Purification of a fragment obtained by autolysis of a PIIIb-SVMP from Bothrops alternatus venom

Snake Venom Metalloproteinases (SVMPs) represent 43.1% of the components in Bothrops alternatus venom and play an important role in envenomation. Disintegrins and disintegrin-like domains are released by proteolytic processing of PII and PIII classes of SVMPs respectively and are potent inhibitors of integrin–ligand interaction. Baltergin is a PIIIb-SVMP isolated from this venom and able to undergo autolysis in vitro, giving rise to a stable disintegrin-like/cystein-rich fragment (baltergin-DC). Conditions of baltergin autolysis were adjusted in order to carry out the purification of baltergin-DC and its effect on cell adhesion was studied. Autolysis was maximal at 37 °C and a pH range of 7.0–8.0. Baltergin-DC amino-terminal sequence begins with IISPPVCGNELLEVGEECDCGTPENCQNECCDAATC, which shows a high degree of homology with other disintegrin-like proteins. Baltergin and purified baltergin-DC were both able to inhibit C2C12 adhesion to fetal bovine serum (FBS) coated plates, indicating that a non-catalytic process is involved, probably mediated by binding to membrane integrins. Baltergin-DC, lacking proteolytic action, becomes an attractive molecule for future studies on blocking integrin–ligand interactions.Fil: Van de Velde, Andrea Carolina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Gay, Claudia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación en Proteínas; ArgentinaFil: Olivera Moritz, Milene Nobrega de. Universidade Federal de São Carlos; BrasilFil: dos Santos, Patty Karina. Universidade Federal de São Carlos; BrasilFil: Bustillo, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Rodríguez, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Acosta, Ofelia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; ArgentinaFil: Biscoglio, Mirtha Josefa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sobreiro Selistre de Araujo, Heloisa. Universidade Federal de São Carlos; BrasilFil: Leiva, Laura Cristina Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; Argentin

Inhibition of αvβ3 integrin induces loss of cell directionality of oral squamous carcinoma cells (OSCC)

The connective tissue formed by extracellular matrix (ECM) rich in fibronectin and collagen consists a barrier that cancer cells have to overpass to reach blood vessels and then a metastatic site. Cell adhesion to fibronectin is mediated by αvβ3 and α5β1 integrins through an RGD motif present in this ECM protein, thus making these receptors key targets for cell migration studies. Here we investigated the effect of an RGD disintegrin, DisBa-01, on the migration of human fibroblasts (BJ) and oral squamous cancer cells (OSCC, SCC25) on a fibronectin-rich environment. Time-lapse images were acquired on fibronectin-coated glassbottomed dishes. Migration speed and directionality analysis indicated that OSCC cells, but not fibroblasts, showed significant decrease in both parameters in the presence of DisBa-01 (1μM and 2μM). Integrin expression levels of the α5, αv and β3 subunits were similar in both cell lines, while β1 subunit is present in lower levels on the cancer cells. Next, we examined whether the effects of DisBa-01 were related to changes in adhesion properties by using paxillin immunostaining and total internal reflection fluorescence TIRF microscopy. OSCCs in the presence of DisBa-01 showed increased adhesion sizes and number of maturing adhesion. The same parameters were analyzed usingβ3-GFP overexpressing cells and showed that β3 overexpression restored cell migration velocity and the number of maturing adhesion that were altered by DisBa-01. Surface plasmon resonance analysis showed that DisBa-01 has 100x higher affinity for αvβ3 integrin than forα5β1 integrin. In conclusion, our results suggest that the αvβ3 integrin is the main receptor involved in cell directionality and its blockage may be an interesting alternative against metastasis

Proteoma da peçonha de Lachesis muta rhombeata

Universidade Federal de Sao CarlosA serpente Lachesis muta rhombeata (Viperidae), popularmente conhecida como surucucu pico-de-jaca, é uma subespécie de Lachesis muta endêmica na Mata Atlântica brasileira, podendo ser encontrada do Ceará até o Rio de Janeiro. O gênero Lachesis apresenta uma peçonha complexa que engloba diferentes proteínas que possuem uma ampla variedade de atividades biológicas, como por exemplo, aquelas que afetam os fatores da coagulação sanguínea, agem na fibrinólise, provocam uma ação inflamatória no local da picada, e induzem um choque hipotensivo. Com o auxílio de técnicas proteômicas e de Bioinformática, este projeto teve como objetivo principal analisar o proteoma da peçonha de L. m. rhombeata, identificando as principais famílias proteicas presentes. Para isso, 200μg da peçonha desta serpente foram aplicados em géis bidimensionais-2D. As proteínas obtidas foram extraídas dos géis, tratadas, e posteriormente identificadas e analisadas por meio de espectrometria de massas (MALDI-TOF/TOF). Os arquivos brutos provenientes dessas análises foram aplicados no programa MASCOT utilizando para a identificação proteica o banco de dados SwissProt e como especificação taxonômica o banco de dados SNAKES contido no site do NCBI. Foram identificadas 11 famílias proteicas: PLA2s (37,93%), inibidores de proteases (22,41%), proteínas de sinalização intracelular (13,79%), NGFs (5,17%), metaloproteases (3,45%), fatores associados à DDB1 e CUL4 (3,45%), oxidorredutases (3,45%), proteínas de transdução de sinal (1,72%), componentes do complemento (1,72%), proteínas NipSnap (1,72%) e proteínas lin-7 (1,72%), das quais a grande maioria é de função metabólica e ajuda na difusão/entrada dos componentes tóxicos no organismo da vítima/presa. Cerca de 1-5mg da mesma peçonha também foram analisados por meio de FPLC e sequenciamento proteico e as proteínas obtidas foram identificadas por meio de busca no banco de dados nr (nonredundant protein sequences) do NCBI utilizando a ferramenta blast-p. As seguintes famílias proteicas foram encontradas: serinoproteases (30,03%), metaloproteases (24,17%), BPPs (19,64%), PLA2s (14,95%), lectinas tipo C (1,94%), LAAOs (1,40%) e metaloendopeptidases (0,73%), ressaltando a toxicidade da peçonha. Os dois resultados obtidos foram comparados com proteomas de outras serpentes do gênero Lachesis encontrados na literatura. Observou-se que há uma grande similaridade entre os proteomas das peçonhas deste gênero, mas que alguns componentes proteicos são diferenciais e assim, podem ajudar na taxonomia destas serpentes, mantendo-as em suas respectivas espécies e subespécies. Além disso, a peçonha também foi analisada por eletroforese unidimensional (SDS-PAGE) para avaliar se o espécime em estudo era um neonato, um juvenil ou um adulto. A presença de uma proteína similar à mutalisina II (metaloprotease) confirmou que a L. m. rhombeata utilizada neste estudo era uma adulta. O conhecimento do proteoma da peçonha de L. m. rhombeata auxiliará (1) os estudos envolvendo a Sistemática do gênero e (2) a produção de novos fármacos que poderão ser desenvolvidos para o tratamento de diversas doenças

Effects of Low-Intensity and Long-Term Aerobic Exercise on the Psoas Muscle of mdx Mice: An Experimental Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a muscle disease characterized by the absence of the protein dystrophin, which causes a loss of sarcolemma integrity, determining recurrent muscle injuries, decrease in muscle function, and progressive degeneration. Currently, there is a need for therapeutic treatments to improve the quality of life of DMD patients. Here, we investigated the effects of a low-intensity aerobic training (37 sessions) on satellite cells, peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α protein (PGC-1α), and different types of fibers of the psoas muscle from mdx mice (DMD experimental model). Wildtype and mdx mice were randomly divided into sedentary and trained groups (n = 24). Trained animals were subjected to 37 sessions of low-intensity running on a motorized treadmill. Subsequently, the psoas muscle was excised and analyzed by immunofluorescence for dystrophin, satellite cells, myosin heavy chain (MHC), and PGC-1α content. The minimal Feret’s diameters of the fibers were measured, and light microscopy was applied to observe general morphological features of the muscles. The training (37 sessions) improved morphological features in muscles from mdx mice and caused an increase in the number of quiescent/activated satellite cells. It also increased the content of PGC-1α in the mdx group. We concluded that low-intensity aerobic exercise (37 sessions) was able to reverse deleterious changes determined by DMD

Alternagin-C binding to α2β1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

<div><p>Abstract Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2β1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2β1 integrin. Herein, we used ALT-C as a α2β1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2β1, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to α2β1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. Conclusion These results demonstrate that α2β1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.</p></div