Publications by authors named "Andrea Soares-Costa"

28 Publications

  • Page 1 of 1

Physiological, nutritional, and molecular responses of Brazilian sugarcane cultivars under stress by aluminum.

PeerJ 2021 28;9:e11461. Epub 2021 Jun 28.

Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

Background: Sugarcane is a crop of global importance and has been expanding to areas with soils containing high levels of exchangeable aluminum (Al), which is a limiting factor for crop development in acidic soils. The study of the sugarcane physiological and nutritional behavior together with patterns of gene expression in response to Al stress may provide a basis for effective strategies to increase crop productivity in acidic soils.

Methods: Sugarcane cultivars were evaluated for physiological parameters (photosynthesis, stomatal conductance, and transpiration), nutrient (N, P, K, Ca, Mg, and S) and Al contents in leaves and roots and gene expression, of the genes , by qPCR, both related to the production of organic acids, and , related to oxidative stress.

Results: Brazilian sugarcane RB867515, RB928064, and RB935744 cultivars exhibited very different responses to induced stress by Al. Exposure to Al caused up-regulation ( and ) or down-regulation (, , and ), depending on the cultivar, Al level, and plant tissue. The RB867515 cultivar was the most Al-tolerant, showing no decline of nutrient content in plant tissue, photosynthesis, transpiration, and stomatal conductance after exposure to Al; it exhibited the highest Al content in the roots, and showed important and gene expression in the roots. RB928064 only showed low expression of in roots and leaves, while RB935744 showed important expression of the gene only in the leaves. Sugarcane cultivars were classified in the following descending Al-tolerance order: RB867515 > RB928064 = RB935744. These results may contribute to the obtention of Al-tolerant cultivars that can play their genetic potential in soils of low fertility and with low demand for agricultural inputs; the selection of potential plants for breeding programs; the elucidation of Al detoxification mechanisms employed by sugarcane cultivars.
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http://dx.doi.org/10.7717/peerj.11461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247702PMC
June 2021

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via β-Catenin involvement.

J Mater Sci Mater Med 2021 Mar 22;32(4):33. Epub 2021 Mar 22.

Department of Restorative Dentistry, School of Dentistry at Araraquara, Sao Paulo State University - UNESP, Araraquara, São Paulo, Brazil.

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.
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http://dx.doi.org/10.1007/s10856-021-06504-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985097PMC
March 2021

hRNAi-mediated knock-down of Sphenophorus levis V-ATPase E in transgenic sugarcane (Saccharum spp interspecific hybrid) affects the insect growth and survival.

Plant Cell Rep 2021 Mar 3;40(3):507-516. Epub 2021 Jan 3.

Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

Key Message: Transgenic sugarcane expressing V-ATPase subunit E dsRNA affects growth and survival of Sphenophorus levis. Plants being sessile organisms are constantly confronted with several biotic and abiotic stresses. Sugarcane (Saccharum spp) is a major tropical crop widely cultivated for its sugar and other by-products. In Brazil, sugarcane plantations account for significant production losses due to Sphenophorus levis (sugarcane weevil) infestations. With the existing control measures being less effective, there arises a necessity for advanced strategies. Our bioassay injection experiments with V-ATPase E dsRNA in S. levis larvae showed significant mortality and reduction in transcription levels. Furthermore, we down-regulated the V-ATPase E gene of S. levis in transgenic sugarcane using an RNAi approach. The resultant RNAi transgenic lines exhibited reduction in larval growth and survival, without compromising plant performance under controlled environment. Our results illustrate that RNAi-mediated down-regulation of key genes is a promising approach in imparting resistance to sugarcane weevil.
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http://dx.doi.org/10.1007/s00299-020-02646-5DOI Listing
March 2021

Sugarcane cystatins: From discovery to biotechnological applications.

Int J Biol Macromol 2021 Jan 4;167:676-686. Epub 2020 Dec 4.

Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil. Electronic address:

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.11.185DOI Listing
January 2021

Gene expression studies and molecular characterization of a cathepsin L-like from the Asian citrus psyllid Diaphorina citri, vector of Huanglongbing.

Int J Biol Macromol 2020 Apr 30;158:375-383. Epub 2020 Apr 30.

Departamento de Biofísica, Universidade Federal de São Paulo, 04039-032 São Paulo, Brazil. Electronic address:

Huanglongbing (HLB) is a devastating citrus disease associated with Candidatus Liberibacter asiaticus (CLas) and is transmitted by the psyllid Diaphorina citri Kuwayama. Diaphorina citri belongs to Hemiptera order, which has cysteine peptidases as the most abundant proteolytic enzymes present in digestive tract. As cysteine peptidases are involved in different insect development processes, this class of enzymes has acquired biotechnological importance. In this context, we identified a cathepsin L-like (DCcathL1) from the Diaphorina citri transcriptome database and expressed the enzyme in E. coli. Quantitative real-time RT-PCR was conducted to determine DCcathL1 gene expression in different parts and developmental phases of the insect. We observed that DCcathL1 expression in the gut was 2.59 and 2.87-fold higher than in the head and carcass, respectively. Furthermore, DCcathL1 expression was greater in eggs than in nymphs and adults, suggesting a putative role of the enzyme in the embryonic development. In addition, enzymatic inhibitory activity using four recombinant Citrus cystatins were performed. Among them, CsinCPI-2 was the strongest DCcathL1 inhibitor with a K value of 0.005 nM. Our results may contribute in the development of strategies for D. citri control, such as silencing the DCcathL1 gene and the use of transgenic plants that overexpress peptidase inhibitors.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.04.070DOI Listing
April 2020

Recombinant expression, characterization and phylogenetic studies of novels cystatins-like proteins of sweet orange (Citrus sinensis) and clementine (Citrus clementina).

Int J Biol Macromol 2020 Jun 25;152:546-553. Epub 2020 Feb 25.

Departamento de Genética e Evolução, Universidade Federal de São Carlos, São Paulo, Brazil. Electronic address:

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.02.280DOI Listing
June 2020

In vivo and in vitro anti-inflammatory and pro-osteogenic effects of citrus cystatin CsinCPI-2.

Cytokine 2019 11 18;123:154760. Epub 2019 Jun 18.

Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Sao Paulo State University - UNESP, Araraquara, São Paulo, Brazil. Electronic address:

Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (p < 0.05) on cathepsin K, cathepsin B, IL-1β, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (p < 0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
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http://dx.doi.org/10.1016/j.cyto.2019.154760DOI Listing
November 2019

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4.

Parasitol Int 2018 Apr 27;67(2):233-236. Epub 2017 Dec 27.

Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil. Electronic address:

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.
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http://dx.doi.org/10.1016/j.parint.2017.12.005DOI Listing
April 2018

A transcriptomic survey of Migdolus fryanus (sugarcane rhizome borer) larvae.

PLoS One 2017 1;12(3):e0173059. Epub 2017 Mar 1.

Department of Genetics and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, São Carlos, Brazil.

Sugarcane, a major crop grown in the tropical and subtropical areas of the world, is produced mainly for sucrose, which is used as a sweetener or for the production of bioethanol. Among the numerous pests that significantly affect the yield of sugarcane, the sugarcane rhizome borer (Migdolus fryanus, a cerambycidae beetle) is known to cause severe damage to the crops in Brazil. The absence of molecular information about this insect reinforces the need for studies and an effective method to control this pest. In this study, RNA-Seq technology was employed to study different parts of M. fryanus larvae. The generated data will help in further investigations about the taxonomy, development, and adaptation of this insect. RNA was extracted from six different parts (head, fat body, integument, hindgut, midgut, and foregut) using Trizol methodology. Using Illumina paired-end sequencing technology and the Trinity platform, trimming and de novo assembly was performed, resulting in 44,567 contigs longer than 200 nt for a reunion of data from all transcriptomes, with a mean length of 1,095.27 nt. Transcripts were annotated using BLAST against different protein databanks (Uniprot/Swissprot, PFAM, KEEG, SignalP 4.1, Gene Ontology, and CAZY) and were compared for similarity using a Venn diagram. Differential expression patterns were studied for select genes through qPCR and FPKM comprising important protein families (digestive peptidases, glucosyl hydrolases, serine protease inhibitors and otopetrin), which allowed a better understanding of the insect's digestion, immunity and gravity sensorial mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173059PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332103PMC
August 2017

Metagenome Sequencing of Prokaryotic Microbiota Collected from Rivers in the Upper Amazon Basin.

Genome Announc 2017 Jan 12;5(2). Epub 2017 Jan 12.

Department of Genetics and Evolution, Center for Biological and Health Sciences, Federal University of São Carlos, São Carlos, São Paulo, Brazil

Tropical freshwater environments, like rivers, are important reservoirs of microbial life. This study employed metagenomic sequencing to survey prokaryotic microbiota in the Solimões, Purus, and Urucu Rivers of the Amazon Basin in Brazil. We report a rich and diverse microbial community.
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http://dx.doi.org/10.1128/genomeA.01450-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256208PMC
January 2017

Metagenomics Analysis of Microorganisms in Freshwater Lakes of the Amazon Basin.

Genome Announc 2016 Dec 22;4(6). Epub 2016 Dec 22.

Department of Genetics and Evolution, Center for Biological and Health Sciences, Federal University of São Carlos, São Carlos, São Paulo, Brazil

The Amazon Basin is the largest hydrographic basin on the planet, and the dynamics of its aquatic microorganisms strongly impact global biogeochemical cycles. However, it remains poorly studied. This metagenome project was performed to obtain a snapshot of prokaryotic microbiota from four important lakes in the Amazon Basin.
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http://dx.doi.org/10.1128/genomeA.01440-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180393PMC
December 2016

Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

Plant Cell Rep 2017 Jan 11;36(1):193-201. Epub 2016 Nov 11.

Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washigton Luis, Km 235, São Carlos, SP, 13565-905, Brazil.

Key Message: Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.
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http://dx.doi.org/10.1007/s00299-016-2071-2DOI Listing
January 2017

Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

PLoS One 2015 30;10(12):e0145132. Epub 2015 Dec 30.

Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145132PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696824PMC
June 2016

Industrial PE-2 strain of Saccharomyces cerevisiae: from alcoholic fermentation to the production of recombinant proteins.

N Biotechnol 2014 Jan 4;31(1):90-7. Epub 2013 Sep 4.

Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luis km 235, 13565-905 São Carlos, SP, Brazil. Electronic address:

Saccharomyces cerevisiae is the most important microorganism used in the ethanol fermentation process. The PE-2 strain of this yeast is widely used to produce alcohol in Brazil due to its high fermentation capacity. The aim of the present study was to develop an expression system for recombinant proteins using the industrial PE-2 strain of S. cerevisiae during the alcoholic fermentation process. The protein chosen as a model for this system was CaneCPI-1, a cysteine peptidase inhibitor. A plasmid containing the CaneCPI-1 gene was constructed and yeast cells were transformed with the pYADE4_CaneCPI-1 construct. To evaluate the effect on fermentation ability, the transformed strain was used in the fermentation process with cell recycling. During the nine-hour fermentative cycles the transformed strain did not have its viability and fermentation ability affected. In the last cycle, when the fermentation lasted longer, the protein was expressed probably at the expense of ethanol once the sugars were exhausted. The recombinant protein was expressed in yeast cells, purified and submitted to assays of activity that demonstrated its functionality. Thus, the industrial PE-2 strain of S. cerevisiae can be used as a viable system for protein expression and to produce alcohol simultaneously. The findings of the present study demonstrate the possibility of producing recombinant proteins with biotechnological applications during the ethanol fermentation process.
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http://dx.doi.org/10.1016/j.nbt.2013.08.005DOI Listing
January 2014

X-ray crystallography and NMR studies of domain-swapped canecystatin-1.

FEBS J 2013 Feb 11;280(4):1028-38. Epub 2013 Jan 11.

Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, Brazil.

The three-dimensional structure of canecystatin-1, a potent inhibitor of cysteine proteases from sugarcane (Saccharum officinarum), has been solved in two different crystal forms. In both cases, it is seen to exist as a domain-swapped dimer, the first such observation for a cystatin of plant origin. Size exclusion chromatography and multidimensional NMR spectroscopy show the dimer to be the dominant species in solution, despite the presence of a measurable quantity of monomer undergoing slow exchange. The latter is believed to be the active species, whereas the domain-swapped dimer is presumably inactive, as its first inhibitory loop has been extended to form part of a long β-strand that forms a double-helical coiled coil with its partner from the other monomer. A similar structure is observed in human cystatin C, but the spatial disposition of the two lobes of the dimer is rather different. Dimerization is presumably a mechanism by which canecystatin-1 can be kept inactive within the plant, avoiding the inhibition of endogenous proteases. The structure described here provides a platform for the rational design of specific cysteine protease inhibitors for biotechnological applications.
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http://dx.doi.org/10.1111/febs.12095DOI Listing
February 2013

Recombinant expression and biochemical characterization of sugarcane legumain.

Plant Physiol Biochem 2012 Aug 29;57:181-92. Epub 2012 May 29.

Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luís, São Carlos SP, Brazil.

Plant legumains, also termed vacuolar processing enzymes (VPEs), are cysteine peptidases that play key roles in plant development, senescence, programmed cell death and defense against pathogens. Despite the increasing number of reports on plant cysteine peptidases, including VPEs, the characterization of sugarcane VPEs and their inhibition by endogenous cystatins have not yet been described. This is the first report of the biochemical characterization of a sugarcane cysteine peptidase. In this work, a recombinant sugarcane legumain was expressed in Pichia pastoris and characterized. Kinetic studies of the recombinant CaneLEG revealed that this enzyme has the main characteristics of VPEs, such as self-activation and activity under acidic pH. CaneLEG activity was strongly inhibited when incubated with sugarcane cystatin 3 (CaneCPI-3). Quantitative analysis of CaneLEG and CaneCPI-3 gene expression indicated a tissue-specific expression pattern for both genes throughout sugarcane growth, with the strong accumulation of CaneLEG transcripts throughout the internode development. Furthermore, the CaneLEG and CaneCPI-3 genes exhibited up-regulation in plantlets treated with abscisic acid (ABA). These results suggest that CaneCPI-3 may be a potential endogenous inhibitor of CaneLEG and these genes may be involved in plant stress response mediated by ABA. Also, the expression analysis provides clues for the putative involvement of CaneLEG and CaneCPI-3 in sugarcane development and phytohormone response.
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http://dx.doi.org/10.1016/j.plaphy.2012.05.020DOI Listing
August 2012

Production of a His-tagged canecystatin in transgenic sugarcane.

Methods Mol Biol 2012 ;847:437-50

Laboratory of Molecular Biology, Department of Genetic and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

Transgenic plants have been widely used as expression systems of recombinant proteins in recent years because it can be an efficient alternative for the large-scale production of proteins. This is an area with great potential but is still not much explored. Indeed, this system can bring a breakthrough in the expression of any protein. The model used here as a protein factory was sugarcane, a crop of great global importance. This chapter describes the system that has been adopted in the routine production of transgenic sugarcane coupled with protein purification protocol. In this chapter, we describe production of transgenic sugarcane expressing a His-tagged cystatin under the control of the maize ubiquitin promoter. A transformed sugarcane plant presented high levels of protein expression and was selected for the purification of this protein through affinity chromatography in a nickel column. These studies demonstrate that sugarcane can be a viable expression system for recombinant protein production and that the His-tag purification strategy used to isolate the purified protein was effective.
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http://dx.doi.org/10.1007/978-1-61779-558-9_34DOI Listing
July 2012

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis.

Insect Biochem Mol Biol 2012 Jan 12;42(1):58-69. Epub 2011 Nov 12.

Department of Genetics and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, 13565-905 São Carlos, Brazil.

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37°C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K(m)=37.53 mMS(-1)), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K(i)=0.196 nM), indicating that this protease is a potential target for pest control.
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http://dx.doi.org/10.1016/j.ibmb.2011.10.008DOI Listing
January 2012

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis.

J Insect Physiol 2011 Apr 20;57(4):462-8. Epub 2011 Jan 20.

Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luis Km 235, São Carlos, 13565-905 São Paulo, Brazil.

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0±1.1 μM(-1)s(-1) and 30.0±0.5 μM(-1)s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.
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http://dx.doi.org/10.1016/j.jinsphys.2011.01.006DOI Listing
April 2011

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling.

BMC Struct Biol 2010 Sep 30;10:30. Epub 2010 Sep 30.

Center for Structural Molecular Biotechnology, Department of Physics and Informatics, Physics Institute of São Carlos, University of São Paulo, Av, Trabalhador são-carlense 400, 13560-970, São Carlos-SP, Brazil.

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis.

Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability.

Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.
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http://dx.doi.org/10.1186/1472-6807-10-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2959088PMC
September 2010

DNA as genetic material: Revisiting classic experiments through a simple, practical class.

Biochem Mol Biol Educ 2009 Sep;37(5):290-5

Department of Genetics and Evolution, Federal University of São Carlos, Rodovia Washington Luis, Km 235, São Carlos, Sao Paulo, Brazil.

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http://dx.doi.org/10.1002/bmb.20309DOI Listing
September 2009

Production of a His-tagged canecystatin in transgenic sugarcane and subsequent purification.

Biotechnol Prog 2008 Sep-Oct;24(5):1060-6

Dept of Genetic and Evolution, Laboratory of Molecular Biology, Federal University of São Carlos, São Carlos, Brazil.

Transgenic plants have been used widely as expression systems of recombinant proteins in recent years. This process can be an efficient alternative for the large-scale production of proteins. In this work, we present the establishment of transgenic sugarcane expressing a His-tagged canecystatin under the control of the maize ubiquitin promoter. A number of studies have demonstrated that cystatins, which are natural inhibitors of cysteine proteinases, can be used for protection against insect attacks. A transformed sugarcane plant that presented high levels of (HIS)CaneCPI-1 expression, was selected for the purification of this protein through affinity chromatography in a nickel column. This purified (HIS)CaneCPI-1 was immunodetected using a polyclonal antibody, which was also able to detect the (HIS)CaneCPI-1 in a crude extract from transgenic plant leaves. Assays of inhibitory activity performed with the purified (HIS)CaneCPI-1 revealed its ability to inhibit the catalytic activity of midgut cysteine proteinase partially purified from the sugarcane weevil Sphenophorus levis and human cathepsin L in nanomolar order. These studies demonstrate that sugarcane is a viable expression system for recombinant protein production.
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http://dx.doi.org/10.1002/btpr.45DOI Listing
April 2009

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum).

Protein Expr Purif 2006 Jun 17;47(2):483-9. Epub 2005 Nov 17.

Laboratório de Biologia Molecular, DGE, Universidade Federal de São Carlos, Rodovia Washington Luis km 235, SP, Brazil.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.
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http://dx.doi.org/10.1016/j.pep.2005.10.026DOI Listing
June 2006

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity.

Biochim Biophys Acta 2006 Jan 11;1764(1):146-52. Epub 2005 Oct 11.

Institute of Physics of São Carlos, University of São Paulo, São Paulo, Brazil.

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).
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http://dx.doi.org/10.1016/j.bbapap.2005.09.011DOI Listing
January 2006

Gene expression profile of human Down syndrome leukocytes.

Croat Med J 2005 Aug;46(4):647-56

Department of Genetics and Evolution, Federal University of Sao Carlos, Rodovia Washington Luis, Km 235, CEP 13565-905, Sao Carlos, SP, Brazil.

Aim: Identification of differences in the gene expression patterns of Down syndrome and normal leukocytes.

Methods: We constructed the first Down syndrome leukocyte serial analysis of gene expression (SAGE) library from a 28 year-old patient. This library was analyzed and compared with a normal leukocyte SAGE library using the eSAGE software. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to validate the results.

Results: We found that a large number of unidentified transcripts were overexpressed in Down syndrome leukocytes and some transcripts coding for growth factors (e.g. interleukin 8, IL-8), ribosomaproteins (e.g. L13a, L29, and L37), and transcription factors (e.g., Jun B, Jun D, and C/EBP beta) were underexpressed. The SAGE data were successfully validated for the genes IL-8, CXCR4, BCL2A1, L13a, L29, L37, and GTF3A using RT-PCR.

Conclusion: Our analysis identified significant changes in the expression pattern of Down syndrome leukocytes compared with normal ones, including key regulators of growth and proliferation, ribosomal proteins, and a large number of overexpressed transcripts that were not matched in UniGene clusters and that may represent novel genes related to Down syndrome. This study offers a new insight into transcriptional changes in Down syndrome leukocytes and indicates candidate genes for further investigations into the molecular mechanism of Down syndrome pathology.
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August 2005

A complete approach for recombinant protein expression training: From gene cloning to assessment of protein functionality*.

Biochem Mol Biol Educ 2005 Jan;33(1):34-40

Department of Genetics and Evolution, Federal University of São Carlos, São Carlos-SP, Brazil.

A practical course was given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering" at the Federal University of São Carlos (UFSCar), São Paulo, Brazil. The goal of the course was to teach current molecular biology tools applied to a real research situation that could be reported by the students themselves. The purpose was to produce a plant recombinant protein and demonstrate a heretofore unreported biological activity. Cystatins, natural inhibitors of cysteine proteases, were proposed for these studies. Initially, the students searched for plant cystatin cDNA sequences in the NCBI databases and selected the Oryzacystatin I gene (ocI) from rice, Oriza sativa, as the target gene for this study. Total RNA was extracted from rice-germinating seeds and primers containing restriction sites for NdeI and EcoRI were designed based on the ocI cDNA sequence and then used to amplify the open reading frame (ORF). RT-PCR amplification provided a band of the expected size for ocI ORF (309 bp). The PCR product was cut with NdeI and EcoRI restriction enzymes and cloned directly in the pET28a expression vector digested with the same enzymes. A pET28-ocI recombinant clone was selected, checked by sequencing, and used to transform Escherichia coli BL21 (DE3) expression strain. After induction of the bacteria with isopropylthiogalactoside and cellular disruption, the His-tagged OCI protein, present mainly in the soluble fraction, was purified by affinity chromatography in a nickel column. The purified protein was successfully used to inhibit fungal growth (Trichoderma reesei). The results were discussed extensively and the students contributed to the writing of this article, of which they are co-authors.
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http://dx.doi.org/10.1002/bmb.2005.494033010418DOI Listing
January 2005

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane.

Biochem Biophys Res Commun 2004 Aug;320(4):1082-6

Department of Biochemistry, Universidade Federal de São Paulo-Escola Paulista de Medicina, Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brazil.

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.
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http://dx.doi.org/10.1016/j.bbrc.2004.06.053DOI Listing
August 2004

A transcript finishing initiative for closing gaps in the human transcriptome.

Genome Res 2004 Jul 14;14(7):1413-23. Epub 2004 Jun 14.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.
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http://dx.doi.org/10.1101/gr.2111304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC442158PMC
July 2004
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