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Daniele Chaves-Moreira
Perelman School of Medicine, University of Pennsylvania

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Preprint content
Published: 10 September 2020
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Worldwide, the number of new ovarian cancer cases approaches 300,000 with more than 180,000 deaths every year. The low survival-rate reflects the limitations of current therapies and highlights the importance of identifying new therapeutic targets. Despite significant recent efforts to identify novel vulnerabilities in ovarian cancer, none have led to effective durable therapies with improvement in overall survival. PAX8, a lineage-transcription factor, whose expression is a major molecular feature of ovarian carcinomas, represents a novel therapeutic target. Herein, we have identified SOX17 as a bona fide PAX8-interacting partner and elucidated the impact of this interaction on the development of ovarian cancer. Importantly, we found that PAX8 and SOX17 regulate tumor angiogenesis in vitro and in vivo. The role of PAX8 and SOX17 in the regulation of angiogenesis reveals a novel function for these factors in regulating the tumor microenvironment and highlight this pathway as a viable therapeutic target.

ACS Style

Daniele Chaves-Moreira; Marilyn A. Mitchell; Cristina Arruza; Priyanka Rawat; Simone Sidoli; Robbin Nameki; Jessica Reddy; Rosario I. Corona; Sisi Ma; Boris Winterhoff; Gottfried E. Konecny; Benjamin A. Garcia; Donita C. Brady; Kate Lawrenson; Patrice J. Morin; Ronny Drapkin. PAX8 orchestrates an angiogenic program through interaction with SOX17. 2020, 1 .

AMA Style

Daniele Chaves-Moreira, Marilyn A. Mitchell, Cristina Arruza, Priyanka Rawat, Simone Sidoli, Robbin Nameki, Jessica Reddy, Rosario I. Corona, Sisi Ma, Boris Winterhoff, Gottfried E. Konecny, Benjamin A. Garcia, Donita C. Brady, Kate Lawrenson, Patrice J. Morin, Ronny Drapkin. PAX8 orchestrates an angiogenic program through interaction with SOX17. . 2020; ():1.

Chicago/Turabian Style

Daniele Chaves-Moreira; Marilyn A. Mitchell; Cristina Arruza; Priyanka Rawat; Simone Sidoli; Robbin Nameki; Jessica Reddy; Rosario I. Corona; Sisi Ma; Boris Winterhoff; Gottfried E. Konecny; Benjamin A. Garcia; Donita C. Brady; Kate Lawrenson; Patrice J. Morin; Ronny Drapkin. 2020. "PAX8 orchestrates an angiogenic program through interaction with SOX17." , no. : 1.

Preprint content
Published: 12 November 2019
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The function of critical developmental regulators can be subverted by cancer cells to control expression of oncogenic transcriptional programs. These "master transcription factors" (MTFs) are often essential for cancer cell survival and represent vulnerabilities that can be exploited therapeutically. The current approaches to identify candidate MTFs examine super-enhancer associated transcription factor-encoding genes with high connectivity in network models. This relies on chromatin immunoprecipitation-sequencing (ChIP-seq) data, which is technically challenging to obtain from primary tumors, and is currently unavailable for many cancer types and clinically relevant subtypes. In contrast, gene expression data are more widely available, especially for rare tumors and subtypes where MTFs have yet to be discovered. We have developed a predictive algorithm called CaCTS (Cancer Core Transcription factor Specificity) to identify candidate MTFs using pan-cancer RNA-sequencing data from The Cancer Genome Atlas. The algorithm identified 273 candidate MTFs across 34 tumor types and recovered known tumor MTFs. We also made novel predictions, including for cancer types and subtypes for which MTFs have not yet been characterized. Clustering based on MTF predictions reproduced anatomic groupings of tumors that share 1-2 lineage-specific candidates, but also dictated functional groupings, such as a squamous group that comprised five tumor subtypes sharing 3 common MTFs. PAX8, SOX17, and MECOM were candidate factors in high-grade serous ovarian cancer (HGSOC), an aggressive tumor type where the core regulatory circuit is currently uncharacterized. PAX8, SOX17, and MECOM are required for cell viability and lie proximal to super-enhancers in HGSOC cells. ChIP-seq revealed that these factors co-occupy HGSOC regulatory elements globally and co-bind at critical gene loci including MUC16 (CA-125). Addiction to these factors was confirmed in studies using THZ1 to inhibit transcription in HGSOC cells, suggesting early down-regulation of these genes may be responsible for cytotoxic effects of THZ1 on HGSOC models. Identification of MTFs across 34 tumor types and 140 subtypes, especially for those with limited understanding of transcriptional drivers paves the way to therapeutic targeting of MTFs in a broad spectrum of cancers.

ACS Style

Jessica Reddy; Marcos A. S. Fonseca; Rosario I Corona; Robbin Nameki; Felipe Segato Dezem; Isaac A. Klein; Heidi Chang; Daniele Chaves-Moreira; Lena Afeyan; Tathiane M Malta; Xianzhi Lin; Forough Abbasi; Alba Font-Tello; Thais Sabedot; Paloma Cejas; Norma Rodríguez-Malavé; Ji-Heui Seo; De-Chen Lin; Ursula Matulonis; Beth Y Karlan; Simon A Gayther; Alexander Gusev; Houtan Noushmehr; Henry Long; Matthew L. Freedman; Ronny Drapkin; Brian J Abraham; Richard A. Young; Kate Lawrenson. Predicting master transcription factors from pan-cancer expression data. 2019, 839142 .

AMA Style

Jessica Reddy, Marcos A. S. Fonseca, Rosario I Corona, Robbin Nameki, Felipe Segato Dezem, Isaac A. Klein, Heidi Chang, Daniele Chaves-Moreira, Lena Afeyan, Tathiane M Malta, Xianzhi Lin, Forough Abbasi, Alba Font-Tello, Thais Sabedot, Paloma Cejas, Norma Rodríguez-Malavé, Ji-Heui Seo, De-Chen Lin, Ursula Matulonis, Beth Y Karlan, Simon A Gayther, Alexander Gusev, Houtan Noushmehr, Henry Long, Matthew L. Freedman, Ronny Drapkin, Brian J Abraham, Richard A. Young, Kate Lawrenson. Predicting master transcription factors from pan-cancer expression data. . 2019; ():839142.

Chicago/Turabian Style

Jessica Reddy; Marcos A. S. Fonseca; Rosario I Corona; Robbin Nameki; Felipe Segato Dezem; Isaac A. Klein; Heidi Chang; Daniele Chaves-Moreira; Lena Afeyan; Tathiane M Malta; Xianzhi Lin; Forough Abbasi; Alba Font-Tello; Thais Sabedot; Paloma Cejas; Norma Rodríguez-Malavé; Ji-Heui Seo; De-Chen Lin; Ursula Matulonis; Beth Y Karlan; Simon A Gayther; Alexander Gusev; Houtan Noushmehr; Henry Long; Matthew L. Freedman; Ronny Drapkin; Brian J Abraham; Richard A. Young; Kate Lawrenson. 2019. "Predicting master transcription factors from pan-cancer expression data." , no. : 839142.

Journal article
Published: 01 October 2019 in Toxicon
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ACS Style

Elidiana De Bona; Pedro Henrique De Caires Schluga; Daniele Moreira; Thiago Beltrami Dias Batista; Hanna Camara Da Justa; Fernando Hitomi Matsubara; Andrea Senff Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. A Novel Isoform of Hyaluronidase of Loxosceles intermedia venom: Production in Baculovirus-Insect Cell Expression System. Toxicon 2019, 168, S12 -S13.

AMA Style

Elidiana De Bona, Pedro Henrique De Caires Schluga, Daniele Moreira, Thiago Beltrami Dias Batista, Hanna Camara Da Justa, Fernando Hitomi Matsubara, Andrea Senff Ribeiro, Luiza Helena Gremski, Silvio Sanches Veiga. A Novel Isoform of Hyaluronidase of Loxosceles intermedia venom: Production in Baculovirus-Insect Cell Expression System. Toxicon. 2019; 168 ():S12-S13.

Chicago/Turabian Style

Elidiana De Bona; Pedro Henrique De Caires Schluga; Daniele Moreira; Thiago Beltrami Dias Batista; Hanna Camara Da Justa; Fernando Hitomi Matsubara; Andrea Senff Ribeiro; Luiza Helena Gremski; Silvio Sanches Veiga. 2019. "A Novel Isoform of Hyaluronidase of Loxosceles intermedia venom: Production in Baculovirus-Insect Cell Expression System." Toxicon 168, no. : S12-S13.

Review
Published: 19 June 2019 in Toxins
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Brown spider envenomation results in dermonecrosis with gravitational spreading characterized by a marked inflammatory reaction and with lower prevalence of systemic manifestations such as renal failure and hematological disturbances. Several toxins make up the venom of these species, and they are mainly peptides and proteins ranging from 5-40 kDa. The venoms have three major families of toxins: phospholipases-D, astacin-like metalloproteases, and the inhibitor cystine knot (ICK) peptides. Serine proteases, serpins, hyaluronidases, venom allergens, and a translationally controlled tumor protein (TCTP) are also present. Toxins hold essential biological properties that enable interactions with a range of distinct molecular targets. Therefore, the application of toxins as research tools and clinical products motivates repurposing their uses of interest. This review aims to discuss possibilities for brown spider venom toxins as putative models for designing molecules likely for therapeutics based on the status quo of brown spider venoms. Herein, we explore new possibilities for the venom components in the context of their biochemical and biological features, likewise their cellular targets, three-dimensional structures, and mechanisms of action.

ACS Style

Daniele Moreira; Fernando Hitomi Matsubara; Zelinda Schemczssen-Graeff; Elidiana De Bona; Vanessa Ribeiro Heidemann; Clara Guerra-Duarte; Luiza Helena Gremski; Carlos Chávez-Olórtegui; Andrea Senff-Ribeiro; Olga Meiri Chaim; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics. Toxins 2019, 11, 355 .

AMA Style

Daniele Moreira, Fernando Hitomi Matsubara, Zelinda Schemczssen-Graeff, Elidiana De Bona, Vanessa Ribeiro Heidemann, Clara Guerra-Duarte, Luiza Helena Gremski, Carlos Chávez-Olórtegui, Andrea Senff-Ribeiro, Olga Meiri Chaim, Raghuvir Krishnaswamy Arni, Silvio Sanches Veiga. Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics. Toxins. 2019; 11 (6):355.

Chicago/Turabian Style

Daniele Moreira; Fernando Hitomi Matsubara; Zelinda Schemczssen-Graeff; Elidiana De Bona; Vanessa Ribeiro Heidemann; Clara Guerra-Duarte; Luiza Helena Gremski; Carlos Chávez-Olórtegui; Andrea Senff-Ribeiro; Olga Meiri Chaim; Raghuvir Krishnaswamy Arni; Silvio Sanches Veiga. 2019. "Brown Spider (Loxosceles) Venom Toxins as Potential Biotools for the Development of Novel Therapeutics." Toxins 11, no. 6: 355.

Meeting report
Published: 01 July 2018 in Molecular and Cellular Biology
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A central problem in the treatment of ovarian cancer remains the heterogeneity among ovarian tumors. DNA and RNA sequencing studies have demonstrated both intertumoral and intratumoral genetic variation. Despite efforts to elucidate common signaling pathways among various ovarian cancer subtypes, few have led to meaningful patient stratification or to truly individualized targeted molecular therapies. Our proposed set of experiments is a radical departure from the conventional approach to treating ovarian cancer. PAX8, a transcription factor that identifies nearly all high-grade serous ovarian cancers (HGSOCs), is also the master regulator of fallopian tube development. Building off observations pointing to the fallopian tube epithelium as a major site of origin for HGSOCs, we propose targeting the fallopian tube developmental program as the basis for new therapies. This possibility is supported by the observation that knockdown of PAX8 leads to apoptosis in ovarian cancer cells. We hypothesize that blocking the ability of PAX8 to influence its gene targets, either by interrupting PAX8 protein-protein interactions or by inhibiting the products of PAX8-driven signaling, has the potential to eliminate the primary growth stimulus for high-grade serous tumors. In order to achieve that goal, we have identified candidate PAX8-interacting partners using gel filtration chromatography and tandem-affinity immunopurification coupled with mass spectrometry analyses. Interactions have been validated by Western blot, immunofluorescence, and proximity ligation assay. We used ovarian cancer (KURAMOCHI, OVSAHO and OVCAR4) and fallopian tube (FT189, FT194 and FT246) cell lines to determine whether PAX8 protein partners changed between benign and malignant cells. Our preliminary results suggest that PAX8 interacts with other transcription factors such as TEAD4 and components of chromatin-remodeling complexes (RUVBL1, RUVBL2 and CHD4). Many of these factors are known to mediate the effects of oncogenes such as YAP, β-catenin and c-Myc. Further studies will be done in order to decipher the functional contribution of each candidate PAX8-interacting protein. Our ultimate goal is to determine how PAX8 regulates expression of its target genes and how PAX8-interacting proteins influence that function. Inhibition of certain protein-protein interactions may provide novel avenues to abrogate PAX8 function in ovarian cancer cells. Citation Format: Daniele Chaves-Moreira, Marilyn Mitchell, Simone Sidoli, Benjamin Garcia, Ronny Drapkin. Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5416.

ACS Style

Daniele Moreira; Marilyn Mitchell; Simone Sidoli; Benjamin Garcia; Ronny Drapkin. Abstract 5416: Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer. Molecular and Cellular Biology 2018, 78, 5416 -5416.

AMA Style

Daniele Moreira, Marilyn Mitchell, Simone Sidoli, Benjamin Garcia, Ronny Drapkin. Abstract 5416: Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer. Molecular and Cellular Biology. 2018; 78 ():5416-5416.

Chicago/Turabian Style

Daniele Moreira; Marilyn Mitchell; Simone Sidoli; Benjamin Garcia; Ronny Drapkin. 2018. "Abstract 5416: Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer." Molecular and Cellular Biology 78, no. : 5416-5416.

Comparative study
Published: 25 April 2017 in Journal of Cellular Biochemistry
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Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from L. intermedia and C. pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. This article is protected by copyright. All rights reserved

ACS Style

Ricardo Mariutti; Daniele Moreira; Larissa Vuitika; Icaro Caruso; Monika A. Coronado; Vasco A. Azevedo; Mario T. Murakami; Silvio Sanches Veiga; Raghuvir K. Arni. Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities. Journal of Cellular Biochemistry 2017, 118, 2053 -2063.

AMA Style

Ricardo Mariutti, Daniele Moreira, Larissa Vuitika, Icaro Caruso, Monika A. Coronado, Vasco A. Azevedo, Mario T. Murakami, Silvio Sanches Veiga, Raghuvir K. Arni. Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities. Journal of Cellular Biochemistry. 2017; 118 (8):2053-2063.

Chicago/Turabian Style

Ricardo Mariutti; Daniele Moreira; Larissa Vuitika; Icaro Caruso; Monika A. Coronado; Vasco A. Azevedo; Mario T. Murakami; Silvio Sanches Veiga; Raghuvir K. Arni. 2017. "Bacterial and Arachnid Sphingomyelinases D: Comparison of Biophysical and Pathological Activities." Journal of Cellular Biochemistry 118, no. 8: 2053-2063.

Review
Published: 08 February 2017 in Journal of Venomous Animals and Toxins including Tropical Diseases
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Brown spiders are venomous arthropods that use their venom for predation and defense. In humans, bites of these animals provoke injuries including dermonecrosis with gravitational spread of lesions, hematological abnormalities and impaired renal function. The signs and symptoms observed following a brown spider bite are called loxoscelism. Brown spider venom is a complex mixture of toxins enriched in low molecular mass proteins (4-40 kDa). Characterization of the venom confirmed the presence of three highly expressed protein classes: phospholipases D, metalloproteases (astacins) and insecticidal peptides (knottins). Recently, toxins with low levels of expression have also been found in Loxosceles venom, such as serine proteases, protease inhibitors (serpins), hyaluronidases, allergen-like toxins and histamine-releasing factors. The toxin belonging to the phospholipase-D family (also known as the dermonecrotic toxin) is the most studied class of brown spider toxins. This class of toxins single-handedly can induce inflammatory response, dermonecrosis, hemolysis, thrombocytopenia and renal failure. The functional role of the hyaluronidase toxin as a spreading factor in loxoscelism has also been demonstrated. However, the biological characterization of other toxins remains unclear and the mechanism by which Loxosceles toxins exert their noxious effects is yet to be fully elucidated. The aim of this review is to provide an insight into brown spider venom toxins and toxicology, including a description of historical data already available in the literature. In this review article, the identification processes of novel Loxosceles toxins by molecular biology and proteomic approaches, their biological characterization and structural description based on x-ray crystallography and putative biotechnological uses are described along with the future perspectives in this field.

ACS Style

Daniele Chaves-Moreira; Andrea Senff-Ribeiro; Ana Carolina Martins Wille; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga. Highlights in the knowledge of brown spider toxins. Journal of Venomous Animals and Toxins including Tropical Diseases 2017, 23, 6 .

AMA Style

Daniele Chaves-Moreira, Andrea Senff-Ribeiro, Ana Carolina Martins Wille, Luiza Helena Gremski, Olga Meiri Chaim, Silvio Sanches Veiga. Highlights in the knowledge of brown spider toxins. Journal of Venomous Animals and Toxins including Tropical Diseases. 2017; 23 (1):6.

Chicago/Turabian Style

Daniele Chaves-Moreira; Andrea Senff-Ribeiro; Ana Carolina Martins Wille; Luiza Helena Gremski; Olga Meiri Chaim; Silvio Sanches Veiga. 2017. "Highlights in the knowledge of brown spider toxins." Journal of Venomous Animals and Toxins including Tropical Diseases 23, no. 1: 6.

Article
Published: 26 October 2016 in Journal of Cellular Biochemistry
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Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 9999: 1–13, 2016.

ACS Style

Daniele Moreira; Fabio Rogerio de Moraes; Icaro Caruso; Olga Chaim; Andrea Senff-Ribeiro; Anwar Ullah; Luciane Sussuchi da Silva; Jorge Chahine; Raghuvir Arni; Silvio Sanches Veiga. Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D. Journal of Cellular Biochemistry 2016, 118, 726 -738.

AMA Style

Daniele Moreira, Fabio Rogerio de Moraes, Icaro Caruso, Olga Chaim, Andrea Senff-Ribeiro, Anwar Ullah, Luciane Sussuchi da Silva, Jorge Chahine, Raghuvir Arni, Silvio Sanches Veiga. Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D. Journal of Cellular Biochemistry. 2016; 118 (4):726-738.

Chicago/Turabian Style

Daniele Moreira; Fabio Rogerio de Moraes; Icaro Caruso; Olga Chaim; Andrea Senff-Ribeiro; Anwar Ullah; Luciane Sussuchi da Silva; Jorge Chahine; Raghuvir Arni; Silvio Sanches Veiga. 2016. "Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D." Journal of Cellular Biochemistry 118, no. 4: 726-738.

Journal article
Published: 01 September 2016 in Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
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Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme’s ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

ACS Style

L. Vuitika; Daniele Moreira; Icaro Caruso; M.A. Lima; F.H. Matsubara; M.T. Murakami; H.K. Takahashi; Marcos Toledo; M.A. Coronado; Helena Nader; Andrea Senff-Ribeiro; Olga Chaim; Raghuvir Arni; S.S. Veiga. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2016, 1861, 970 -979.

AMA Style

L. Vuitika, Daniele Moreira, Icaro Caruso, M.A. Lima, F.H. Matsubara, M.T. Murakami, H.K. Takahashi, Marcos Toledo, M.A. Coronado, Helena Nader, Andrea Senff-Ribeiro, Olga Chaim, Raghuvir Arni, S.S. Veiga. Active site mapping of Loxosceles phospholipases D: Biochemical and biological features. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 2016; 1861 (9):970-979.

Chicago/Turabian Style

L. Vuitika; Daniele Moreira; Icaro Caruso; M.A. Lima; F.H. Matsubara; M.T. Murakami; H.K. Takahashi; Marcos Toledo; M.A. Coronado; Helena Nader; Andrea Senff-Ribeiro; Olga Chaim; Raghuvir Arni; S.S. Veiga. 2016. "Active site mapping of Loxosceles phospholipases D: Biochemical and biological features." Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1861, no. 9: 970-979.

Journal article
Published: 03 August 2016 in Journal of Molecular Modeling
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Inhibitor cystine knots (ICKs) are a family of structural peptides with a large number of cysteine residues that form intramolecular disulfide bonds, resulting in a knot. These peptides are involved in a variety of biological functions including predation and defense, and are found in various species, such as spiders, scorpions, sea anemones, and plants. The Loxosceles intermedia venom gland transcriptome identified five groups of ICK peptides that represent more than 50 % of toxin-coding transcripts. Here, we describe the molecular cloning of U2-Sicaritoxin-Lit2 (U2-SCRTX-Lit2), bioinformatic characterization, structure prediction, and molecular dynamic analysis. The sequence of U2-SCRTX-Lit2 obtained from the transcriptome is similar to that of μ-Hexatoxin-Mg2, a peptide that inhibits the insect Nav channel. Bioinformatic analysis of sequences classified as ICK family members also showed a conservation of cysteine residues among ICKs from different spiders, with the three dimensional molecular model of U2-SCRTX-Lit2 similar in structure to the hexatoxin from μ-hexatoxin-Mg2a. Molecular docking experiments showed the interaction of U2-SCRTX-Lit2 to its predictable target-the Spodoptera litura voltage-gated sodium channel (SlNaVSC). After 200 ns of molecular dynamic simulation, the final structure of the complex showed stability in agreement with the experimental data. The above analysis corroborates the existence of a peptide toxin with insecticidal activity from a novel ICK family in L. intermedia venom and demonstrates that this peptide targets Nav channels.

ACS Style

Gabriel Meissner; Pedro Túlio De Resende Lara; Luis Paulo Barbour Scott; Antônio Sérgio Kimus Braz; Daniele Chaves-Moreira; Fernando Hitomi Matsubara; Eduardo Mendonça Soares; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio. Sanches Veiga; Olga Meiri Chaim. Molecular cloning and in silico characterization of knottin peptide, U2-SCRTX-Lit2, from brown spider (Loxosceles intermedia) venom glands. Journal of Molecular Modeling 2016, 22, 1 -13.

AMA Style

Gabriel Meissner, Pedro Túlio De Resende Lara, Luis Paulo Barbour Scott, Antônio Sérgio Kimus Braz, Daniele Chaves-Moreira, Fernando Hitomi Matsubara, Eduardo Mendonça Soares, Dilza Trevisan-Silva, Luiza Helena Gremski, Silvio. Sanches Veiga, Olga Meiri Chaim. Molecular cloning and in silico characterization of knottin peptide, U2-SCRTX-Lit2, from brown spider (Loxosceles intermedia) venom glands. Journal of Molecular Modeling. 2016; 22 (9):1-13.

Chicago/Turabian Style

Gabriel Meissner; Pedro Túlio De Resende Lara; Luis Paulo Barbour Scott; Antônio Sérgio Kimus Braz; Daniele Chaves-Moreira; Fernando Hitomi Matsubara; Eduardo Mendonça Soares; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio. Sanches Veiga; Olga Meiri Chaim. 2016. "Molecular cloning and in silico characterization of knottin peptide, U2-SCRTX-Lit2, from brown spider (Loxosceles intermedia) venom glands." Journal of Molecular Modeling 22, no. 9: 1-13.

Book chapter
Published: 06 January 2016 in Venom Genomics and Proteomics
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Brown spiders (Loxosceles genus) are venomous arthropods that use venom for predation and defense. These spiders have also been associated with human accidents, and the primary clinical manifestations are dermonecrosis with gravitational lesion spreading, hematological disturbances, and acute renal failure. Loxosceles venom comprises a complex mixture of toxins enriched in low molecular mass proteins (5–40 kDa). Characterization of this venom revealed three highly expressed protein classes: phospholipase-D family proteins, astacin-like proteases, and inhibitor cystine knot (ICK) peptides. A recent study also showed the presence of several other venom proteins, such as serine proteases, protease inhibitors, hyaluronidases, allergen-like toxins, and translationally controlled tumor protein (TCTP), expressed at low levels in Loxosceles venom. The Brown spider phospholipase-D family proteins have been well studied, and these toxins alone induce inflammatory responses, dermonecrosis, hemolysis, thrombocytopenia, and renal failure. In addition, the functional role of hyaluronidases as spreading factors in loxoscelism has been demonstrated. However, the biological characterization of other toxins has not been reported. Nevertheless, the mechanism by which Loxosceles toxins exert noxious effects is not fully elucidated. The aim of this chapter is to provide insights into Brown spider toxins, including the identification of novel toxins using molecular and proteomics approaches, and the biological characterization and structural description of toxins using X-ray crystallography. Putative biotechnological uses of Loxosceles toxins and future perspectives in this field will also be discussed.

ACS Style

Daniele Chaves-Moreira; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio Sanches Veiga. Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins. Venom Genomics and Proteomics 2016, 125 -147.

AMA Style

Daniele Chaves-Moreira, Dilza Trevisan-Silva, Luiza Helena Gremski, Silvio Sanches Veiga. Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins. Venom Genomics and Proteomics. 2016; ():125-147.

Chicago/Turabian Style

Daniele Chaves-Moreira; Dilza Trevisan-Silva; Luiza Helena Gremski; Silvio Sanches Veiga. 2016. "Brown Spider Venom: The Identification and Biotechnological Potential of Venom Toxins." Venom Genomics and Proteomics , no. : 125-147.

Comparative study
Published: 01 December 2015 in Toxicon
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Loxosceles spiders are responsible for serious human envenomations worldwide. The collection of symptoms found in victims after accidents is called loxoscelism and is characterized by two clinical conditions: cutaneous loxoscelism and systemic loxocelism. The only specific treatment is serum therapy, in which an antiserum produced with Loxosceles venom is administered to the victims after spider accidents. Our aim was to improve our knowledge, regarding the immunological relationship among toxins from the most epidemiologic important species in Brazil (Loxosceles intermedia, Loxosceles gaucho and Loxosceles laeta). Immunoassays using spider venoms and L. intermedia recombinant toxins were performed and their cross-reactivity assessed. The biological conservation of the main Loxosceles toxins (Phospholipases-D, Astacin-like metalloproteases, Hyaluronidase, ICK-insecticide peptide and TCTP-histamine releasing factor) were investigated. An in silico analysis of the putative epitopes was performed and is discussed on the basis of the experimental results. Our data is an immunological investigation in light of biological conservation throughout the Loxosceles genus. The results bring out new insights on brown spider venom toxins for study, diagnosis and treatment of loxoscelism and putative biotechnological applications concerning immune conserved features in the toxins.

ACS Style

Daniela Regina Buch; Fernanda Nunes Souza; Gabriel Meissner; Adriano Marcelo Morgon; Luiza Gremski; Valéria Pereira Ferrer; Dilza Trevisan-Silva; Fernando Hitomi Matsubara; Marianna Boia Ferreira; Youssef Sade; Daniele Moreira; Waldemiro Gremski; Silvio Sanches Veiga; Olga Chaim; Andrea Senff-Ribeiro. Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity. Toxicon 2015, 108, 154 -166.

AMA Style

Daniela Regina Buch, Fernanda Nunes Souza, Gabriel Meissner, Adriano Marcelo Morgon, Luiza Gremski, Valéria Pereira Ferrer, Dilza Trevisan-Silva, Fernando Hitomi Matsubara, Marianna Boia Ferreira, Youssef Sade, Daniele Moreira, Waldemiro Gremski, Silvio Sanches Veiga, Olga Chaim, Andrea Senff-Ribeiro. Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity. Toxicon. 2015; 108 ():154-166.

Chicago/Turabian Style

Daniela Regina Buch; Fernanda Nunes Souza; Gabriel Meissner; Adriano Marcelo Morgon; Luiza Gremski; Valéria Pereira Ferrer; Dilza Trevisan-Silva; Fernando Hitomi Matsubara; Marianna Boia Ferreira; Youssef Sade; Daniele Moreira; Waldemiro Gremski; Silvio Sanches Veiga; Olga Chaim; Andrea Senff-Ribeiro. 2015. "Brown spider (Loxosceles genus) venom toxins: Evaluation of biological conservation by immune cross-reactivity." Toxicon 108, no. : 154-166.

Journal article
Published: 28 August 2015 in Current Protein & Peptide Science
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Phospholipases D (PLDs), the major dermonecrotic factors from brown spider venoms, trigger a range of biological reactions both in vitro and in vivo. Despite their clinical relevance in loxoscelism, structural data is restricted to the apo-form of these enzymes, which has been instrumental in understanding the functional differences between the class I and II spider PLDs. The crystal structures of the native class II PLD from Loxosceles intermedia complexed with myo-inositol 1-phosphate and the inactive mutant H12A complexed with fatty acids indicate the existence of a strong ligand-dependent conformation change of the highly conserved aromatic residues, Tyr 223 and Trp225 indicating their roles in substrate binding. These results provided insights into the structural determinants for substrate recognition and binding by class II PLDs.

ACS Style

M.A. Coronado; Anwar Ullah; Luciane Sussuchi da Silva; Daniele Moreira; L. Vuitika; Olga Chaim; S.S. Veiga; Jorge Chahine; M.T. Murakami; R.K. Arni. Structural Insights into Substrate Binding of Brown Spider Venom Class II Phospholipases D. Current Protein & Peptide Science 2015, 16, 768 -774.

AMA Style

M.A. Coronado, Anwar Ullah, Luciane Sussuchi da Silva, Daniele Moreira, L. Vuitika, Olga Chaim, S.S. Veiga, Jorge Chahine, M.T. Murakami, R.K. Arni. Structural Insights into Substrate Binding of Brown Spider Venom Class II Phospholipases D. Current Protein & Peptide Science. 2015; 16 (8):768-774.

Chicago/Turabian Style

M.A. Coronado; Anwar Ullah; Luciane Sussuchi da Silva; Daniele Moreira; L. Vuitika; Olga Chaim; S.S. Veiga; Jorge Chahine; M.T. Murakami; R.K. Arni. 2015. "Structural Insights into Substrate Binding of Brown Spider Venom Class II Phospholipases D." Current Protein & Peptide Science 16, no. 8: 768-774.

Meeting report
Published: 06 July 2015 in Molecular Regulation of the PI3K-mTOR Network
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Structural biology has yielded many significant insights into inhibitor binding and isoform selectivity, directing the design of many important clinical candidates. To date, structural studies have focused on the ATP-binding site and ATP-competitive inhibitors. We have recently determined the structure of PI3Kα; in complex with the soluble lipid substrate mimetic, diC4-PIP2. The substrate binds in a positively charged pocket, defined by the activation and P-loops of the kinase domain, and the iSH2 domain of p85. The positively charged residues responsible for binding PIP2 that are present on the activation loop of Class I PI3K are missing in Class II and III PI3K. This explains why these two classes have lower affinity for PIP2. The complex structure provides insights into the catalysis and regulation of PI3K. Key interactions between the activation loop and nSH2 domain may modulate active and inactive conformations of the enzyme upon the binding of phosphorylated receptor tyrosine kinases. In addition, a second lipid substrate was identified in the structure. It binds in a hydrophobic pocket between the Adaptor Binding Domain (ABD), kinase domain and iSH2 domain of p85. Fluorescence quenching studies confirm the ability of PI3K to bind an additional PIP2 molecule. This abstract is also being presented as Poster B17. Citation Format: Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Targeting PI3K: The PIP2 binding site. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr PR02.

ACS Style

Michelle Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Chaves-Moreira; Marc Allaire; Kenneth W. Kinzler; Ian G. Jennings; Philip E. Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. Abstract PR02: Targeting PI3K: The PIP2 binding site. Molecular Regulation of the PI3K-mTOR Network 2015, 14, PR02 -PR02.

AMA Style

Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Abstract PR02: Targeting PI3K: The PIP2 binding site. Molecular Regulation of the PI3K-mTOR Network. 2015; 14 (7):PR02-PR02.

Chicago/Turabian Style

Michelle Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Chaves-Moreira; Marc Allaire; Kenneth W. Kinzler; Ian G. Jennings; Philip E. Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. 2015. "Abstract PR02: Targeting PI3K: The PIP2 binding site." Molecular Regulation of the PI3K-mTOR Network 14, no. 7: PR02-PR02.

Conference paper
Published: 30 September 2014 in Cancer Chemistry
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PI3Kα is a well-established target for the development of novel cancer therapeutics. Currently, all inhibitors in clinical trials target the highly conserved ATP-binding site, which has made the development of PI3K selective inhibitors difficult, with many inhibitors displaying cross-reactivity against protein kinases. We have used X-ray crystallography and fluorescence quenching studies to characterize the lipid-binding site of PI3Kα and the structural basis of regulation by nSH2. A newly determined p110α/niSH2 crystal structure is the first to reveal the nSH2 domain in complex with wild-type p110α, allowing investigation of the mechanisms of nSH2 regulation. Key interactions between the nSH2 domain and the activation loop suggest a mechanism by which the kinase domain is kept in an inactive conformation until activation by phosphopeptide binding. Key differences in nSH2 domain binding to p110α were identified between the wild-type and oncogenic mutant, p110αH1047R. Increased buried surface area and two unique salt-bridges are suggestive of tighter regulatory control in the wild-type PI3Kα compared with the oncogenic mutant. A second structure reveals the details of PI3K binding to a lipid substrate mimetic, diC4-PIP2. Unexpectedly, we found an additional lipid-binding site, and this striking observation was confirmed by fluorescence quenching experiments. The identification of multiple lipid binding sites provides additional targets that may enable more selective inhibition among the various isoforms, or even between mutant and wild-type forms of PI3Kα. Citation Format: Michelle S. Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Structural basis of lipid-binding and regulation in PI3Kα. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-326. doi:10.1158/1538-7445.AM2014-LB-326

ACS Style

Michelle S. Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Chaves-Moreira; Marc Allaire; Kenneth W. Kinzler; Ian G. Jennings; Philip E. Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. Abstract LB-326: Structural basis of lipid-binding and regulation in PI3Kα. Cancer Chemistry 2014, 74, LB-326 -LB-326.

AMA Style

Michelle S. Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Chaves-Moreira, Marc Allaire, Kenneth W. Kinzler, Ian G. Jennings, Philip E. Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Abstract LB-326: Structural basis of lipid-binding and regulation in PI3Kα. Cancer Chemistry. 2014; 74 ():LB-326-LB-326.

Chicago/Turabian Style

Michelle S. Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Chaves-Moreira; Marc Allaire; Kenneth W. Kinzler; Ian G. Jennings; Philip E. Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. 2014. "Abstract LB-326: Structural basis of lipid-binding and regulation in PI3Kα." Cancer Chemistry 74, no. : LB-326-LB-326.

Journal article
Published: 25 July 2014 in Oncotarget
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We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

ACS Style

Michelle Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Moreira; Marc Allaire; Kenneth W. Kinzler; Ian Jennings; Philip Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. Structural basis of nSH2 regulation and lipid binding in PI3Kα. Oncotarget 2014, 5, 5198 -5208.

AMA Style

Michelle Miller, Oleg Schmidt-Kittler, David M. Bolduc, Evan T. Brower, Daniele Moreira, Marc Allaire, Kenneth W. Kinzler, Ian Jennings, Philip Thompson, Philip A. Cole, L. Mario Amzel, Bert Vogelstein, Sandra B. Gabelli. Structural basis of nSH2 regulation and lipid binding in PI3Kα. Oncotarget. 2014; 5 (14):5198-5208.

Chicago/Turabian Style

Michelle Miller; Oleg Schmidt-Kittler; David M. Bolduc; Evan T. Brower; Daniele Moreira; Marc Allaire; Kenneth W. Kinzler; Ian Jennings; Philip Thompson; Philip A. Cole; L. Mario Amzel; Bert Vogelstein; Sandra B. Gabelli. 2014. "Structural basis of nSH2 regulation and lipid binding in PI3Kα." Oncotarget 5, no. 14: 5198-5208.

Journal article
Published: 09 January 2014 in Biophysical Reviews
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PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP2) to phosphoinositide-3,4,5-trisphosphate (PIP3), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP2, a membrane lipid.

ACS Style

Sandra B. Gabelli; Ignacia Echeverria; Megan Alexander; Krisna Duong-Ly; Daniele Moreira; Evan T. Brower; B. Vogelstein; L. Mario Amzel. Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects. Biophysical Reviews 2014, 6, 89 -95.

AMA Style

Sandra B. Gabelli, Ignacia Echeverria, Megan Alexander, Krisna Duong-Ly, Daniele Moreira, Evan T. Brower, B. Vogelstein, L. Mario Amzel. Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects. Biophysical Reviews. 2014; 6 (1):89-95.

Chicago/Turabian Style

Sandra B. Gabelli; Ignacia Echeverria; Megan Alexander; Krisna Duong-Ly; Daniele Moreira; Evan T. Brower; B. Vogelstein; L. Mario Amzel. 2014. "Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects." Biophysical Reviews 6, no. 1: 89-95.

Journal article
Published: 13 September 2013 in Journal of Cellular Biochemistry
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Brown spider (Loxosceles genus) bites have been reported worldwide. The venom contains a complex composition of several toxins, including phospholipases-D. Native or recombinant phospholipase-D toxins induce cutaneous and systemic loxoscelism, particularly necrotic lesions, inflammatory response, renal failure, and hematological disturbances. Herein, we describe the cloning, heterologous expression and purification of a novel phospholipase-D toxin, LiRecDT7 in reference to six other previously described in phospholipase-D toxin family. The complete cDNA sequence of this novel brown spider phospholipase-D isoform was obtained and the calculated molecular mass of the predicted mature protein is 34.4 kDa. Similarity analyses revealed that LiRecDT7 is homologous to the other dermonecrotic toxin family members particularly to LiRecDT6, sharing 71% sequence identity. LiRecDT7 possesses the conserved amino acid residues involved in catalysis except for a conservative mutation (D233E) in the catalytic site. Purified LiRecDT7 was detected as a soluble 36 kDa protein using anti-whole venom and anti-LiRecDT1 sera, indicating immunological cross-reactivity and evidencing sequence-epitopes identities similar to those of other phospholipase-D family members. Also, LiRecDT7 exhibits sphingomyelinase activity in a concentration dependent-manner and induces experimental skin lesions with swelling, erythema and dermonecrosis. In addition, LiRecDT7 induced a massive inflammatory response in rabbit skin dermis, which is a hallmark of brown spider venom phospholipase-D toxins. Moreover, LiRecDT7 induced in vitro hemolysis in human erythrocytes and increased blood vessel permeability. These features suggest that this novel member of the brown spider venom phospholipase-D family, which naturally contains a mutation (D233E) in the catalytic site, could be useful for future structural and functional studies concerning loxoscelism and lipid biochemistry. 1- Novel brown spider phospholipase-D recombinant toxin contains a conservative mutation (D233E) on the catalytic site. 2-LiRecDT7 shares high identity level with isoforms of Loxosceles genus. 3-LiRecDT7 is a recombinant protein immunodetected by specific antibodies to native and recombinant phospholipase-D toxins. 4-LiRecDT7 shows sphingomyelinase-D activity in a concentration-dependent manner, but less intense than other isoforms. 5-LiRecDT7 induces dermonecrosis and inflammatory response in rabbit skin. 6-LiRecDT7 increases vascular permeability in mice. 7-LiRecDT7 triggers direct complement-independent hemolysis in erythrocytes.

ACS Style

Larissa Vuitika; Luiza Helena Gremski; Valéria Pereira Ferrer; Matheus Regis Belisário‐Ferrari; Daniele Chaves‐Moreira; Andrea Senff‐Ribeiro; Olga Meiri Chaim; Silvio Sanches Veiga. Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. Journal of Cellular Biochemistry 2013, 114, 2479 -2492.

AMA Style

Larissa Vuitika, Luiza Helena Gremski, Valéria Pereira Ferrer, Matheus Regis Belisário‐Ferrari, Daniele Chaves‐Moreira, Andrea Senff‐Ribeiro, Olga Meiri Chaim, Silvio Sanches Veiga. Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. Journal of Cellular Biochemistry. 2013; 114 (11):2479-2492.

Chicago/Turabian Style

Larissa Vuitika; Luiza Helena Gremski; Valéria Pereira Ferrer; Matheus Regis Belisário‐Ferrari; Daniele Chaves‐Moreira; Andrea Senff‐Ribeiro; Olga Meiri Chaim; Silvio Sanches Veiga. 2013. "Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization." Journal of Cellular Biochemistry 114, no. 11: 2479-2492.

Journal article
Published: 24 February 2013 in Toxicon
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The mechanism through which brown spiders (Loxosceles genus) cause dermonecrosis, dysregulated inflammatory responses, hemolysis and platelet aggregation, which are effects reported following spider bites, is currently attributed to the presence of phospholipase-D in the venom. In the present investigation, through two-dimensional immunoblotting, we observed immunological cross-reactivity for at least 25 spots in crude Loxosceles intermedia venom, indicating high expression levels for different isoforms of phospholipase-D. Using a recombinant phospholipase-D from the venom gland of L. intermedia (LiRecDT1) in phospholipid-degrading kinetic experiments, we determined that this phospholipase-D mainly hydrolyzes synthetic sphingomyelin in a time-dependent manner, generating ceramide 1-phosphate plus choline, as well as lysophosphatidylcholine, generating lysophosphatidic acid plus choline, but exhibits little activity against phosphatidylcholine. Through immunofluorescence assays with antibodies against LiRecDT1 and using a recombinant GFP-LiRecDT1 fusion protein, we observed direct binding of LiRecDT1 to the membrane of B16-F10 cells. We determined that LiRecDT1 hydrolyzes phospholipids in detergent extracts and from ghosts of B16-F10 cells, generating choline, indicating that the enzyme can access and modulate and has activity against membrane phospholipids. Additionally, using Fluo-4, a calcium-sensitive fluorophore, it was shown that treatment of cells with phospholipase-D induced an increase in the calcium concentration in the cytoplasm, but without altering viability or causing damage to cells. Finally, based on the known endogenous activity of phospholipase-D as an inducer of cell proliferation and the fact that LiRecDT1 binds to the cell surface, hydrolyzing phospholipids to generate bioactive lipids, we employed LiRecDT1 as an exogenous source of phospholipase-D in B16-F10 cells. Treatment of the cells was effective in increasing their proliferation in a time- and concentration-dependent manner, especially in the presence of synthetic sphingomyelin in the medium. The results described herein indicate the ability of brown spider phospholipase-D to induce the generation of bioactive phospholipids, calcium influx into the cytoplasm and cell proliferation, suggesting that this molecule can be used as a bioactive tool for experimental protocols in cell biology.

ACS Style

Ana Carolina Martins Wille; Daniele Moreira; Dilza Trevisan-Silva; Mariana Gabriel Magnoni; Marianna Boia Ferreira; Luiza Gremski; Waldemiro Gremski; Olga Chaim; Andrea Senff-Ribeiro; Silvio Sanches Veiga. Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom. Toxicon 2013, 67, 17 -30.

AMA Style

Ana Carolina Martins Wille, Daniele Moreira, Dilza Trevisan-Silva, Mariana Gabriel Magnoni, Marianna Boia Ferreira, Luiza Gremski, Waldemiro Gremski, Olga Chaim, Andrea Senff-Ribeiro, Silvio Sanches Veiga. Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom. Toxicon. 2013; 67 ():17-30.

Chicago/Turabian Style

Ana Carolina Martins Wille; Daniele Moreira; Dilza Trevisan-Silva; Mariana Gabriel Magnoni; Marianna Boia Ferreira; Luiza Gremski; Waldemiro Gremski; Olga Chaim; Andrea Senff-Ribeiro; Silvio Sanches Veiga. 2013. "Modulation of membrane phospholipids, the cytosolic calcium influx and cell proliferation following treatment of B16-F10 cells with recombinant phospholipase-D from Loxosceles intermedia (brown spider) venom." Toxicon 67, no. : 17-30.

Journal article
Published: 01 November 2011 in Vaccine
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An NC-1 mimotope from Taenia solium cysticerci can help identify patients with neurocysticercosis through immunoassay. After chemical synthesis, an NC-1 peptide was coupled to bovine serum albumin (NC-1/BSA) for used as an immunogen in murine Taenia crassiceps cysticercosis, which is an experimental model of cysticercosis caused by T. solium. NC-1/BSA immunisation decreased parasitaemia by inducing 74% protection compared to the 77% protection obtained with T. crassiceps crude antigen. The influence of immunisation was also observed on the size and stage of development of the parasite. Antibodies from NC-1/BSA-immunised mice recognised proteins from the tegument and from the buddings, and intense immunostaining was observed in the final stage of the metacestode. The capacity of NC-1/BSA to induce protective antibodies which are reactive to proteins from the tegument of the metacestode suggests that this mimotope is a potential candidate for a vaccine against human and animal cysticercosis

ACS Style

Janaína Capelli-Peixoto; Carlos Chávez-Olórtegui; Daniele Chaves-Moreira; João Carlos Minozzo; Juarez Gabardo; Kádima Nayara Teixeira; Vanete Thomaz-Soccol; Larissa Magalhães Alvarenga; Juliana de Moura. Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis. Vaccine 2011, 29, 9473 -9479.

AMA Style

Janaína Capelli-Peixoto, Carlos Chávez-Olórtegui, Daniele Chaves-Moreira, João Carlos Minozzo, Juarez Gabardo, Kádima Nayara Teixeira, Vanete Thomaz-Soccol, Larissa Magalhães Alvarenga, Juliana de Moura. Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis. Vaccine. 2011; 29 (51):9473-9479.

Chicago/Turabian Style

Janaína Capelli-Peixoto; Carlos Chávez-Olórtegui; Daniele Chaves-Moreira; João Carlos Minozzo; Juarez Gabardo; Kádima Nayara Teixeira; Vanete Thomaz-Soccol; Larissa Magalhães Alvarenga; Juliana de Moura. 2011. "Evaluation of the protective potential of a Taenia solium cysticercus mimotope on murine cysticercosis." Vaccine 29, no. 51: 9473-9479.