Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization.

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

Novel cysteine protease inhibitor derived from the Haementeria vizottoi leech: recombinant expression, purification, and characterization

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

Data on global expression of non-coding RNome in mice gastrocnemius muscle exposed to jararhagin, snake venom metalloproteinase.

This article describes the data on the global expression profile of small RNA (smRNAs) molecules in mice gastrocnemius muscle exposed to jararhagin, snake venom metalloproteinase. The data include smRNAs in mice gastrocnemius muscle challenged with Jararhagin (Jar; n=4) in the right paw or phosphate-buffered saline (PBS; control; n=4) in the left paw. smRNA-Seq libraries were generated after 24 h of exposure to PBS or jararhagin. The expression profiles of smRNAs including microRNA and snoRNA were compared between both groups. The sequencing data from both groups have been uploaded to Zenodo http://dx.doi.org/10.5281/zenodo.56492.

Purificação da Jararagina-C a partir do veneno de Bothrops Jararaca e avaliação da atividade biológica

Dentre todas as toxinas presentes na complexa mistura de um veneno botrópico, sabe-se que as SVMPs (Snake Venom Metalloproteinases) são importantes toxinas envolvidas na patogênese induzida pelo envenenamento. A jararagina-C é uma proteína derivada da autólise da jararagina (SVMP isolada a partir do veneno de Bothrops jararaca) que contém apenas os domínios tipo disintegrina/rico em cisteínas e é capaz de desencadear, em modelo experimental in vivo, os eventos iniciais da resposta inflamatória, como aumento no numero de leucócitos em rolamento na parede do endotélio e liberação local de citocinas pró-inflamatórias. Estudos realizados mais recentemente por nosso grupo com a jararagina-C, em modelo in vitro, demonstram sua ação na migração de células endoteliais e na regulação da expressão de genes envolvidos com o processo de regeneração tecidual, sugerindo um possível papel desta toxina na angiogênese. Na literatura poucas proteínas tipo-disintegrinas estão descritas por apresentar esta atividade angiogênica. O objetivo deste trabalho foi purificar a jararagina-C a partir do veneno de Bothrops jararaca, para avaliar o seu efeito na angiogênese. Para a obtenção desta proteína na sua forma nativa, foram realizados inúmeros procedimentos de purificação por cromatografias de interação hidrofóbica e por troca iônica. A ação biológica da proteína nativa foi analisada em modelos experimentais, como ensaio de ligação ao colágeno em fase sólida e inibição da agregação plaquetária induzida por colágeno. A quantidade de jararagina-C obtida após o passo de purificação corresponde a aproximadamente 0,3% do veneno bruto, de modo que é necessário realizar várias vezes este processo de purificação para obtenção de uma quantidade significativa desta proteína para conduzir os futuros experimentos de estudo da angiogênese.