The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

O trabalho ora apresentado foi concebido como tendo dois objetivos. O primeiro, mais geral, foi implementar uma nova metodologia para o estudo de mudanças conformacionais em proteínas, ou seja, de sua dinâmica estrutural. A técnica de marcação de spin sítio dirigida aliada à ressonância paramagnética eletrônica (SDSL-RPE) são os pilares desse novo método que faz, agora, parte do conjunto de técnicas disponíveis no Grupo de Biofísica Molecular Sérgio Mascarenhas do Instituto de Física de São Carlos (USP). O segundo objetivo, mais específico, representou o caminho efetivamente tomado para que se alcançasse o objetivo geral. Para isso, foi proposto o estudo da correlação estrutura e função de dois sistemas biológicos muito interessantes. O primeiro deles envolveu o estudo do movimento das hélices que compõem a estrutura da proteína ligante de cálcio S100A12 humana (HS100A12) induzido pelos íons cálcio e zinco. Sabendo que a proteína S100A12 humana além de ligar íons Ca+2, apresenta afinidade por outros metais divalentes, como os íons Zn+2 e Cu+2, e que a formação de diferentes oligômeros da proteína é governada pela concentração dos íons Ca+2 e Zn+2, realizamos estudos espectroscópicos utilizando a técnica de dicroísmo circular a fim de investigarmos a estabilidade térmica da proteína HS100A12 na presença e ausência dos íons cálcio e zinco. Mudanças conformacionais na estrutura da HS100A12 foram monitoradas através da construção de uma série de mutantes (simples e duplos) em que resíduos nas hélices B, C e D foram trocados por cisteínas, subsequentemente marcadas com a sonda magnética MTSSL e submetidas às análises de SDSL-RPE. Estas consistiram na medida do espectro de RPE dos vários mutantes em temperatura ambiente para estudarmos os efeitos da presença dos íons sobre a dinâmica experimentada pela sonda nas diversas posições. Além disso, efetuamos medidas de distância entre duas sondas seletivamente inseridas na estrutura protéica, procurando assim complementar o entendimento acerca do efeito da presença dos íons sobre a proteína. Por fim, devido ao fato da proteína HS100A12 estar envolvida em alguns eventos de sinalização celular e interação com o receptor para produtos de glicosilação (RAGE), decidimos também, estudar a interação da proteína com modelos de biomembranas, utilizando monocamadas de Langmuir. O outro problema de interesse utilizou a lizosima do fago T4, uma proteína padrão, da qual uma variedade de mutantes é produzida rotineiramente a fim de obtermos mais detalhes a respeito da sua correlação estrutura e função e tornar mais sólido o entendimento da técnica SDSL. Inicialmente, realizamos um estudo com a suposta criação de uma cavidade no \"core\" hidrofóbico da porção C-terminal da enzima, quando mutamos a Leu 133 por Ala e/ou Gly, ou seja, quando trocamos um resíduo grande por um de menor volume, pois se acredita que a proteína sofra um reajuste estrutural com o intuito de preencher o espaço vazio criado por essa substituição. Para isso, propusemos estudar por SDSL o movimento da α-hélice H inserindo o marcador de spin na posição vizinha ao resíduo mutado. Adicionalmente, realizamos um experimento de \"transmutação\" com a enzima T4L, a fim de investigar a natureza das contribuições para os diferentes modos dinâmicos experimentados pelo marcador de spin quando introduzido em sítios topologicamente semelhantes. / The work presented here was conceived with two main objectives. The first one, more general, involved the implementation of a new methodology for the study of conformational changes in proteins, i.e., its structural dynamics. The technique of Site-directed Spin Labeling combined with Electronic Paramagnetic Resonance (SDSL-EPR) are the pillars of this new method, which is now part of the set of techniques available at the Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos (USP). The second objective, more specific, represented the path actually taken to achieve the overall goal. Therefore, it was proposed to study the structure-function correlation in two interesting biological systems. The first involved the study of the movement of the helices that form the structure of the human calcium binding protein S100A12 (HS100A12) induced by calcium and zinc ions. Knowing that, besides Ca+2, human S100A12 has also affinity for other divalent metals, such as Zn+2 and Cu+2 ions, and that the formation of different protein oligomers is governed by the concentration of Ca+2 and Zn+2, we performed spectroscopic studies using circular dichroism (CD) to investigate the thermal stability of protein HS100A12 in the presence and absence of calcium and zinc. Conformational changes in the structure of HS100A12 were monitored by producing a series of mutants (singles and doubles) in which residues in helices B, C and D were replaced by cysteine and subsequently labeled with a magnetic probe MTSSL and then analyzed via SDSL-EPR. The latter consisted of the EPR spectra measurement of many mutants at room temperature to study the effects of the presence of ions on the dynamics experienced by the probe in different positions. In addition, we performed measurements of the distance between two probes inserted in the protein structure, thereby, seeking to improve the understanding of the effect of the ions presence on the protein. Finally, due to the fact that HS100A12 is involved in some events of cell signaling and interaction with the Receptor for Advanced Glycation End Products (RAGE), we also decided to study the interaction of protein with models of biomembranes using Langmuir monolayers. In the other problem of interest, we used a variety of mutants of the enzyme T4 lysozyme, a protein standard, in order to obtain more details about its structure-function correlation and make more solid the understanding of SDSL technique. Initially, we conducted a study about the alleged creation of a cavity in the hydrophobic C-terminal portion of the enzyme, when we replaced the Leu 133 by Ala and/or Gly, or when we changed a large residue for a smaller one, because it is believed that the protein undergoes a structural adjustment in order to fill the gap created by this substitution. For this, we studied by SDSL the α-helix H motion, inserting the spin label in a neighbor position of the mutated residue. Additionally, we performed an experiment of \"transmutation\" with the enzyme T4L in order to investigate the nature of contributions for different dynamic modes experienced by the spin label when it is introduced in topologically similar sites.

O trabalho ora apresentado foi concebido como tendo dois objetivos. O primeiro, mais geral, foi implementar uma nova metodologia para o estudo de mudanças conformacionais em proteínas, ou seja, de sua dinâmica estrutural. A técnica de marcação de spin sítio dirigida aliada à ressonância paramagnética eletrônica (SDSL-RPE) são os pilares desse novo método que faz, agora, parte do conjunto de técnicas disponíveis no Grupo de Biofísica Molecular Sérgio Mascarenhas do Instituto de Física de São Carlos (USP). O segundo objetivo, mais específico, representou o caminho efetivamente tomado para que se alcançasse o objetivo geral. Para isso, foi proposto o estudo da correlação estrutura e função de dois sistemas biológicos muito interessantes. O primeiro deles envolveu o estudo do movimento das hélices que compõem a estrutura da proteína ligante de cálcio S100A12 humana (HS100A12) induzido pelos íons cálcio e zinco. Sabendo que a proteína S100A12 humana além de ligar íons Ca+2, apresenta afinidade por outros metais divalentes, como os íons Zn+2 e Cu+2, e que a formação de diferentes oligômeros da proteína é governada pela concentração dos íons Ca+2 e Zn+2, realizamos estudos espectroscópicos utilizando a técnica de dicroísmo circular a fim de investigarmos a estabilidade térmica da proteína HS100A12 na presença e ausência dos íons cálcio e zinco. Mudanças conformacionais na estrutura da HS100A12 foram monitoradas através da construção de uma série de mutantes (simples e duplos) em que resíduos nas hélices B, C e D foram trocados por cisteínas, subsequentemente marcadas com a sonda magnética MTSSL e submetidas às análises de SDSL-RPE. Estas consistiram na medida do espectro de RPE dos vários mutantes em temperatura ambiente para estudarmos os efeitos da presença dos íons sobre a dinâmica experimentada pela sonda nas diversas posições. Além disso, efetuamos medidas de distância entre duas sondas seletivamente inseridas na estrutura protéica, procurando assim complementar o entendimento acerca do efeito da presença dos íons sobre a proteína. Por fim, devido ao fato da proteína HS100A12 estar envolvida em alguns eventos de sinalização celular e interação com o receptor para produtos de glicosilação (RAGE), decidimos também, estudar a interação da proteína com modelos de biomembranas, utilizando monocamadas de Langmuir. O outro problema de interesse utilizou a lizosima do fago T4, uma proteína padrão, da qual uma variedade de mutantes é produzida rotineiramente a fim de obtermos mais detalhes a respeito da sua correlação estrutura e função e tornar mais sólido o entendimento da técnica SDSL. Inicialmente, realizamos um estudo com a suposta criação de uma cavidade no \"core\" hidrofóbico da porção C-terminal da enzima, quando mutamos a Leu 133 por Ala e/ou Gly, ou seja, quando trocamos um resíduo grande por um de menor volume, pois se acredita que a proteína sofra um reajuste estrutural com o intuito de preencher o espaço vazio criado por essa substituição. Para isso, propusemos estudar por SDSL o movimento da α-hélice H inserindo o marcador de spin na posição vizinha ao resíduo mutado. Adicionalmente, realizamos um experimento de \"transmutação\" com a enzima T4L, a fim de investigar a natureza das contribuições para os diferentes modos dinâmicos experimentados pelo marcador de spin quando introduzido em sítios topologicamente semelhantes. / The work presented here was conceived with two main objectives. The first one, more general, involved the implementation of a new methodology for the study of conformational changes in proteins, i.e., its structural dynamics. The technique of Site-directed Spin Labeling combined with Electronic Paramagnetic Resonance (SDSL-EPR) are the pillars of this new method, which is now part of the set of techniques available at the Grupo de Biofísica Molecular Sérgio Mascarenhas, Instituto de Física de São Carlos (USP). The second objective, more specific, represented the path actually taken to achieve the overall goal. Therefore, it was proposed to study the structure-function correlation in two interesting biological systems. The first involved the study of the movement of the helices that form the structure of the human calcium binding protein S100A12 (HS100A12) induced by calcium and zinc ions. Knowing that, besides Ca+2, human S100A12 has also affinity for other divalent metals, such as Zn+2 and Cu+2 ions, and that the formation of different protein oligomers is governed by the concentration of Ca+2 and Zn+2, we performed spectroscopic studies using circular dichroism (CD) to investigate the thermal stability of protein HS100A12 in the presence and absence of calcium and zinc. Conformational changes in the structure of HS100A12 were monitored by producing a series of mutants (singles and doubles) in which residues in helices B, C and D were replaced by cysteine and subsequently labeled with a magnetic probe MTSSL and then analyzed via SDSL-EPR. The latter consisted of the EPR spectra measurement of many mutants at room temperature to study the effects of the presence of ions on the dynamics experienced by the probe in different positions. In addition, we performed measurements of the distance between two probes inserted in the protein structure, thereby, seeking to improve the understanding of the effect of the ions presence on the protein. Finally, due to the fact that HS100A12 is involved in some events of cell signaling and interaction with the Receptor for Advanced Glycation End Products (RAGE), we also decided to study the interaction of protein with models of biomembranes using Langmuir monolayers. In the other problem of interest, we used a variety of mutants of the enzyme T4 lysozyme, a protein standard, in order to obtain more details about its structure-function correlation and make more solid the understanding of SDSL technique. Initially, we conducted a study about the alleged creation of a cavity in the hydrophobic C-terminal portion of the enzyme, when we replaced the Leu 133 by Ala and/or Gly, or when we changed a large residue for a smaller one, because it is believed that the protein undergoes a structural adjustment in order to fill the gap created by this substitution. For this, we studied by SDSL the α-helix H motion, inserting the spin label in a neighbor position of the mutated residue. Additionally, we performed an experiment of \"transmutation\" with the enzyme T4L in order to investigate the nature of contributions for different dynamic modes experienced by the spin label when it is introduced in topologically similar sites.

Rheumatoid arthritis is a chronic inflammatory autoimmune disease. Measurement of the level of serum markers (sRAGE, S100A9, S100A8 and S100A12) and genetic testing for the presence of the PTPN22 genetic polymorphism could help elucidate the underlying cause of inflammation and complications in RA, such as atherosclerosis. Therefore, serum levels of sRAGE, S100A9, S100A8 and S100A12 were measured by ELISA in patients with established RA (n=138). The associations between the serum levels of these molecules; and inflammatory markers and RA complications were analysed by multiple linear regression modelling. Established RA patients (n=192) were investigated for the PTPN22 C1858T genetic polymorphism by PCR-RFLP. Multiple logistic regression modelling was used to examine the association between PTPN22 C1858T genetic polymorphism and inflammatory markers and RA complications. In RA patients, we found that serum levels of S100A9 were associated with the body mass index (BMI); and the presence of S100A8 and S100A12. The serum levels of S100A8 in RA patients were associated with the presence of anti-citrullinated peptide antibodies, rheumatoid factor and S100A9. The serum levels of S100A12 in RA patients were associated with the presence of anti-citrullinated peptide antibodies and S100A9; and a history of diabetes. Inflammatory markers and RA complications were not associated with the PTPN22 genetic polymorphism in established RA patients; serum level of triglyceride was the only variable associated with PTPN22 C1858T in multiple logistic regression analysis. Taken together, these data suggest that serum levels of sRAGE, S100A9 and S100A12 protein may be useful correlates of inflammation and autoantibody production in RA patients. Further studies are recommended to determine whether these markers predict clinical outcomes when measured at the onset of RA.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand". Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand”. Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

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Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...

Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...