Caracterização bioquímica de uma lipase recombinante de Staphylococus xylosus e detecção dos mecanismos estruturais envolvidos em sua termotolerância

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-graduação em Biotecnologia, Florianópolis, 2010As lipases são enzimas altamente versáteis que catalisam reações químicas com diversos substratos em diferentes condições e possuem características tanto bioquímicas quanto estruturais que lhes conferem resistência e termotolerância, além de serem empregadas em vários bioprocessos industriais como na produção de alimentos e biocombustíveis. A lipase de S. xylosus (AF208229) foi recentemente isolada e inicialmente caracterizada por nosso grupo de pesquisa e colaboradores. Essa proteína possui uma alta homologia e identidade estrutural com diversas outras lipases da família I.5, e apresenta atividade enzimática sobre p-nitrofenil-ésteres. Apesar de ser considerada uma enzima moderadamente termoestável e ter atividade enzimática sobre diferentes ésteres, o mecanismo de ação catalítica ainda permanece desconhecido, e principalmente, os mecanismos estruturais envolvidos em sua estabilização térmica nunca foram descritos. O objetivo deste trabalho foi caracterizar bioquimicamente a lipase recombinante AF208229 de S. xylosus recentemente clonada e expressa heterologamente, e investigar os possíveis mecanismos envolvidos em sua termotolerância por meio de análises de espectroscopia de dicroísmo circular (DC). Nesse trabalho a lipase de S. xylosus (AF208229) foi expressa e purificada, submetida a ensaios enzimáticos em três temperaturas (25°C, 37°C e 42°C), e três pH (7,0 8,0 e 9,0), sua especificidade catalítica foi analisada utilizando-se diferentes p-nitrofenil-ésteres como substratos (de 2 a 18 carbonos) e sua termoestabilidade foi acompanhada durante 10, 20 e 30 minutos em 95°C. Para as análises espectroscópicas, a enzima foi submetida a um tratamento de remoção de íons (produzindo-se a apo enzima) e sua atividade enzimática e sua termotolerância foram reavaliadas. Por fim o espectro de dicroísmo circular (DC) da apo e da holo_enzima foram analisados em diferentes condições. Os resultados obtidos apresentam pela primeira vez o perfil estrutural e os mecanismos de estabilidade 8 térmica dessa lipase. O perfil de DC da holo_enzima foi avaliado nas mesmas temperaturas descritas para os ensaios enzimáticos, bem como sua capacidade de re-enovelamento. O perfil de DC e a capacidade de re-enovelamento também foram observados para a apo_enzima. O efeito dos cofatores metálicos Zn2+ e Ca2+ foram avaliados nos ensaios enzimáticos da apo_enzima, na sua termotolerância, no seu perfil de DC e na sua capacidade de re-enovelamento. Observou-se que a holo_enzima apresenta sua maior atividade em pH 9,0 e 42°C, que tem preferência por substratos de cadeias curtas (pNP-acetato, 2 carbonos) e que retem 77% de sua atividade enzimática após 10 minutos de tratamento térmico (95°C). A holo_enzima é capaz de se re-enovelar, readquirindo sua conformação estruturalmente estável e ativa. A apo_enzima também é capaz de se re-enovelar após o tratamento térmico, porém perde quase totalmente sua atividade. Observou-se que na presença do íon Zn2+ a apo_enzima se re-enovelou e reteve sua atividade enzimática. Com base nesses resultados poderia afirmar-se que a lipase de S. xylosus (AF208229) é uma metaloenzima dependente de zinco e que esse íon possivelmente localizado em um motivo estrutural rico em -hélices próximo ao ativo site.Lipases are highly versatile enzymes, which catalyze many chemical reactions with different substrates in different conditions. These enzymes have particular biochemical and structural characteristiscs which promote stability and thermal resistance. Besides, they are used by industries, i.e: food technology, detergents and biofuels. S. xylosus lipase (AF208229) was recently isolated and characterized by our group. This enzyme shares a high homology with other lipases among I.5 lipases family and has activity on p-nitrophenyl esters. Although considered a moderated thermostable lipase, its catalytic function remains unclear and its structural mechanisms for thermal stabilization have never been described. This work assessed the enzymatic activity of the recombinant lipase from S. xylosus in three different temperatures (25°C, 37°C and 42°C) and pH (7.0, 8.0 and 9.0), and the thermostability (incubation at 95°C for 10, 20 and 30 minutes). In order to investigate the mechanism involved in enzymatic activation, the enzyme was submitted to an ion removal treatment (to obtain an apo_- enzyme) and its activity and themotolerance were evaluated in the presence and absence of Zn2+ and Ca2+. The structural profile of apo-_enzyme and holo_enzyme were assessed by circular dichroism (CD) at different conditions in the presence an in the absence of metal cofactors as: Zn2+ and Ca2+. These results represent the first insights on S. xylosus lipases secondary structure and its mechanism for thermal stabilization. S. xylosus lipase (AF208229) was more active in pH 9.0, 42°C with specificity for short-chain substrates (pNP-acetate C2). In addition, this enzyme was able to maintain 77% of its activity after a thermal treatment (95°C for 10 min) and to refold to its stable conformation. apo_enzyme showed the same ability to refold as seen to holo_enzyme, but was not able to keep its activity after thermal treatment, which indicate the need of metal for its activity stabilization. Interesting, in the presence of Zn2+ the apo_enzyme was able to acquire a more stable conformation after thermal treatment and still keep residual activity. 10 These results propose that S. xylosus lipase (AF208229) is a metalloenzyme which uses Zn2+ as its metal coordinating, and the Zn2+ is possibly located in a-helical rich motif close to the active site

Vitamin D supplementation in the prevention and management of major chronic diseases not related to mineral homeostasis in adults : research for evidence and a scientific statement from the European society for clinical and economic aspects of osteoporosis and osteoarthritis (ESCEO)

Introduction: Optimal vitamin D status promotes skeletal health and is recommended with specific treatment in individuals at high risk for fragility fractures. A growing body of literature has provided indirect and some direct evidence for possible extraskeletal vitamin D-related effects. Purpose and Methods: Members of the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis have reviewed the main evidence for possible proven benefits of vitamin D supplementation in adults at risk of or with overt chronic extra-skeletal diseases, providing recommendations and guidelines for future studies in this field. Results and conclusions: Robust mechanistic evidence is available from in vitro studies and in vivo animal studies, usually employing cholecalciferol, calcidiol or calcitriol in pharmacologic rather than physiologic doses. Although many cross-sectional and prospective association studies in humans have shown that low 25-hydroxyvitamin D levels (i.e., 50 nmol/L, did not simultaneously assess multiple outcomes, and did not report overall safety (e.g., falls). Thus, no recommendations can be made to date for the use of vitamin D supplementation in general, parental compounds, or non-hypercalcemic vitamin D analogs in the prevention and treatment of extra-skeletal chronic diseases. Moreover, attainment of serum 25-hydroxyvitamin D levels well above the threshold desired for bone health cannot be recommended based on current evidence, since safety has yet to be confirmed. Finally, the promising findings from mechanistic studies, large cohort studies, and small clinical trials obtained for autoimmune diseases (including type 1 diabetes, multiple sclerosis, and systemic lupus erythematosus), cardiovascular disorders, and overall reduction in mortality require further confirmation

Structural stability of Staphylococcus xylosus lipase is modulated by Zn2+ ions

AbstractLipases are well-known enzymes extensively used in industrial biotransformation processes. Besides, their structural and catalytic characteristics have attracted increasing attention of several industries in the last years. In this work, we used biophysical and molecular modeling tools to assess structural properties of Staphylococcus xylosus lipase (SXL). We studied the thermal unfolding of this protein and its zinc-dependent thermotolerance. We demonstrated that SXL is able to be active and stable at moderate temperatures, but this feature is only acquired in the presence of Zn2+. Such characteristic indicates SXL as a zinc-dependent metallolipase

Protein S-nitrosylation: specificity and identification strategies in plants

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the Biotin Switch Technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed

Synthetic compounds from an in house library as inhibitors of falcipain-2 from Plasmodium falciparum

Falcipain-2 (FP-2) is a key cysteine protease from the malaria parasite Plasmodium falciparum. Many previous studies have identified FP-2 inhibitors; however, none has yet met the criteria for an antimalarial drug candidate. In this work, we assayed an in-house library of non-peptidic organic compounds, including (E)-chalcones, (E)-N'-benzylidene-benzohydrazides and alkyl-esters of gallic acid, and assessed the activity toward FP-2 and their mechanisms of inhibition. The (E)-chalcones 48, 54 and 66 showed the lowest IC50 values (8.5±0.8μM, 9.5±0.2μM and 4.9±1.3μM, respectively). The best inhibitor (compound 66) demonstrated non-competitive inhibition, and using mass spectrometry and fluorescence spectroscopy assays, we suggest a potential allosteric site for the interaction of this compound, located between the catalytic site and the hemoglobin binding arm in FP-2. We combined structural biology tools and mass spectrometry to characterize the inhibition mechanisms of novel compounds targeting FP-2.Fil: Bertoldo, Jean Borges. Universidade Federal de Santa Catarina; BrasilFil: Chiaradia Delatorre, Louise Domeneghini. Universidade Federal de Santa Catarina; BrasilFil: Mascarello, Alessandra. Universidade Federal de Santa Catarina; BrasilFil: Leal, Paulo César. Universidade Federal de Santa Catarina; BrasilFil: Sechini Cordeiro, Marlon Norberto. Universidade Federal de Santa Catarina; BrasilFil: Nunes, Ricardo José. Universidade Federal de Santa Catarina; BrasilFil: Salas Sarduy, Emir. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de La Habana; CubaFil: Rosenthal, Philip Jon. University of California; Estados UnidosFil: Terenzi, Hernán. Universidade Federal de Santa Catarina; Brasi

Chemo- and Regioselective Lysine Modification on Native Proteins.

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Ka was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality