Divergent cellulosome architecture in rumen bacteria : structure and function studies in cohesin-dockerin complexes of Ruminococcus flavefaciens

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Produção AnimalProtein-protein interactions play a vital role in many cellular processes as exemplified by the assembly of the cellulosome, a bacterial multi-enzyme complex that efficiently degrades cellulose and hemicellulose. Cellulosome assembly involves the high-affinity binding of type I enzyme-borne dockerins to repeated cohesin modules located on non-catalytic structural proteins termed scaffoldins. In addition, the complex is anchored into the bacterial surface through the binding of a scaffoldin type II dockerin to cell-bound cohesins. Initially, the architecture and organization of cellulosomes was thought to rely uniquely on type I and type II cohesin-dockerin interactions. It was recently suggested that cellulosomes from rumen bacteria are organized through different mechanisms involving a third type of cohesin-dockerin complexes. Thus, the genome of the major ruminal bacterium Ruminococcus flavefaciens FD-1 revealed a particularly elaborate cellulosome system that is assembled from a library of more than 200 different components through divergent cohesin-dockerin pairs. Providing structural insights for the specificity displayed by the increasing repertoire of cohesin-dockerin interaction is not only of fundamental importance but essential for the development of novel cellulosome based tools. The present work aimed to identify the molecular basis for the organization of R. flavefaciens cellulosome by dissecting the structural basis of cohesin-dockerin specificity in cellulosomes of rumen bacteria. The data revealed a collection of unique cohesin-dockerin interactions, supporting the functional relevance of dockerin classification in groups based on primary sequence similarity. In addition, R. flavefaciens cellulosome is assembled through a mechanism involving single but not dual-binding mode dockerins. This contrasts with the majority of the cellulosomes described to date where dockerins generally present two similar cohesin-binding interfaces, supporting a dual-binding mode. To illustrate this, the structures of two cohesin-dockerin complexes containing an Acetivibrio cellulolyticus dual-binding mode dockerin were solved. Finally, structural information was used to engineer a dockerin presenting a dual cohesin specificity, revealing the plasticity of the cohesin-dockerin platform to design novel protein-protein interactions.RESUMO - Arquitetura celulossomal divergente em bactérias ruminais: estudos de estrutura e função em complexos coesina-doquerina do Ruminococcus flavefaciens - As interacções proteína-proteína desempenham um papel essencial em vários processos celulares, sendo exemplo disso a estruturação do celulosoma, um complexo bacteriano multienzimático altamente eficiente na degradação da celulose e hemicelulose. A montagem do celulosoma envolve interações de alta afinidade entre doquerinas do tipo I, presentes em enzimas, e os módulos coesina presentes em proteínas estruturais não catalíticas denominadas de escafoldinas. Adicionalmente, todo o complexo é ancorado à superfície bacteriana através da ligação de uma dockerina do tipo II, presente numa escafoldina, a coesinas ligadas à célula. Inicialmente, pensava-se que a arquitectura e organização dos celulosomas assentava exclusivamente em interacções coesina-doquerina do tipo I e II. Recentemente, foi sugerido que a microbiota ruminal contém bactérias produtoras de celulossoma com diferentes mecanismos de organização, envolvendo um terceiro tipo de complexos coesina-dockerina. O genoma da bactéria ruminal Ruminococcus flavefaciens FD-1, revelou um sistema celulossomal particularmente elaborado, montado a partir de uma biblioteca com mais de 200 componentes, através de complexos coesina-doquerina do tipo III. Estabelecer uma base estrutural para a especificidade exibida pelo crescente repertório de pares coesina-doquerina é não só fundamentalmente importante mas também essencial para o desenvolvimento de novas ferramentas com base no celulossoma. O presente trabalho teve como objetivo identificar a base estrutural para a especificidade coesina-doquerina do R. flavefaciens, permitindo descortinar os mecanismos por detrás da montagem dos celulosomas ruminais. Os dados obtidos revelaram uma colecção de interacções coesina-doquerina única, suportando a relevância funcional da classificação das doquerinas em grupos com base na homologia da sua estrutura primária. Mostraram ainda que o celulossoma do R. flavefaciens é montado através de um mecanismo envolvendo doquerinas com modo de ligação único mas não duplo. Isto contrasta com a maioria dos celulosomas descritos até à data, em que as doquerinas geralmente apresentam duas interfaces semelhantes de ligação à coesina, suportando um modo de ligação dupla. Tal é ilustrado pela estrutura de dois complexos coesina-doquerina do Acetivibrio cellulolyticus, envolvendo uma doquerina com modo de ligação dupla. Finalmente, esta informação estrutural foi usada para desenhar uma doquerina com dupla especificidade, mostranto a plasticidade da plataforma coesina-doquerina para o desenvolvimento de novas interações proteína:proteína.N/

Traumatismo craniano : contribuição da craniectomia descompressiva para a sobrevivência de pacientes caninos com hipertensão intracraniana traumática refractária ao tratamento médico : estudo retrospectivo

Dissertação de Mestrado Integrado em Medicina VeterináriaO traumatismo crânio-encefálico (TCE) grave está associado a uma taxa de mortalidade muito elevada e é cada vez mais comum na clínica de pequenos animais. Isto deve-se em grande parte ao crescente número de animais domésticos e consequentemente de acidentes rodoviários envolvendo esses mesmos animais. A lesão cerebral causada pelo TCE tem duas componentes essenciais: a primária que diz respeito à lesão mecânica causada pelas forças de aceleração, desaceleração, rotação e compressão que actuam sobre o crânio e cérebro no momento do impacto e a secundária que se refere a um número de alterações metabólicas e bioquímicas que se auto-perpetuam e agravam o quadro inicial. A formação de hematomas e edema cerebral de origem vascular e citotóxica vão aumentar o volume do conteúdo intracraniano o que, devido à natureza não expansível do crânio, vai levar ao aumento da pressão intracraniana (PIC) e compromisso da perfusão e oxigenação cerebrais. Quando não controlada a hipertensão intracraniana leva à hérnia cerebral e à morte do animal. A abordagem terapêutica à vítima de TCE e hipertensão intracraniana ainda é algo controversa pois não existem evidências clínicas e experimentais suficientes para elaborar um protocolo definitivo. É no entanto inquestionável que esta deverá ser expediente e agressiva. No estudo retrospectivo aqui apresentado procura-se avaliar a eficácia de uma técnica de descompressão cirúrgica como meio de reduzir a PIC e assim contribuir para a sobrevivência de pacientes vítimas de hipertensão intracraniana traumática. Foram para isso recolhidos os dados de 86 indivíduos de espécie canina vítimas de TCE fechado grave, tendo 40 deles sido submetidos a uma craniectomia descompressiva de urgência e os restantes 46 tratados apenas de forma conservativa, com recurso à hiperventilação controlada e à terapêutica hiperosmolar. As taxas de sobrevivência acumulada às 72 horas foram de 4,3% e 15% respectivamente para os grupos de controlo e cirúrgico. Não foi no entanto encontrada relação estatisticamente significativa entre a realização da craniectomia e a probabilidade de sobrevivência dos indivíduos. Concluiu-se então que não houve relação entre a realização de craniectomia descompressiva de urgência e a sobrevivência dos pacientes sendo no entanto necessários mais estudos para que se possam fazer afirmações neste sentido.ABSTRACT - THE CONTRIBUTION OF DECOMPRESSIVE CRANIECTOMY FOR THE SURVIVAL OF CANINE PATIENTS SUFFERING FROM REFRACTORY TRAUMATIC INTRACRANIAL HYPERTENSION - Severe traumatic brain injury (TBI) is associated with a very high mortality rate and its expression in small animal practice is becoming progressively higher. This is in great measure due to the always increasing number of both domestic animals and motor vehicles which translates into more road accidents involving those animals. Traumatic brain injury has two main components: the primary injury, which refers to the mechanical lesions caused by the acceleration, deceleration, rotation and compression forces that act on the brain and cranium at the moment of impact and the secondary injury, which refers to a number of self-perpetuating biochemical and metabolic changes that and aggravate the prognosis. The development of hematomas, citotoxic and vasogenic edema causes an increase in the intracranial volume that, due to the inexpansible nature of the cranium, will lead to an increased intracranial pressure (ICP) and compromised brain perfusion and oxygenation. If unattended, intracranial hypertension will lead to brain herniation and death. There is still a lot of debate surrounding the therapeutic approach to the TBI and intracranial hypertension mainly because there’s not enough clinical and experimental evidence to elaborate a standard protocol. However, few would argue that it should be expedient and aggressive. The present study aims to evaluate the role of a surgical decompression technique in reducing ICP and therefore contribute to an increased survival of patients suffering from traumatic intracranial hypertension. In order to achieve this, data from 86 canine patients suffering from severe TBI was collected. 40 of those 86 were submitted to an emergency decompressive craniectomy and the other 46 were treated conservatively using controlled hyperventilation and hyperosmolar therapy. The 72 hours post-admission cumulative survival rates where 4, 3% and 15% for the control group and surgical group, respectively. However, no statistical significant relation was found between the surgical procedure and the probability of survival. It was concluded that the emergency decompressive craniectomy did not contribute to the survival of the patients, thought further investigation is needed before any affirmations concerning this matter can be made

Chemokine-directed tumor microenvironment modulation in cancer immunotherapy

Research Areas: Biochemistry & Molecular Biology. ChemistryChemokines are a large family of small chemotactic cytokines that coordinates immune cell trafficking. In cancer, they have a pivotal role in the migration pattern of immune cells into the tumor, thereby shaping the tumor microenvironment immune profile, often towards a pro-tumorigenic state. Furthermore, chemokines can directly target non-immune cells in the tumor microenvironment, including cancer, stromal and vascular endothelial cells. As such, chemokines participate in several cancer development processes such as angiogenesis, metastasis, cancer cell proliferation, stemness and invasiveness, and are therefore key determinants of disease progression, with a strong influence in patient prognosis and response to therapy. Due to their multifaceted role in the tumor immune response and tumor biology, the chemokine network has emerged as a potential immunotherapy target. Under the present review, we provide a general overview of chemokine effects on several tumoral processes, as well as a description of the currently available chemokine-directed therapies, highlighting their potential both as monotherapy or in combination with standard chemotherapy or other immunotherapies. Finally, we discuss the most critical challenges and prospects of developing targeted chemokines as therapeutic options.info:eu-repo/semantics/publishedVersio

An individual alginate lyase is effective in the disruption of Laminaria digitata recalcitrant cell wall

Research Areas: Science & Technology - Other TopicsIn the present study, 199 pre-selected Carbohydrate-Active enZymes (CAZymes) and sulfatases were assessed, either alone or in combination, to evaluate their capacity to disrupt Laminaria digitata cell wall, with the consequent release of interesting nutritional compounds. A previously characterized individual alginate lyase, belonging to the family 7 of polysaccharide lyases (PL7) and produced by Saccharophagus degradans, was shown to be the most efcient in the in vitro degradation of L. digitata cell wall. The alginate lyase treatment, compared to the control, released up to 7.11 g/L of reducing sugars (p< 0.001) and 8.59 mmol/100 g dried alga of monosaccharides (p< 0.001), and reduced cell wall fuorescence intensity by 39.1% after staining with Calcofuor White (p= 0.001). The hydrolysis of gel-forming polymer alginate by the alginate lyase treatment could prevent the trapping of fatty acids and release benefcial monounsaturated fatty acids, particularly 18:1c9 (p < 0.001), to the extracellular medium. However, no liberation of proteins (p > 0.170) or pigments (p > 0.070) was observed. Overall, these results show the ability of an individual alginate lyase, from PL7 family, to partially degrade L. digitata cell wall under physiological conditions. Therefore, this CAZyme can potentially improve the bioavailability of L. digitata bioactive compounds for monogastric diets, with further application in feed industry.info:eu-repo/semantics/publishedVersio

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

This work was supported in part by the European Union, Area NMP.2013.1.1–2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530, and by the EU Seventh Framework Programme (FP7 2007–2013) under the WallTraC project (Grant Agreement no 263916), and BioStruct-X (grant agreement no 283570). This paper reflects the author’s views only. The European Community is not liable for any use that may be made of the information contained herein. CMGAF is also supported by Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) through grants PTDC/BIA-PRO/103980/2008 and EXPL/BIA-MIC/1176/2012. EAB is also funded by a grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel and by a grant (No. 2013284) from the U.S.-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.Peer reviewedPublisher PD

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies

the Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS) grant LA/P/0059/2020. LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Publisher Copyright: © 2022 The Author(s)The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.publishersversionepub_ahead_of_prin

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Research Areas: Science & TechnologyAnti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.info:eu-repo/semantics/publishedVersio

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin- dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dualbinding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.publishersversionpublishe

Telerreabilitação em tempo de pandemia COVID 19:Revisão Integrativa da Literatura

Introduction: COVID-19 pandemic is having an unprecedented impact on people's lives around the world. To protect vulnerable people from the virus, conventional organized rehabilitation programs offered in rehabilitation centers have been suspended in most countries. In this context, Telehealth emerges as a safe solution for patients, families, and employees. Objective: to identify the state of knowledge about the use of telerehabilitation in patients with chronic illness in the context of COVID 19 pandemic. Method: integrative literature review, carried out in June 2020. The following databases were used for data collection: SCOPUS, MEDLINE, and Academic Google. The inclusion and exclusion criteria were applied. Results: of the 364 articles identified, 15 articles were selected, all published in 2020. The data suggest benefits related to risk factors’ reduction, optimization of physical and cognitive functionality, reduction of disability, improvement social functionality and psychological well-being and prevention of isolation. Conclusions: telehealth helps to promote early rehabilitation, increases confidence in recovery and improves self-management resources from the disease and contributes to decrease of isolation