In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>The major Dengue virus vector <it>Aedes aegypti </it>requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of <it>Ae. aegypti </it>midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.</p> <p>Results</p> <p>We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k_cat/K_M) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.</p> <p>Conclusions</p> <p>These data show that <it>in vitro </it>activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.</p

A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues

Pathogen and Circadian Controlled 1 (PCC1) Protein Is Anchored to the Plasma Membrane and Interacts with Subunit 5 of COP9 Signalosome in Arabidopsis

The Pathogen and Circadian Controlled 1 (PCC1) gene, previously identified and further characterized as involved in defense to pathogens and stress-induced flowering, codes for an 81-amino acid protein with a cysteine-rich C-terminal domain. This domain is essential for homodimerization and anchoring to the plasma membrane. Transgenic plants with the ß- glucuronidase (GUS) reporter gene under the control of 1.1 kb promoter sequence of PCC1 gene display a dual pattern of expression. At early post-germination, PCC1 is expressed only in the root vasculature and in the stomata guard cells of cotyledons. During the transition from vegetative to reproductive development, PCC1 is strongly expressed in the vascular tissue of petioles and basal part of the leaf, and it further spreads to the whole limb in fully expanded leaves. This developmental pattern of expression together with the late flowering phenotype of long-day grown RNA interference (iPCC1) plants with reduced PCC1 expression pointed to a regulatory role of PCC1 in the photoperiod-dependent flowering pathway. iPCC1 plants are defective in light perception and signaling but are not impaired in the function of the core CO-FT module of the photoperiod-dependent pathway. The regulatory effect exerted by PCC1 on the transition to flowering as well as on other reported phenotypes might be explained by a mechanism involving the interaction with the subunit 5 of the COP9 signalosome (CSN).This work was funded by grants BIO2008-00839, BIO2011-27526 and CSD2007-0057 from Ministerio de Ciencia e Innovacion of Spain to J.L. A fellowship/contract of the FPU program of the Ministerio de Educacion y Ciencia (Spain) funded R.M. work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Mir Moreno, R.; Leon Ramos, J. (2014). Pathogen and Circadian Controlled 1 (PCC1) Protein Is Anchored to the Plasma Membrane and Interacts with Subunit 5 of COP9 Signalosome in Arabidopsis. PLoS ONE. 1(9):1-14. https://doi.org/10.1371/journal.pone.0087216S11419Sauerbrunn, N., & Schlaich, N. L. (2004). PCC1 : a merging point for pathogen defence and circadian signalling in Arabidopsis. Planta, 218(4), 552-561. doi:10.1007/s00425-003-1143-zSEGARRA, S., MIR, R., MARTÍNEZ, C., & LEÓN, J. (2009). Genome-wide analyses of the transcriptomes of salicylic acid-deficient versus wild-type plants uncover Pathogen and Circadian Controlled 1 (PCC1) as a regulator of flowering time in Arabidopsis. Plant, Cell & Environment, 33(1), 11-22. doi:10.1111/j.1365-3040.2009.02045.xVenancio, T. M., & Aravind, L. (2009). CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across eukaryotes. Bioinformatics, 26(2), 149-152. doi:10.1093/bioinformatics/btp647Lau, O. S., & Deng, X. W. (2010). Plant hormone signaling lightens up: integrators of light and hormones. Current Opinion in Plant Biology, 13(5), 571-577. doi:10.1016/j.pbi.2010.07.001Seo, M., Nambara, E., Choi, G., & Yamaguchi, S. (2008). Interaction of light and hormone signals in germinating seeds. Plant Molecular Biology, 69(4), 463-472. doi:10.1007/s11103-008-9429-yDe Lucas, M., Davière, J.-M., Rodríguez-Falcón, M., Pontin, M., Iglesias-Pedraz, J. M., Lorrain, S., … Prat, S. (2008). A molecular framework for light and gibberellin control of cell elongation. Nature, 451(7177), 480-484. doi:10.1038/nature06520Feng, S., Martinez, C., Gusmaroli, G., Wang, Y., Zhou, J., Wang, F., … Deng, X. W. (2008). Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature, 451(7177), 475-479. doi:10.1038/nature06448Mutasa-Gottgens, E., & Hedden, P. (2009). Gibberellin as a factor in floral regulatory networks. Journal of Experimental Botany, 60(7), 1979-1989. doi:10.1093/jxb/erp040Bastian, R., Dawe, A., Meier, S., Ludidi, N., Bajic, V. B., & Gehring, C. (2010). Gibberellic acid and cGMP-dependent transcriptional regulation inArabidopsis thaliana. Plant Signaling & Behavior, 5(3), 224-232. doi:10.4161/psb.5.3.10718Yu, S., Galvão, V. C., Zhang, Y.-C., Horrer, D., Zhang, T.-Q., Hao, Y.-H., … Wang, J.-W. (2012). Gibberellin Regulates the Arabidopsis Floral Transition through miR156-Targeted SQUAMOSA PROMOTER BINDING–LIKE Transcription Factors. The Plant Cell, 24(8), 3320-3332. doi:10.1105/tpc.112.101014Arc, E., Galland, M., Cueff, G., Godin, B., Lounifi, I., Job, D., & Rajjou, L. (2011). Reboot the system thanks to protein post-translational modifications and proteome diversity: How quiescent seeds restart their metabolism to prepare seedling establishment. PROTEOMICS, 11(9), 1606-1618. doi:10.1002/pmic.201000641Dill, A., Thomas, S. G., Hu, J., Steber, C. M., & Sun, T. (2004). The Arabidopsis F-Box Protein SLEEPY1 Targets Gibberellin Signaling Repressors for Gibberellin-Induced Degradation. The Plant Cell, 16(6), 1392-1405. doi:10.1105/tpc.020958Wang, F., & Deng, X. W. (2011). Plant ubiquitin-proteasome pathway and its role in gibberellin signaling. Cell Research, 21(9), 1286-1294. doi:10.1038/cr.2011.118Hotton, S. K., & Callis, J. (2008). Regulation of Cullin RING Ligases. Annual Review of Plant Biology, 59(1), 467-489. doi:10.1146/annurev.arplant.58.032806.104011Cope, G. A. (2002). Role of Predicted Metalloprotease Motif of Jab1/Csn5 in Cleavage of Nedd8 from Cul1. Science, 298(5593), 608-611. doi:10.1126/science.1075901Gusmaroli, G., Figueroa, P., Serino, G., & Deng, X. W. (2007). Role of the MPN Subunits in COP9 Signalosome Assembly and Activity, and Their Regulatory Interaction with Arabidopsis Cullin3-Based E3 Ligases. The Plant Cell, 19(2), 564-581. doi:10.1105/tpc.106.047571Serino, G., & Deng, X.-W. (2003). THECOP9 SIGNALOSOME: Regulating Plant Development Through the Control of Proteolysis. Annual Review of Plant Biology, 54(1), 165-182. doi:10.1146/annurev.arplant.54.031902.134847Stratmann, J. W., & Gusmaroli, G. (2012). Many jobs for one good cop – The COP9 signalosome guards development and defense. Plant Science, 185-186, 50-64. doi:10.1016/j.plantsci.2011.10.004Lozano-Juste, J., & León, J. (2011). Nitric Oxide Regulates DELLA Content and PIF Expression to Promote Photomorphogenesis in Arabidopsis. Plant Physiology, 156(3), 1410-1423. doi:10.1104/pp.111.177741Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., … Kimura, T. (2007). Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Journal of Bioscience and Bioengineering, 104(1), 34-41. doi:10.1263/jbb.104.34Fromont-Racine, M., Rain, J.-C., & Legrain, P. (1997). Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nature Genetics, 16(3), 277-282. doi:10.1038/ng0797-277Belda-Palazón B, Ruiz L, Martí E, Tárraga S, Tiburcio AF, et al.. (2012) Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells. PLoS One 7(10), e46907.Simon, R., Igeño, M. I., & Coupland, G. (1996). Activation of floral meristem identity genes in Arabidopsis. Nature, 384(6604), 59-62. doi:10.1038/384059a0Martínez, C., Pons, E., Prats, G., & León, J. (2003). Salicylic acid regulates flowering time and links defence responses and reproductive development. The Plant Journal, 37(2), 209-217. doi:10.1046/j.1365-313x.2003.01954.xKyte, J., & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157(1), 105-132. doi:10.1016/0022-2836(82)90515-0Marmagne, A., Rouet, M.-A., Ferro, M., Rolland, N., Alcon, C., Joyard, J., … Ephritikhine, G. (2004). Identification of New Intrinsic Proteins inArabidopsisPlasma Membrane Proteome. Molecular & Cellular Proteomics, 3(7), 675-691. doi:10.1074/mcp.m400001-mcp200Nühse, T. S., Stensballe, A., Jensen, O. N., & Peck, S. C. (2004). Phosphoproteomics of the Arabidopsis Plasma Membrane and a New Phosphorylation Site Database. The Plant Cell, 16(9), 2394-2405. doi:10.1105/tpc.104.023150Kobayashi, Y., & Weigel, D. (2007). Move on up, it’s time for change mobile signals controlling photoperiod-dependent flowering. Genes &amp; Development, 21(19), 2371-2384. doi:10.1101/gad.1589007Jaeger, K. E., & Wigge, P. A. (2007). FT Protein Acts as a Long-Range Signal in Arabidopsis. Current Biology, 17(12), 1050-1054. doi:10.1016/j.cub.2007.05.008Mathieu, J., Warthmann, N., Küttner, F., & Schmid, M. (2007). Export of FT Protein from Phloem Companion Cells Is Sufficient for Floral Induction in Arabidopsis. Current Biology, 17(12), 1055-1060. doi:10.1016/j.cub.2007.05.009Mir, R., Hernández, M. L., Abou-Mansour, E., Martínez-Rivas, J. M., Mauch, F., Métraux, J.-P., & León, J. (2013). Pathogen and Circadian Controlled 1 (PCC1) regulates polar lipid content, ABA-related responses, and pathogen defence in Arabidopsis thaliana. Journal of Experimental Botany, 64(11), 3385-3395. doi:10.1093/jxb/ert177Nordgård, O., Dahle, Ø., Andersen, T. Ø., & Gabrielsen, O. S. (2001). JAB1/CSN5 interacts with the GAL4 DNA binding domain: A note of caution about two-hybrid interactions. Biochimie, 83(10), 969-971. doi:10.1016/s0300-9084(01)01329-3Kwok, S. F., Staub, J. M., & Deng, X.-W. (1999). Characterization of two subunits of Arabidopsis 19S proteasome regulatory complex and its possible interaction with the COP9 complex 1 1Edited by J. Karn. Journal of Molecular Biology, 285(1), 85-95. doi:10.1006/jmbi.1998.2315Nezames, C. D., & Deng, X. W. (2012). The COP9 Signalosome: Its Regulation of Cullin-Based E3 Ubiquitin Ligases and Role in Photomorphogenesis. Plant Physiology, 160(1), 38-46. doi:10.1104/pp.112.198879Moon, J., Parry, G., & Estelle, M. (2004). The Ubiquitin-Proteasome Pathway and Plant Development. The Plant Cell, 16(12), 3181-3195. doi:10.1105/tpc.104.161220Dreher, K., & Callis, J. (2007). Ubiquitin, Hormones and Biotic Stress in Plants. Annals of Botany, 99(5), 787-822. doi:10.1093/aob/mcl255Parry, G., & Estelle, M. (2004). Regulation of cullin-based ubiquitin ligases by the Nedd8/RUB ubiquitin-like proteins. Seminars in Cell & Developmental Biology, 15(2), 221-229. doi:10.1016/j.semcdb.2003.12.003Wee, S., Geyer, R. K., Toda, T., & Wolf, D. A. (2005). CSN facilitates Cullin–RING ubiquitin ligase function by counteracting autocatalytic adapter instability. Nature Cell Biology, 7(4), 387-391. doi:10.1038/ncb1241Kuramata, M., Masuya, S., Takahashi, Y., Kitagawa, E., Inoue, C., Ishikawa, S., … Kusano, T. (2008). Novel Cysteine-Rich Peptides from Digitaria ciliaris and Oryza sativa Enhance Tolerance to Cadmium by Limiting its Cellular Accumulation. Plant and Cell Physiology, 50(1), 106-117. doi:10.1093/pcp/pcn175Zeng, W., Melotto, M., & He, S. Y. (2010). Plant stomata: a checkpoint of host immunity and pathogen virulence. Current Opinion in Biotechnology, 21(5), 599-603. doi:10.1016/j.copbio.2010.05.006Wigge, P. A. (2011). FT, A Mobile Developmental Signal in Plants. Current Biology, 21(9), R374-R378. doi:10.1016/j.cub.2011.03.038Kardailsky, I. (1999). Activation Tagging of the Floral Inducer FT. Science, 286(5446), 1962-1965. doi:10.1126/science.286.5446.1962Srikanth, A., & Schmid, M. (2011). Regulation of flowering time: all roads lead to Rome. Cellular and Molecular Life Sciences, 68(12), 2013-2037. doi:10.1007/s00018-011-0673-yGalvao, V. C., Horrer, D., Kuttner, F., & Schmid, M. (2012). Spatial control of flowering by DELLA proteins in Arabidopsis thaliana. Development, 139(21), 4072-4082. doi:10.1242/dev.080879Cerdán, P. D., & Chory, J. (2003). Regulation of flowering time by light quality. Nature, 423(6942), 881-885. doi:10.1038/nature01636Guo, H. (1998). Regulation of Flowering Time by Arabidopsis Photoreceptors. Science, 279(5355), 1360-1363. doi:10.1126/science.279.5355.1360Liu, B., Zuo, Z., Liu, H., Liu, X., & Lin, C. (2011). Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes & Development, 25(10), 1029-1034. doi:10.1101/gad.2025011Weidler, G., zur Oven-Krockhaus, S., Heunemann, M., Orth, C., Schleifenbaum, F., Harter, K., … Batschauer, A. (2012). Degradation of Arabidopsis CRY2 Is Regulated by SPA Proteins and Phytochrome A. The Plant Cell, 24(6), 2610-2623. doi:10.1105/tpc.112.09821

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

Background: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. Results: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Conclusions: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a ‘One Health’ approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans

Freqüência e variáveis associadas ao aleitamento materno em crianças com até 12 meses de idade no município de Araçatuba, São Paulo, Brazil Frequency and associated variables to breastfeeding among infant up to 12 months of age in Araçatuba, State of São Paulo, Brazil

OBJETIVOS: avaliar a prevalência do aleitamento materno em crianças assistidas em Araçatuba, São Paulo, Brasil; verificar a associação com variáveis materno-infantis e o conhecimento das mães sobre a relação da amamentação com saúde bucal. MÉTODOS: estudo transversal. Os dados foram coletados durante a Campanha Nacional de Vacinação, em 2005. Foram entrevistadas 100 mães de crianças com até 12 meses de idade. A freqüência do aleitamento foi estimada por meio da análise de sobrevivência, e foram realizadas análises estatísticas para verificação da associação entre aleitamento e variáveis independentes. RESULTADOS: a prevalência do aleitamento aos 6 e 12 meses foi de 22,2% (exclusivo) e 65% (total). A duração mediana da amamentação exclusiva foi de 3,65 meses. As variáveis associadas ao desmame foram uso de mamadeira (&#967;2=9,537; p=0,002) e chupeta (&#967;2= 14,667; p=0,001). Poucas mães (33) demonstraram saber a influência do aleitamento sobre a saúde bucal de seus filhos, sendo o cirurgião-dentista o profissional mais citado como responsável por essa informação. CONCLUSÕES: a prevalência da amamentação foi satisfatória, porém foram baixas as taxas de aleitamento exclusivo, e como fatores determinantes destacaram-se o uso de mamadeiras e chupetas associado ao desmame. É dever de órgãos governamentais, meios de comunicação e profissionais de saúde compactuarem ações efetivas em prol do aleitamento.<br>OBJECTIVES: to evaluate the prevalence of breastfeeding among infants receiving care in Araçatuba, in the State of São Paulo, Brazil; to assess associated variables relating to mother and child and the mother's knowledge regarding the relationship between breastfeeding and oral health. METHODS: cross-sectional study. Data was collected during the National Immunization Campaign, in 2005. One hundred mothers of children up to 12 months of age were interviewed. The frequency of breastfeeding was estimated using a survival analysis and statistical analyses were carried out to assess the association between breastfeeding and independent variables. RESULTS: the prevalence of breastfeeding, at 6 and 12 months was 22.2% (exclusive) and 65% (total). The mean duration of exclusive breastfeeding was 3.65 months. The variables associated with weaning were use of a bottle (&#967;2=9.537; p=0.002) and of a pacifier (&#967;2=14.667; p=0.001). Few mothers (33) demonstrated knowledge of the influence of breastfeeding on their children's oral health, the dentist being more often mentioned as responsible for providing this information. CONCLUSIONS: the prevalence of breastfeeding was satisfactory, although rates for exclusive breastfeeding were low. The use of a bottle and a pacifier were positively associated as determining factors with weaning. The government, media and health professionals must improve effective actions to promote breastfeeding