegreffii extracts indicates the presence of tropomyosin and cysteine protease inhibitor proteins of 44 and 12.8 kDa molecular weight, respectively. These results are similar to the allergenic tropomyosin and cysteine protease inhibitor of Anisakis simplex. Also, strong reactivity with human serum designated these proteins as allergens in Anisakis pegreffii spp. The molecular sequencing was successful for tropomyosin and cysteine protease inhibitor genes from Anisakis pegreffii, and demonstrated that they are closely related to the Anisakis simplex proteins previously sequenced and submitted in the GenBank database. Our results indicated that, the cDNA sequence similarity searches using the BLAST program and phylogenetic analysis (Clustal W programme) revealed that tropomyosin of A. pegreffii L3 has the highest homology to α tropomyosins isoform from A. simplex L3 (98%) and A. lumbricoides (84%), respectively. In addition, cysteine-protease inhibitor of A. pegreffii L3 has the highest identity to cysteine-protease inhibitor isoform from A. simplex L3 (98%) using the BLAST program and 100% using phylogenetic analysis (Clustal W programme). This analysis highlights that this nematode tropomyosin and cysteine protease inhibitor share high sequence identity with other parasitic nematodes, house dust and crustacean tropomyosin, respectively

Structural and Functional Analysis of Extracts in Plants

Structural and Functional Analysis of Extracts in Plants collects 1 editorial, 3 reviews, and 26 research articles reporting recent research findings which cover several aspects of plant-derived bioactive compounds, to correlate extraction techniques with the chemical composition of extracts and their bioactivity for identifying molecules that might be used as active substances in a wide variety of areas.This book is a valuable resource for members of the scientific community wishing to further explore plants and the therapeutic applications of their bioactive compounds. It will appeal to scholars, teachers and scientists involved in plant product research, and facilitate the development of innovative new drugs

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin