Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Several pathogens, including spirochetes, have been shown to express surface proteins that interact with the extracellular matrix (ECM). This adhesin-mediated binding process seems to be a crucial step in the colonization of host tissues. This study examined the interaction of putative leptospiral outer membrane proteins with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. Six predicted coding sequences selected from the Leptospira interrogans serovar Copenhageni genome were cloned, and proteins were expressed, purified by metal affinity chromatography, and characterized by circular dichroism spectroscopy. Their capacity to mediate attachment to ECM components was evaluated by binding assays. We have identified a leptospiral protein encoded by LIC12906, named Lsa24 (leptospiral surface adhesin; 24 kDa) that binds strongly to laminin. Attachment of Lsa24 to laminin was specific, dose dependent, and saturable. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. Triton X-114-solubilized extract of L. interrogans and phase partitioning showed that Lsa24 was exclusively in the detergent phase, indicating that it is a component of the leptospiral membrane. Moreover, Lsa24 partially inhibited leptospiral adherence to immobilized laminin. This newly identified membrane protein may play a role in mediating adhesion of L. interrogans to the host. To our knowledge, this is the first leptospiral adhesin with laminin-binding properties reported to date.

SUMMARYInterleukin-8 (IL-8) is regarded as an important mediator of inflammation because of its potent and specific chemotactic activity on neutrophils. In the present investigation, human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce IL-8, in a dose-and timedependent manner. After stimulation with 10 U/ml thrombin for 24 hr, the level of IL-8 in the conditioned medium was 14 ng/ml, or enough to elicit PMN chemotaxis in vitro. Northern blot analysis revealed that thrombin as well as IL-1b elevates the level of IL-8 mRNA preceding the formation of IL-8 protein.A synthetic peptide SFLLRN [human thrombin receptor-activating peptide (TRAP)] was found to mimic the action of thrombin. Preincubation with anti-thrombin compounds such as hirudin and antithrombin-III-heparin almost completely suppressed the action of thrombin without affecting the actions of other stimuli including IL-1b, phorbol 12-myristate 13-acetate (PMA) and TRAP. Diisopropylfluorophosphate-treated thrombin did not stimulate IL-8 production. Calphostin-C, a protein kinase C (PKC) inhibitor, attenuated the production of IL-8 by thrombin, TRAP and PMA, but left the action of IL-1b unchanged. These results strongly suggest that catalytic activation of thrombin receptor by thrombin results in PKC-dependent IL-8 production accompanied by an increase in IL-8 mRNA level.

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Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.

AgradecimentosÀ Denise de Castro Fernandes pela orientação, inspiração, compreensão e apoio em todos os momentos nessa fase, exemplo de pessoa que sempre irei me inspirar.Ao Prof. Francisco Laurindo pelo apoio e as conversas científicas, que me inspiraram sobre o que é realmente fazer ciência.À minha família (Cecília, Eduardo, Marcela, Yoshio e Ytiro) pelo apoio e compreensão incondicional.Ao Danilo por estar ao meu lado, com carinho e compreensão, nesta etapa que certamente será importante para futuras etapas em nossa vida.Ao João Wosniak Jr pelo apoio e ajuda cultivo celular, na otimização de condições experimentais e em todas as minhas apresentações.À Maria Bertoline, que sempre me ajudou no HPLC e pela carinhosa companhia no laboratório.Aos colegas de laboratório: Ana Moretti, Angélica, Carol, Daniela, Jéssyca, Júlia, Laura, Leonora, Luciana, Raquel, Renata, Phelipe, Thais, Thalita, Vanda, Victor. substantially from in vitro studies using purified PDI and chimeras. In these experimental scenarios, PDI reductase and chaperone are readily approachable. LISTA DE ABREVIATURASHowever, isomerase activity, the hallmark of PDI family, is significantly complex.Assessment of PDI roles in cells and tissues mainly relies on gain-or loss-of-function experiments. However, there is limited information regarding correlation of these results with PDI activities. In this manuscript, we put together the main methods described for measuring the four PDI activities: thiol reductase, thiol oxidase, thiol isomerase and chaperone, with emphasis on controls and critical interferents, such as detergentcontaining buffers. We also discuss the transposition of these methods from purified PDI to cellular or in vivo samples, with critical thoughts about the interpretation of results.Keywords: Protein disulfide isomerase, Chaperone, Isomerization, Oxidation. INTRODUÇÃO Estrutura da Dissulfeto Isomerase ProteicaA Dissulfeto Isomerase Proteíca -PDI (também denominada PDIA1 ou no genoma humano, gene P4HB) é uma chaperona redox pertencente ao grupo de ditiol-proteínas da superfamília da tiorredoxina ( Fig. 1 A PDI é uma proteína de 55 kDa formada por quatro domínios tiorredoxina (denominados a-b-b'-a '), sendo que os sítios catalíticos contém 2 cisteínas (motivo WCGHC) e estão presentes nos dois domínios a e a' (Fig. 1 Estudos de cristalização da PDI de levedura realizados em diferentes temperaturas (4°C versus 22°) (Tian e col., 2006; Tian e col., 2008) descreveram uma proteína altamente flexível, que pode portanto, ajustar sua estrutura para ligar substratos de diferentes tamanhos e conformações, de tal forma que permite a acessibilidade dos sítios redox da PDI para tióis críticos dos substratos. Nestes trabalhos observou-se que a PDI de levedura pode apresentar duas distintas conformações, dependendo da temperatura de cristalização: i) uma forma em "U", na qual os dois sítios catalíticos estão frente a frente e os resíduos hidrofóbicos estão na base rígida do "U" (Fig. 3) ou ii) uma forma aberta, denominada "forma de barco". Além disso, ...