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Made available in DSpace on 2022-04-29T08:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 Background: We evaluated the impacts of corona protein (CP) formation on the alternating current biosusceptometry (ACB) signal intensity and in vivo circulation times of three differently coated magnetic nanoparticles (MNP): bare, citrate-coated and bovine serum albumin-coated MNPs. Methods: We employed the ACB system, gel electrophoresis and mass spectrometry analysis. Results: Higher CP formation led to a greater reduction in the in vitro ACB signal intensity and circulation time. We found fewer proteins forming the CP for the bovine serum albumin-coated MNPs, which presented the highest circulation time in vivo among the MNPs studied. Conclusion: These data showed better biocompatibility, stability and magnetic signal uniformity in biological media for bovine serum albumin-coated MNPs than for citrate-coated MNPs and bare MNPs. Department of Biophysics and Pharmacology São Paulo State University, São Paulo Museum National d'Histoire Naturelle Institut de Minéralogie Physique des Matériaux et Cosmochimie IMPMC Sorbonne Université UMR 7590 CNRS Graduate Program in Tropical Diseases Botucatu Medical School (FMB) São Paulo State University (UNESP), São Paulo Biotechnology Institute São Paulo State University, São Paulo Institute of Physics and CNanoMed Federal University of Goiás Department of Biophysics and Pharmacology São Paulo State University, São Paulo Graduate Program in Tropical Diseases Botucatu Medical School (FMB) São Paulo State University (UNESP), São Paulo Biotechnology Institute São Paulo State University, São Paulo

Varespladib (LY315920) is a potent inhibitor of human group IIA phospholipase A

Made available in DSpace on 2021-06-25T10:12:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT,which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results. Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Laboratoire de Biologie et de Pharmacologie Appliquée École Normale Supérieure Paris Saclay UMR 8113 CNRS, 4 Avenue des Sciences Departamento de Física Experimental Instituto de Física Universidade de São Paulo (USP) Departamento de Biotecnologia e Bioprocessos Faculdade de Ciências Agronômicas Universidade Estadual Paulista (UNESP) Instituto de Biotecnologia Universidade Estadual Paulista (UNESP) Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Biotecnologia e Bioprocessos Faculdade de Ciências Agronômicas Universidade Estadual Paulista (UNESP) Instituto de Biotecnologia Universidade Estadual Paulista (UNESP) CNPq: 302883/2017-7 CNPq: 401190/2017-0 CAPES: 88881.134154/2016-01 CAPES: 88882.183567/2007-01

Made available in DSpace on 2020-12-12T02:24:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Post-translational modifications provide suitable mechanisms for cellular adaptation to environmental changes. Lysine acetylation is one of these modifications and occurs with the addition of an acetyl group to Nϵ-amino chain of this residue, eliminating its positive charge. Recently, we found distinct acetylation profiles of procyclic and bloodstream forms of Trypanosoma brucei, the agent of African Trypanosomiasis. Interestingly, glycolytic enzymes were more acetylated in the procyclic, which develops in insects and uses oxidative phosphorylation to obtain energy, compared with the bloodstream form, whose main source of energy is glycolysis. Here, we investigated whether acetylation regulates the T. brucei fructose 1,6-bisphosphate aldolase. We found that aldolase activity was reduced in procyclic parasites cultivated in the absence of glucose and partial recovered by in vitro deacetylation. Similarly, acetylation of protein extracts from procyclics cultivated in glucose-rich medium, caused a reduction in the aldolase activity. In addition, aldolase acetylation levels were higher in procyclics cultivated in the absence of glucose compared with those cultivated in the presence of glucose. To further confirm the role of acetylation, lysine residues near the catalytic site were substituted by glutamine in recombinant T. brucei aldolase. These replacements, especially K157, inhibited enzymatic activity, changed the electrostatic surface potential, decrease substrate binding and modify the catalytic pocket structure of the enzyme, as predicted by in silico analysis. Taken together, these data confirm the role of acetylation in regulating the activity of an enzyme from the glycolytic pathway of T. brucei, expanding the factors responsible for regulating important pathways in this parasite. Departamento de Microbiologia Escola Paulista de Medicina Universidade Federal de Saõ Paulo Imunologia e Parasitologia Departamento de Biofísica e Farmacologia Instituto de Biociências-UNESP-Universidade Estadual Paulista Departamento de Biofísica e Farmacologia Instituto de Biociências-UNESP-Universidade Estadual Paulista

Made available in DSpace on 2022-04-28T19:44:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fonds Wetenschappelijk Onderzoek Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) KU Leuven Vlaamse regering PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent. School of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo, Av. do Café s/n° University of Vila Velha, Av. Comissário José Dantas de Melo, 21, Boa Vista II Department of Biophysics and Pharmacology Institute of Biosciences São Paulo State University (UNESP) Toxicology and Pharmacology KU Leuven, O&N II Herestraat 49 - PO box 922 Institute of Biotechnology Federal University of Uberlandia Department of Biophysics and Pharmacology Institute of Biosciences São Paulo State University (UNESP) FAPESP: 2011/23236-4 FAPESP: 2015/17286-0 FAPESP: 2015/18432-0 CNPq: 302883/2017-7 CNPq: 307155/2017-0 KU Leuven: CELSA 17/047 Vlaamse regering: GOA4919N Vlaamse regering: GOC2319N Vlaamse regering: GOE7120N KU Leuven: PDM/19/164

Made available in DSpace on 2022-04-28T19:41:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-31 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Snakebite envenoming is the cause of an ongoing health crisis in several regions of the world, particularly in tropical and neotropical countries. This scenario creates an urgent necessity for new practical solutions to address the limitations of current therapies. The current study investigated the isolation, phytochemical characterization, and myotoxicity inhibition mechanism of gallic acid (GA), a myotoxin inhibitor obtained from Anacardium humile. The identification and isolation of GA was achieved by employing analytical chromatographic separation, which exhibited a compound with retention time and nuclear magnetic resonance spectra compatible with GA's commercial standard and data from the literature. GA alone was able to inhibit the myotoxic activity induced by the crude venom of Bothrops jararacussu and its two main myotoxins, BthTX-I and BthTX-II. Circular dichroism (CD), fluorescence spectroscopy (FS), dynamic light scattering (DLS), and interaction studies by molecular docking suggested that GA forms a complex with BthTX-I and II. Surface plasmon resonance (SPR) kinetics assays showed that GA has a high affinity for BthTX-I with a KD of 9.146 × 10−7 M. Taken together, the two-state reaction mode of GA binding to BthTX-I, and CD, FS and DLS assays, suggest that GA is able to induce oligomerization and secondary structure changes for BthTX-I and -II. GA and other tannins have been shown to be effective inhibitors of snake venoms' toxic effects, and herein we demonstrated GA's ability to bind to and inhibit a snake venom PLA2, thus proposing a new mechanism of PLA2 inhibition, and presenting more evidence of GA's potential as an antivenom compound. Instituto de Genética e Bioquímica Universidade Federal de Uberlândia UFU Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista UNESP Laboratório de Biotecnologia de Proteínas e Compostos Bioativos LABIOPROT Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Laboratório de Imunologia Celular Aplicada à Saúde Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis IBAMA Departamento de Química Universidade Federal de Lavras UFLA Instituto Federal Goiano IF Goiano Faculty of Chemical Sciences University of Cuenca LAQV/Requimte Faculty of Sciences University of Porto Centro Universitário São Lucas UniSL Instituto Nacional de Ciência e Tecnologia em Epidemiologia da Amazônia Ocidental (INCT-EpiAmO) Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista UNESP

Made available in DSpace on 2022-04-28T19:41:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 The classical nuclear import pathway is mediated by importin (Impα and Impβ), which recognizes the cargo protein by its nuclear localization sequence (NLS). NLSs have been extensively studied resulting in different proposed consensus; however, recent studies showed that exceptions may occur. This mechanism may be also dependent on specific characteristics of different Impα. Aiming to better understand the importance of specific residues from consensus and adjacent regions of NLSs, we studied different mutations of a high-affinity NLS complexed to Impα by crystallography and calorimetry. We showed that although the consensus sequence allows Lys or Arg residues at the second residue of a monopartite sequence, the presence of Arg is very important to its binding in major and minor sites of Impα. Mutations in the N or C-terminus (position P1 or P6) of the NLS drastically reduces their affinity to the receptor, which is corroborated by the loss of hydrogen bonds and hydrophobic interactions. Surprisingly, a mutation in the far Nterminus of the NLS led to an increase in the affinity for both binding sites, corroborated by the structure with an additional hydrogen bond. The binding of NLSs to the human variant Impα1 revealed that these are similar to those found in structures presented here. For human variant Impα3, the bindings are only relevant for the major site. This study increases understanding of specific issues sparsely addressed in previous studies that are important to the task of predicting NLSs, which will be relevant in the eventual design of synthetic NLSs. Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista

Made available in DSpace on 2021-06-25T10:28:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. Methods: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. Results: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. Conclusion: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. General significance: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily. Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Instituto Clodomiro Picado Facultad de Microbiología Universidad de Costa Rica Center for Exploration and Travel Health California Academy of Sciences Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) FAPESP: 2015/17286-0 FAPESP: 2016/24191-8 CNPq: 302883/2017-7

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95–235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

Made available in DSpace on 2019-10-06T17:01:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America, Bothrops snakes are responsible for the majority of the snakebite envenomings that are not efficiently treated by conventional serum therapy. Thus, the search for simple and efficient inhibitors to complement this therapy is a promising research area, and a combination of functional and structural assays have been used to test candidate ligands against specific ophidian venom compounds. Herein, we tested a commercial drug (acetylsalicylic acid, ASA) and a plant compound with antiophidian properties (rosmarinic acid, RA) using myographic, crystallographic and bioinformatics experiments with a phospholipase A 2 -like toxin, MjTX-II. MjTX-II/RA and MjTX-II/ASA crystal structures were solved at high resolution and revealed the presence of ligands bound to different regions of the toxin. However, in vitro myographic assays showed that only RA is able to prevent the myotoxic effects of MjTX-II. In agreement with functional results, molecular dynamics simulations showed that the RA molecule remains tightly bound to the toxin throughout the calculations, whereas ASA molecules tend to dissociate. This approach aids the design of effective inhibitors of PLA 2 -like toxins and, eventually, may complement serum therapy. Depto. de Física e Biofísica Instituto de Biociências UNESP – Universidade Estadual Paulista Depto. de Farmacologia UFMG - Universidade Federal de Minas Gerais Depto. de Farmacologia Instituto de Biociências UNESP – Universidade Estadual Paulista Depto. de Física e Biofísica Instituto de Biociências UNESP – Universidade Estadual Paulista Depto. de Farmacologia Instituto de Biociências UNESP – Universidade Estadual Paulista FAPESP: 2015/17286-0 FAPESP: 2015/24167-7

Background Specific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A2-like (PLA2-like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA2-like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently. Methods Myographic and histological techniques were used to evaluate the inhibitory effect of chicoric acid (CA) against BthTX-I myotoxin. Isothermal titration calorimetry assays were used to measure the affinity between the inhibitor and the toxin. X-ray crystallography was used to reveal details of this interaction. Results CA prevented the blockade of indirectly evoked muscle contraction and inhibited muscle damage induced by BthTX-I. The inhibitor binds to the toxin with the highest affinity measured for a natural compound in calorimetric assays. The crystal structure and molecular dynamics simulations demonstrated that CA binds at the entrance of the hydrophobic channel of the toxin and binds to one of the clusters that participates in membrane disruption. Conclusions CA prevents the myotoxic activity of the toxin, preventing its activation by simultaneous binding with two critical regions. General significance CA is a potential myotoxic inhibitor to other PLA2-like proteins and a possible candidate to complement serum therapy.

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins., The authors express their gratitude for the valuable discussions with Dr. Juan J. Calvete and Dr. Fábio Florença Cardoso. R.J.B. received a fellowship from FAPESP [grant number 16/24191-8], MRMF received a fellowship from CNPq [302883/2017-7] and IU was supported by PGC [PGC2018-101370-BI00 MICINN/AEI/FEDER/UE]. The research was financed by FAPESP [2015/17286-0, 2019/05958-4 and 2020/10143-7].

Made available in DSpace on 2021-06-25T10:18:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Snakebite envenomation has been categorized by World Health Organization as a category A neglected tropical disease, since it causes chronic psychological disorders, physical disablement and death. Ophidian accidents may cause local myonecrosis that cause drastic sequelae, which are not efficiently neutralized via serum therapy. Phospholipase A2-like (PLA2-like) myotoxins have a major role in the local effects caused by several snake venoms. We previously demonstrated that chicoric acid (CA) is an efficient inhibitor of the BthTX-I myotoxin and solved the X-ray structure of complex. Herein, we assess the oligomeric behavior of the BthTX-I/CA complex in solution under different physical-chemical conditions and using toxin obtained by two different biochemical methodologies to fully elucidate structural bases of inhibition of myotoxins by CA. We demonstrated the ability of PLA2-like proteins to form different oligomeric assemblies in the presence of certain inhibitors, which can also be modulated by buffer polarity change. In the presence of ethanol, BthTX-I/CA remains predominantly in a monomeric conformation, which prevents it from being in its active form (dimeric conformation). In contrast, in the absence of ethanol, the tetramer assembly was observed, which hid key regions of the protein responsible for docking and disruption of the muscle membrane. Therefore, the “plasticity” of these proteins with regard to their abilities to form oligomeric assemblies is a key issue for the future development of therapeutic agents to complement of serum therapy. Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Instituto de Física Universidade de São Paulo (USP) Departamento de Biofísica e Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP)

Made available in DSpace on 2020-12-12T01:29:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Secretaría Nacional de Ciencia, Tecnología e Innovación A bioactive compound isolated from the stem extract of Aristolochia sprucei through High Performance Liquid Chromatography (HPLC) was identified via Nuclear Magnetic Resonance (NMR) as the aristolochic acid (AA). This compound showed an inhibitory effect over the myotoxic activity of Bothrops jararacussu and Bothrops asper venoms, being also effective against the indirect hemolytic activity of B. asper venom. Besides, AA also inhibited the myotoxic activity of BthTX-I and MTX-II with an efficiency greater than 60% against both myotoxins. Docking predictions revealed an interesting mechanism, through which the AA displays an interaction profile consistent with its inhibiting abilities, binding to both active and putative sites of svPLA2. Overall, the present findings indicate that AA may bind to critical regions of myotoxic Asp 49 and Lys49-PLA2s from snake venoms, highlighting the relevance of domains comprising the active and putative sites to inhibit these toxins. Faculdade de Ciências Farmacêuticas de Ribeirão Preto FCFRP Universidade de São Paulo USP Laboratório de Biotecnologia de Proteínas e Compostos Bioativos da Amazônia Ocidental LaBioProt Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia e Universidade Federal de Rondônia UNIR Centro de Informaciones e Investigaciones Toxicológicas y Químicas Aplicadas (CEIITOXQUIA) and Departamento de Química FCNYE Universidad Autónoma de Chiriquí UNACHI Instituto Brasileiro Do Meio Ambiente e Dos Recursos Naturais Renováveis IBAMA Unidade de Biotecnologia Universidade de Ribeirão Preto UNAERP Instituto Federal de Goiás IFG Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Laboratório de Imunologia Celular Aplicada a Saúde Fundação Oswaldo Cruz FIOCRUZ Unidade Rondônia Centro Universitário São Lucas UniSL Instituto Nacional de Ciência e Tecnologia Em Epidemiologia da Amazônia Ocidental INCT – EpiAmO Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP

Made available in DSpace on 2021-06-25T10:33:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Laboratório Nacional de Luz Síncrotron Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P–I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming. Departamento de Biofísica e Farmacologia Instituto de Biociências UNESP – Universidade Estadual Paulista, SP Graduate Program in Tropical Diseases Faculdade de Medicina de Botucatu (FMB) and Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) UNESP – Universidade Estadual Paulista, SP Departamento de Biofísica e Farmacologia Instituto de Biociências UNESP – Universidade Estadual Paulista, SP Graduate Program in Tropical Diseases Faculdade de Medicina de Botucatu (FMB) and Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP) UNESP – Universidade Estadual Paulista, SP FAPESP: 16/24191-8

17 pags., 11 figs., 8 tabs., Fragment-based molecular-replacement methods can solve a macromolecular structure quasi-ab initio. ARCIMBOLDO, using a common secondary-structure or tertiary-structure template or a library of folds, locates these with Phaser and reveals the rest of the structure by density modification and autotracing in SHELXE. The latter stage is challenging when dealing with diffraction data at lower resolution, low solvent content, high β-sheet composition or situations in which the initial fragments represent a low fraction of the total scattering or where their accuracy is low. SEQUENCE SLIDER aims to overcome these complications by extending the initial polyalanine fragment with side chains in a multisolution framework. Its use is illustrated on test cases and previously unknown structures. The selection and order of fragments to be extended follows the decrease in log-likelihood gain (LLG) calculated with Phaser upon the omission of each single fragment. When the starting substructure is derived from a remote homolog, sequence assignment to fragments is restricted by the original alignment. Otherwise, the secondary-structure prediction is matched to that found in fragments and traces. Sequence hypotheses are trialled in a brute-force approach through side-chain building and refinement. Scoring the refined models through their LLG in Phaser may allow discrimination of the correct sequence or filter the best partial structures for further density modification and autotracing. The default limits for the number of models to pursue are hardware dependent. In its most economic implementation, suitable for a single laptop, the main-chain trace is extended as polyserine rather than trialling models with different sequence assignments, which requires a grid or multicore machine. SEQUENCE SLIDER has been instrumental in solving two novel structures: that of MltC from 2.7 Å resolution data and that of a pneumococcal lipoprotein with 638 residues and 35% solvent content., RJB received a fellowship from FAPESP (16/24191-8 and 17/13485-3) and MS from Marie Skłodowska-Curie Actions (H2020-MSCA-IF-2017, No. 790122). This work was supported by grants BIO2015-64216-P, PGC2018-101370-B-I00, BFU2017-90030-P and MDM2014-0435-01 from the Spanish Ministry of Science and Innovation and EU FEDER funds and 2017SGR-1192 from Generalitat de Catalunya.

Made available in DSpace on 2020-12-12T02:33:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Envenoming by snakebite is an important global health issue that has received little attention, leading the World Health Organization to naming it as neglected tropical disease. Several snakebites present serious local symptoms manifested on victims that may not be efficiently neutralized by serum therapy. Phospholipase A2-like (PLA2-like) toxins are present in Viperidae venoms and are responsible for local myotoxic activity. Herein, we investigated the association between BthTX-I toxin and caftaric acid (CFT), a molecule present in plants. CFT neutralized neuromuscular blocking and muscle-damaging activities promoted by BthTX-I. Calorimetric and light-scattering assays demonstrated that CFT inhibitor interacted with dimeric BthTX-I. Bioinformatics simulations indicated that CFT inhibitor binds to the toxin's hydrophobic channel (HCh). According to the current myotoxic mechanism, three different regions of PLA2-like toxins have specific tasks: protein allosteric activation (HCh), membrane dockage (MDoS), and membrane rupture (MDiS). We propose CFT inhibitor interferes with the allosteric activation, which is related to the conformation change leading to the exposure/alignment of MDoS/MDiS region. This is the first report of a PLA2-like toxin fully inhibited by a compound that interacts only with its HCh region. Thus, CFT is a novel candidate to complement serum therapy and improve the treatment of snakebite. Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Morfologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Morfologia Instituto de Biociências Universidade Estadual Paulista (UNESP) CAPES: 1592/2011 FAPESP: 2012/07112-6 FAPESP: 2015/17286-0 CNPq: 302883/2017-7 CNPq: 401190/2017-7

The Author(s) 2022., Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources., São Paulo Research Foundation [FAPESP, 2015/17286–0, 2019/05958-4, 2020/10143–7]; FAPESP [2016/24191–8 to R.J.B.]; Ministerio de Ciencia e Innovación [PGC2018-101370-BI00 AEI/FEDER/UE]; CNPq [301974/2019–5, 302883/2017–7]. Funding for open access charge: FAPESP [2016/24191–8 and 2020/10143–7]; Ministerio de Ciencia e Innovación [PGC2018-101370-BI00 AEI/FEDER/UE].

Made available in DSpace on 2020-12-12T02:01:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Centre National de la Recherche Scientifique Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Institut National de la Santé et de la Recherche Médicale Muséum National d'Histoire Naturelle Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Background: Telomeres are chromosome end structures important in the maintenance of genome homeostasis. They are replenished by the action of telomerase and associated proteins, such as the OB (oligonucleotide/oligosaccharide-binding)-fold containing telomere-end binding proteins (TEBP) which plays an essential role in telomere maintenance and protection. The nature of TEBPs is well known in higher and some primitive eukaryotes, but it remains undetermined in trypanosomatids. Previous in silico searches have shown that there are no homologs of the classical TEPBs in trypanosomatids, including Leishmania sp. However, Replication Protein A subunit 1 (RPA-1), an OB-fold containing DNA-binding protein, was found co-localized with trypanosomatids telomeres and showed a high preference for the telomeric G-rich strand. Methods and results: We predicted the absence of structural homologs of OB-fold containing TEBPs in the Leishmania sp. genome using structural comparisons. We demonstrated by molecular docking that the ssDNA binding mode of LaRPA-1 shares features with the higher eukaryotes POT1 and RPA-1 crystal structures ssDNA binding mode. Using fluorescence spectroscopy, protein-DNA interaction assays, and FRET, we respectively show that LaRPA-1 shares some telomeric functions with the classical TEBPs since it can bind at least one telomeric repeat, protect the telomeric G-rich DNA from 3′-5′ Exonuclease I digestion, and unfold telomeric G-quadruplex. Conclusions: Our results suggest that RPA-1 emerges as a TEBP in trypanosomatids, and in this context, we present two possible evolutionary landscapes of trypanosomatids RPA-1 that could reflect upon the evolution of OB-fold containing TEBPs from all eukaryotes. Department of Biophysics and Pharmacology Biosciences Institute São Paulo State University (UNESP) – Botucatu Laboratoire de Biologie et Pharmacologie Appliquée École Normale Supérieure Paris-Saclay Department of Chemical and Biological Sciences São Paulo State University (UNESP) – Botucatu MNHN CNRS UMR 7196 INSERM U1154, 43 rue Cuvier Sorbonne Université UFR927, 4 place Jussieu Department of Biophysics and Pharmacology Biosciences Institute São Paulo State University (UNESP) – Botucatu Department of Chemical and Biological Sciences São Paulo State University (UNESP) – Botucatu CAPES: 171895/2018-01 FAPESP: 2015/17286-0 FAPESP: 2015/18641-5 FAPESP: 2018/0395-6 FAPESP: 2018/04375-2 FAPESP: 2019/11496-3 CNPq: 305300/2006-7 CAPES: 88881

Color Index (C.I.) Disperse Red 1 (DR1) is a widely used textile azo dye found in rivers. As it may not be completely removed by conventional treatments, humans can be exposed through drinking water. Studies have supported in vitro toxicity and mutagenicity of commercial DR1. This study aimed to investigate the mutagenic and toxicogenomic effects of commercial DR1 in multiple tissues/organs of Swiss male mice. For that, animals were orally exposed to the dye (by gavage), at single doses of 0.0005, 0.005, 0.5, 50, or 500 mg/kg bw. The two lowest doses were equivalent to the ones found in two Brazilian rivers receiving influx of textile discharges. Cytotoxicity, micronucleated cell frequencies (for all doses tested), primary DNA damage (comet assay), and gene expression profiling of (0.0005 and 0.005 mg/kg of bw) were investigated 24 hr after animal exposure to commercial DR1. Data showed increased frequencies of micronucleated polychromatic erythrocytes in bone marrow cells after treatment with 0.5 and 50 mg/kg bw. At 0.005 mg/kg bw, commercial DR1 induced an increase of primary DNA damage in liver, but not in kidney cells. Additionally, upregulation of genes involved in the inflammatory process (IL1B) (0.0005 and 0.005 mg/kg bw) and cell-cycle control (CDKN1A) in liver cells, and apoptosis (BCL2 and BAX) in leukocytes (0.005 mg/kg bw) were also detected. In conclusion, the commercial DR1 was genotoxic (chromosome aberrations and primary DNA damage) and modulated gene expression in mice, and such effects were dependent on the doses and tissues analyzed. Environ. Mol. Mutagen. 59:822-828, 2018. © 2018 Wiley Periodicals, Inc.

The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP–NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Background Leishmania spp. telomeres are composed of 5′-TTAGGG-3′ repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Methods and results Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3′G-overhang, and DNA binding is calcium dependent. Conclusions We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. General significance LCalA is the first reported calmodulin that binds in vivo telomeric DNA.

There are 2.5 million cases of snakebite per year and approximately 100,000 to 150,000 deaths. Thus, it is considered an important public health problem by the World Health Organization. Snakes from the Bothrops genus may cause severe local effects in the victims, so it is important to develop inhibitors to treat local effects in patients. In addition, approximately 30 different species of bothropic snakes have been described that may present differences in their venom composition. Small structural differences in the venom proteins may result in different ligands binding. Herein, BnSP-7, a PLA 2 -like protein that causes local myotoxic effects, was analyzed using different biophysical techniques. Crystal structures of BnSP-7 binding to three different cinnamic acid derivates were solved showing that the ligands bind in the membrane-dockage region (MDoS) of the protein. Spectroscopy fluorescence and microscale thermophoresis (MST) assays showed that these ligands also bind to BnSP-7 in solution and provide comparative information about their affinity to BnSP-7. MST experiments also showed that hydroxyl radicals of the ligands, involved in their binding with the MDoS region of BnSP-7, are essential to increase their affinity with the protein. As this region has been indicated as essential for the myotoxic mechanism, the ligands could potentially be used as inhibitors for BnSP-7. These results provide relevant insights to understand the PLA 2 -like proteins myotoxic mechanism and may eventually lead to design of new inhibitors for these toxins. Furthermore, a comparative structural analysis of BnSP-7 with other PLA 2 -like proteins showed that BnSP-7 has an atypical quaternary conformation, suggesting an intermediate state that is unlike other PLA 2 -like proteins. This information, combined with the absence or partial occupancy of molecules in their hydrophobic channel and the misaligned membrane-disruption region, led us to hypothesize that the protein is not able to fully exert its myotoxic activity like other PLA 2 -like proteins.

Made available in DSpace on 2018-12-11T16:47:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107–116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. Centro de Estudos de Biomoléculas Aplicadas a Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Fiocruz Rondônia and Departamento de Medicina Universidade Federal de Rondônia UNIR Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP Center for the Study of Biological Complexity Life Sciences Virginia Commonwealth University PVE-CNPq Centro Universitário São Lucas UNISL Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista UNESP

Made available in DSpace on 2018-12-11T16:47:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100 μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14 ± 0.08 and 6.41 ± 0.64 μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64 ± 7.94 and >150 μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. Centro de Estudos de Biomoléculas Aplicadas à Saúde CEBio Fundação Oswaldo Cruz FIOCRUZ Fiocruz Rondônia Departamento de Medicina Universidade Federal de Rondônia UNIR Centro para el Desarrollo de la Investigación Científica CEDIC Laboratório da Plataforma de Bioensaios de Malária e Leishmaniose Fiocruz Rondônia Faculdade São Lucas FSL Departamento de Física e Biofísica Universidade Estadual Paulista UNESP Laboratório de Bioquímica e Biofísica Instituto Butantan Departamento de Física e Biofísica Universidade Estadual Paulista UNESP

Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a “myotoxic cluster”, which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms. Fundação de Amparo à Pesquisa do Estado de São Paulo/[2015/17286-0]/FAPESP/Brasil Fundação de Amparo à Pesquisa do Estado de São Paulo/[2015/24167-7]/FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico/[300596/2013-8]/CNPq/Brasil Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior/[23038.006271/2011-16]/CAPES/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Made available in DSpace on 2020-12-12T02:25:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-20 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil; University of Leuven, Belgium; School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil; Department of Biochemistry and Immunology, Medical School, University of São Paulo, Brazil; Medical School of Roraima, Federal University of Roraima, Brazil; Institute of Biosciences, São Paulo State University (UNESP), Botucatu, Brazil; School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Brazil

Made available in DSpace on 2020-12-12T02:37:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) KU Leuven Fonds Wetenschappelijk Onderzoek Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel. School of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo Av. do Café s/n° Toxicology and Pharmacology KU Leuven, O&N II Herestraat 49, PO 922 Medical School of Roraima Federal University of Roraima Av. Capitão Ene Garcez 2413 Bairro Aeroporto Institute of Biosciences São Paulo State University (UNESP) Rua Prof. Dr. Antonio Celso Wagner Zanin 250 University of Vila Velha Av. Comissário José Dantas de Melo 21 Boa Vista II Institute of Biosciences São Paulo State University (UNESP) Rua Prof. Dr. Antonio Celso Wagner Zanin 250 FAPESP: 2011/23236-4 FAPESP: 2015/00740-0 FAPESP: 2015/17286-0 FAPESP: 2016/04761-4 FAPESP: 2016/24191-8 FAPESP: 2017/14035-1 FAPESP: 2018/14158-9 CNPq: 307155/2017-0 CAPES: 88881.186830/2018-01 KU Leuven: CELSA 17/047 Fonds Wetenschappelijk Onderzoek: GOA4919N

Made available in DSpace on 2019-10-06T15:48:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-01 Natural Sciences and Engineering Research Council of Canada The tumor suppressor activity of maspin (mammary serine protease inhibitor) has been associated with its nuclear localization. In this study we explore the regulation of maspin nuclear translocation. An in vitro nuclear import assay suggested that maspin can passively enter the nucleus. However, in silico analysis identified a putative maspin nuclear localization signal (NLS), which was able to mediate the nuclear translocation of a chimeric protein containing this NLS fused to five green fluorescent protein molecules in tandem (5GFP). Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N suppressed this process. Unexpectedly, the full-length maspin fused to 5GFP failed to enter the nucleus. As maspin's putative NLS is partially hidden in its three-dimensional structure, we suggest that maspin nuclear transport could be conformationally regulated. Our results suggest that maspin nuclear translocation involves both passive and active mechanisms. Department of Cell and Developmental Biology Institute of Biomedical Science of University of São Paulo Department of Zoology University of British Columbia Department of Physics and Biophysics Institute of Biosciences São Paulo State University (UNESP) Department of Physics and Biophysics Institute of Biosciences São Paulo State University (UNESP) Natural Sciences and Engineering Research Council of Canada: RGPIN 227926-11 Natural Sciences and Engineering Research Council of Canada: RGPIN-2017-04600

Several snake species possess, in their circulating blood, endogenous PLA2 inhibitors (sbPLIs) with the primary function of natural protection against toxic enzymes from homologous and heterologous venoms. Among the three structural classes of sbPLIs – named α, β, and γ − the β class (sbβPLIs) is the least known with only four identified sequences, so far. The last class of inhibitors encompass molecules with leucine rich repeats (LRRs) motifs containing repeating amino acid segments. In the present study, we identified and characterized putative sbβPLIs from the liver and venom glands of six Latin American pit vipers belonging to Bothrops and Crotalus genera. The inhibitor from Crotalus durissus terrificus snakes (CdtsbβPLI) was chosen as a reference for the construction of the first in silico structural model for this class of inhibitors, using molecular modeling and molecular dynamics simulations. Detailed analyses of the electrostatic surface of the CdtsbβPLI model and protein-protein docking with crotoxin B from homologous venoms predict the interacting surface between these proteins., Highlights • Transcripts of phospholipases A2 inhibitors from the β-class (sbβPLIs) were identified in Latin American pit vipers. • Structural features of sbβPLIs were compared and discussed, including their characteristic leucine-rich repeats (LRRs). • One sbβPLI (CdtsbβPLI) was chosen for the in silico construction of the first structural model of a sbβPLI. • A possible mechanism of interaction between sbβPLIs and basic snake venom PLA2s was suggested. • Docking predictions between CdtsbβPLI and crotoxin B highlighted the amino acids residues at the interaction surfaces.

Made available in DSpace on 2020-12-12T01:05:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 The World Health Organization recently listed snakebite envenoming as a Neglected Tropical Disease, proposing strategies to significantly reduce the global burden of this complex pathology by 2030. In this context, effective adjuvant treatments to complement conventional antivenom therapy based on inhibitory molecules for specific venom toxins have gained renewed interest. Varespladib (LY315920) is a synthetic molecule clinically tested to block inflammatory cascades of several diseases associated with elevated levels of secreted phospholipase A2 (sPLA2). Most recently, Varespladib was tested against several whole snake venoms and isolated PLA2 toxins, demonstrating potent inhibitory activity. Herein, we describe the first structural and functional study of the complex between Varespladib and a PLA2-like snake venom toxin (MjTX-II). In vitro and in vivo experiments showed this compound’s capacity to inhibit the cytotoxic and myotoxic effects of MjTX-II from the medically important South American snake, Bothrops moojeni. Crystallographic and bioinformatics analyses revealed interactions of Varespladib with two specific regions of the toxin, suggesting inhibition occurs by physical blockage of its allosteric activation, preventing the alignment of its functional sites and, consequently, impairing its ability to disrupt membranes. Furthermore, based on the analysis of several crystallographic structures, a distinction between toxin activators and inhibitors is proposed. Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) Instituto Clodomiro Picado Facultad de Microbiología Universidad de Costa Rica Center for Exploration and Travel Health California Academy of Sciences Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP)

Made available in DSpace on 2018-12-11T17:29:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-01 The tobacco calmodulin-like protein rgs-CaM is involved in host defense against virus and is reported to possess an associated RNA silencing suppressor activity. Rgs-CaM is also believed to act as an antiviral factor by interacting and targeting viral silencing suppressors for autophagic degradation. Despite these functional data, calcium interplay in the modulation of rgs-CaM is still poorly understood. Here we show that rgs-CaM displays a prevalent alpha-helical conformation and possesses three functional Ca2+-binding sites. Using computational modeling and molecular dynamics simulation, we demonstrate that Ca2+ binding to rgs-CaM triggers expansion of its tertiary structure with reorientation of alpha-helices within the EF-hands. This conformational change leads to the exposure of a large negatively charged region that may be implicated in the electrostatic interactions between rgs-CaM and viral suppressors. Moreover, the kd values obtained for Ca2+ binding to the three functional sites are not within the affinity range of a typical Ca2+ sensor. Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Física e Biofísica Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Genética Universidade Estadual Paulista (UNESP) Instituto de Biociências Departamento de Física e Biofísica

12 pags, 6 figs, 3 tabs, Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin-α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG., This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil) and Spanish Ministerio de Economía y Competitividad (BFU2013-47064-P to A.V.C.). B.K. is an NHMRC (National Health and Medical Research Council) Research Fellow (1003325), A.A.S.T. is a CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) Research Fellow and M.R.M.F. is a CNPq Research Fellow. We acknowledge the use of the Laboratório Nacional de Luz Síncrotron (Brazil) and National Synchrotron Light Source (New York, USA).

Made available in DSpace on 2018-12-11T16:40:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-03-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named α, β and γ. In the present study, we identified, characterized and performed structural and evolutionary analyses of sbαPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbαPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbαPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbαPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbαPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources. Fundação Ezequiel Dias (FUNED) Departamento de Bioquímica e Imunologia Universidade Federal de Minas Gerais (UFMG) Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) CNPq: 1810/11

Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones – a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs – and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms.

Made available in DSpace on 2021-06-25T10:26:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Teaching and learning strategies that use innovating technologies, such as the development of software or an understanding of the theoretical concepts of analytical equipment, can be essential tools for the construction of knowledge. In this way, this work describes the construction of an innovative and lowcost colorimeter using a RGB LED and a microcontroller board as a guide for teachers of undergraduate classes to prepare pedagogical activities using the proposed device as a learning object. The device was applied in experiments using methylene blue (MB), methylene orange (MO) and cobalt chloride (CC) aqueous solutions of different concentrations. Each one of these solutions has absorption peaks near the RGB colors. The results demonstrated that this lowcost device was able to identify unambiguous absorption peaks in the expected range of the MB, MO and CC absorption spectrums, allowing us to determine a feasible calibration curve for each tested solution. Universidade Estadual Paulista “Júlio de Mesquista Filho” (UNESP). Professor Assistente Doutor Departamento de Bioprocessos e Biotecnologia São Paulo State University (UNESP) School of Agriculture Department of Bioprocess and Biotechnology Avenida Universitária São Paulo State University (UNESP) School of Medicine Avenida Prof. Mário Rubens Guimarães, Montenegro s/n São Paulo State University (UNESP) Institute of Biosciences Department of Physics and Biophysics Rua Prof. Dr. Antônio Celso Wagner Zanin Universidade de São Paulo (USP) Instituto de Física de São Carlos, Caixa Postal 369 Universidade Estadual Paulista “Júlio de Mesquista Filho” (UNESP). Professor Assistente Doutor Departamento de Bioprocessos e Biotecnologia São Paulo State University (UNESP) School of Agriculture Department of Bioprocess and Biotechnology Avenida Universitária São Paulo State University (UNESP) School of Medicine Avenida Prof. Mário Rubens Guimarães, Montenegro s/n São Paulo State University (UNESP) Institute of Biosciences Department of Physics and Biophysics Rua Prof. Dr. Antônio Celso Wagner Zanin

Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies suggest that subcellular localization plays an essential role on maspin tumor suppression activity. In this study we investigated the molecular mechanisms underlying maspin nucleocytoplasmic shuttling. Anin vitronuclear-import assay using digitonin-permeabilized HeLa cells demonstrated that maspin enters the nucleus by an energy-and carrier-independent mechanism. However, previous studies indicated that maspin subcellular localization is regulated in the cell. Using a nuclear localization signal (NLS) prediction software, we identified a putative NLS in the maspin amino acid sequence. To distinguish between passive and regulated nuclear translocation, maspinNLS or the full-length protein (MaspinFL) were fused to 5GFP, rendering the construct too large to enter the nucleus passively. Unexpectedly, 5GFP-maspinNLS, but not maspinFL-5GFP, entered the nucleus of HeLa cells. Dominant-negative Ran-GTPase mutants RanQ69L or RanT24N, suppressed 5GFP-maspinNLS nuclear localization. In summary, we provide evidence that maspin translocates to the nucleus passively. In addition, we identified a peptide in the maspin protein sequence, which is able to drive a 5GFP construct to the nucleus in an energy-dependent manner.

Made available in DSpace on 2018-12-11T17:37:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy. Universidade Estadual Paulista (UNESP) Instituto de Biociências Dep. de Física e Biofísica Universidade Federal de Minas Gerais (UFMG) Instituto de Ciências Biológicas Dep. de Farmacologia Universidade Estadual Paulista (UNESP) Instituto de Biociências Dep. de Farmacologia Universidade Estadual Paulista (UNESP) Instituto de Biociências Dep. de Física e Biofísica Universidade Estadual Paulista (UNESP) Instituto de Biociências Dep. de Farmacologia

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

Made available in DSpace on 2018-12-11T17:34:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Phospholipase A2-like (PLA2-like) proteins contribute to the development of muscle necrosis in Viperidae snake bites and are not efficiently neutralized by current antivenom treatments. The toxic mechanisms of PLA2-like proteins are devoid of catalytic activity and not yet fully understood, although structural and functional experiments suggest a dimeric assembly and that the C-terminal residues are essential to myotoxicity. Herein, we characterized the functional mechanism of bothropic PLA2-like structures related to global and local measurements using the available models in the Protein Data Bank and normal mode molecular dynamics (NM-MD). Those measurements include: (i) new geometric descriptions between their monomers, based on Euler angles; (ii) characterizations of canonical and non-canonical conformations of the C-terminal residues; (iii) accessibility of the hydrophobic channel; (iv) inspection of ligands; and (v) distance of clustered residues to toxin interface of interaction. Thus, we described the allosteric activation of PLA2-like proteins and hypothesized that the natural movement between monomers, calculated from NM-MD, is related to their membrane disruption mechanism, which is important for future studies of the inhibition process. These methods and strategies can be applied to other proteins to help understand their mechanisms of action. Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP) Departamento de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP)

Made available in DSpace on 2018-12-11T17:14:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação para a Ciência e a Tecnologia Background and purpose Crotoxin (CTX), a heterodimeric phospholipase A2 (PLA2) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. Experimental approach Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [3H]-acetylcholine ([3H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. Key results Both CTX (5 μg/mL) and its basic PLA2 subunit (CB, 20 μg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [3H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. Conclusion and implications Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer. Departamento de Farmacologia Instituto de Ciências Biológicas, UFMG, Av. Antônio Carlos Laboratório de Farmacologia e Neurobiologia Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), R. Jorge Viterbo Ferreira, 228 Center for Drug Discovery and Innovative Medicines (MedInUP) Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), R. Jorge Viterbo Ferreira, 228 Departamento de Física e Biofísica Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Farmacologia Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Física e Biofísica Instituto de Biociências UNESP, Distrito de Rubião Jr. Departamento de Farmacologia Instituto de Biociências UNESP, Distrito de Rubião Jr. CAPES: 1592/2011 CNPq: 300596/2013-8 Fundação para a Ciência e a Tecnologia: OE/SAU/UI0215/2014 Fundação para a Ciência e a Tecnologia: UID/BIM/4308/2016

Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a heterotrimeric complex formed by RPA-1, RPA-2 and RPA-3. RPA is a fundamental player in replication, repair, recombination and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruzi RPA presents canonical functions being involved with DNA replication and DNA damage response. Here we found by FISH/IF assays that T. cruzi RPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA-1. Telomeric DNA induces different secondary structural modifications on RPA-1 in comparison with other types of DNA. In addition, RPA-1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region. This article is protected by copyright. All rights reserved.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A 2 inhibitor (γPLI) from Crotalus durissus collilineatus ( Cdc ) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1–Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA 2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA 2 -TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA 2 s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA 2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA 2 s may be involved.