Cristalogénesis biológica y cristalografía en la elucidación de la estructura tridimensional de las proteínas. Revisión narrativa / Protein tridimensional structure by biological crystallogenesis and crystallography. Narrative review

La obtención de información estructural tridimensional de una proteína es de suma importancia en campos tan variados como la bioquímica funcional, las ciencias de materiales o biomédicas. Siendo actualmente la difracción de rayos X de monocristal el estándar de oro para la consecución de este objetivo, la obtención de dicho monocristal sigue siendo un cuello de botella desde el punto de vista práctico, y poco entendido desde el punto de vista teórico. En este artículo se revisa desde la perspectiva estructural de la proteína la forma en que los rayos X permiten obtener la información estructural y las condiciones fisicoquímicas que permiten la formación de un cristal adecuado para estos experimentos.

Obtaining three-dimensional structural information of a protein is of utmost importance in various fields such as functional biochemistry, materials science, or biomedical sciences. Even though single crystal X-ray diffraction is currently the gold standard for this purpose, growing said single crystal is still a bottleneck from a practical viewpoint, and not fully understood from a theoretical point of view. In this article, we review, from a protein structure perspective, the way X-rays provide structural information, and the physicochemical conditions that promote the formation of an adequate crystal for these experiments.

In vitro enzyme activity and in vivo healing activity of the protein extract from pineapple peel

The pineapple is a fruit that has wide acceptance worldwide both in natural form, as industrialized. Your peel is a residue generated by food industries and from this residue can obtain a protein extract which is a good source of bromelain. This study aimed to obtain a protein extract from pineapple peel, evaluate its enzyme activity and its healing properties in skin lesions in rats. Seven animal groups were used: control, treated with 5% of protein extract, 10% of protein extract and pure protein extract; 5% of commercial bromelain, 10% of commercial bromelain and pure commercial bromelain. The animals were subjected to a tissue incision and treated for 21 days. Proteolytic and specific activities of the protein extract were 1.30 U mg-1 and 45 x 10-3 U μg-1 and, for commercial bromelain, 1.04 U mg-1 and 6 x 10-3 U μg-1, respectively. In the histology of the lesion, there was no significant difference between the control and treated groups; however, macroscopically, the prepared topical formulations assisted in the recovery of skin lesions, providing a significant reduction in their width, in the groups treated with pure protein extract, 5 and 10% commercial bromelain, and pure bromelain.

Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

Background:Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.Methods:The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.Results:V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.Conclusion:The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.

Reconhecimento molecular na doença de chagas do ponto de vista do parasita e do hospedeiro / Molecular recognition in Chagas disease from the point of view of the parasite and the host

A doença de Chagas, causada pelo parasita protozoário Trypanosoma cruzi, afeta milhões de pessoas, a maioria delas vivendo na América latina. Apesar dos avanços da medicina e da biotecnologia, ainda existem poucas opções de tratamento para indivíduos com a doença. Assim, é importante compreendermos os detalhes moleculares da infecção parasitária, para que novas alternativas terapêuticas e de diagnóstico possam ser desenvolvidas para esses pacientes. Neste trabalho estudamos esta doença em duas frentes, uma do ponto de vista do parasita, e a outra, da resposta do hospedeiro. Utilizando bioinformática, identifcamos um peptídeo conservado (denominado TS9) presente nas proteínas de superfície gp85/transsialidases do parasita. Este peptídeo é capaz de promover adesão celular e, na sua forma sintética, inibe a entrada do T. cruzi na célula hospedeira. Análise da estrutura proteica revelou que o peptídeo TS9 encontra-se num domínio do tipo laminina-G, lado-a-lado com o peptídeo FLY, outro peptídeo conservado desta grande família, previamente descrito pelo nosso grupo. Juntos, eles formam um sítio de adesão a citoqueratinas e proteínas de flamento intermediário. Na segunda parte, investigamos os antígenos e epítopos reconhecidos pelas imunoglobulinas de pacientes portadores da doença nas suas diferentes formas clínicas: assintomática e cardiomiopatias, leve ou grave. Criamos uma biblioteca de phage display contendo, virtualmente, todos os fragmentos proteicos existentes no T. cruzi, que foi varrida contra imunoglobulinas para a construção de um mapa da resposta humoral dos pacientes com a doença de Chagas. Nossos resultados mostram que a resposta dos pacientes é complexa, e mais de dois mil epítopos foram mapeados. Muitos deles, como os antígenos B13, SAPA e FRA já foram previamente descritos, validando nosso método. Porém, um grande número de novos epítopos, inclusive contra proteína descritas como hipotéticas ou sem função conhecida, também foram encontrados. Seus papéis na infecção e resposta imune da doença merecem, portanto, atenção. Em resumo, as abordagens e técnicas utilizadas nesta tese são inovadoras, e permitiram a identificação de peptídeos e moléculas que poderão ser úteis para o desenvolvimento de novos métodos diagnósticos e terapêuticos para a doença de Chagas

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, afects millions of people, most of them living in Latin America. Despite advances in medicine and biotechnology, there are still few treatment options for individuals with the disease. Thus, it is important to understand the molecular details of the parasitic infection, so that new therapeutic and diagnostic alternatives can be developed for these patients. In this work, we study this disease in two fronts, one from the point of view of the parasite, and the other, of the response of the host. Using bioinformatics, we identifed a conserved peptide (called TS9) present in the surface proteins gp85 / trans-sialidases of the parasite. This peptide is capable of promoting cell adhesion and, in its synthetic form, inhibits the entry of T. cruzi into the host cell. Analysis of the protein structure revealed that the TS9 peptide is in a laminin-G-like domain, side-by-side with the peptide FLY, another conserved peptide of this large family, previously described by our group. Together, they form an adhesion site to cytokeratins and intermediate flament proteins. In the second part, we investigated the antigens and epitopes recognized by the immunoglobulins of patients with the disease in their diferent clinical forms: asymptomatic and cardiomyopathies, mild or severe. We created a phage display library containing virtually all existing protein fragments in T. cruzi. This library was screened against immunoglobulins for the construction of a humoral response map of patients with Chagas disease. Our results show that the response of the patients is complex, and more than 2,000 epitopes have been mapped. Many of them, such as the B13, SAPA and FRA antigens have been previously described, validating our method. However, a large number of new epitopes, including many against proteins described as hypothetical or with no known function, were also found. Their roles in infection and immune response of the disease deserve, therefore, attention. In summary, the approaches and techniques used in this thesis are innovative and have allowed the identifcation of new peptides and molecules that may be useful for the development of new diagnostic and therapeutic methods for Chagas disease

Enfermedades Autoinflamatorias / Autoinflammatory Diseases

Presentamos un artículo de revisión sobre las enfermedades autoinflamatorias, narrando su origen histórico y describiendo la estructura proteica y molecular del Inflamosoma, la clasificación actual de los trastornos autoinflamatorios y una descripción de las características inmunogenéticas y clínicas más sobresalientes de cada enfermedad.

We present a review article on the autoinflammatory diseases, narrating its historical origin and describing the protein and molecular structure of the Inflammasome, the current classification of the autoinflammatory diseases and a description of the immunogenetics and clinical characteristics more important of every disease.