Updating the role of matrix metalloproteinases in mineralized tissue and related diseases.

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of "smart" medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer.

Proteomic analysis of the acquired enamel pellicle formed on human and bovine tooth: a study using the Bauru in situ pellicle model (BISPM).

OBJECTIVES: The acquired enamel pellicle (AEP) is an organic film, bacteria-free, formed in vivo as a result of the selective adsorption of salivary proteins and glycoproteins to the solid surfaces exposed to the oral environment. Objective: This study aimed to compare the proteomic profile of AEP formed in situ on human and bovine enamel using a new intraoral device (Bauru in situ pellicle model - BISPM). MATERIAL AND METHODS: Material and Methods: One hundred and eight samples of human and bovine enamel were prepared (4×4 mm). Nine subjects with good oral conditions wore a removable jaw appliance (BISPM) with 6 slabs of each substrate randomly allocated. The AEP was formed during the morning, for 120 minutes, and collected with an electrode filter paper soaked in 3% citric acid. This procedure was conducted in triplicate and the pellicle collected was processed for analysis by LC-ESI-MS/MS. The obtained mass spectrometry MS/MS spectra were searched against human protein database (SWISS-PROT). RESULTS: Results: The use of BISPM allowed the collection of enough proteins amount for proper analysis. A total of 51 proteins were found in the AEP collected from the substrates. Among them, 15 were common to both groups, 14 were exclusive of the bovine enamel, and 22 were exclusive of the human enamel. Proteins typically found in the AEP were identified, such as Histatin-1, Ig alpha-1, Ig alpha 2, Lysozyme C, Statherin and Submaxillary gland androgen-regulated protein 3B. Proteins not previously described in the AEP, such as metabolism, cell signaling, cell adhesion, cell division, transport, protein synthesis and degradation were also identified. CONCLUSIONS: Conclusion: These results demonstrate that the proteins typically found in the AEP appeared in both groups, regardless the substrate. The BISPM revealed to be a good device to be used in studies involving proteomic analysis of the AEP.

Standardization of a protocol for shotgun proteomic analysis of saliva.

INTRODUCTION: Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. OBJECTIVE: This study compared methodologies to extract salivary proteins for proteomic analysis. MATERIAL AND METHODS: Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. RESULTS: In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. CONCLUSIONS: The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.

Standardization of a protocol for shotgun proteomic analysis of saliva

Abstract Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. Objective This study compared methodologies to extract salivary proteins for proteomic analysis. Material and Methods Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. Results In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. Conclusions The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.

The proteomic profile of the acquired enamel pellicle according to its location in the dental arches.

OBJECTIVE: This study evaluated the variation in the protein profile of the acquired enamel pellicle (AEP) formed in vivo according to its location in the dental arches. DESIGN: The AEP was formed for 120min in 9 volunteers. Pellicle formed at upper+lower anterior facial (ULAFa; teeth 13-23 and 33-43), upper anterior palatal (UAPa; teeth 13-23), lower anterior lingual (LALi; teeth 33-43), upper+lower posterior facial (ULPFa; teeth 14-17 24-27, 34-37 and 44-47), upper posterior palatal (UPPa; teeth 14-17 and 24-27) and lower posterior lingual (LPLi; teeth 34-37 and 44-47) regions were collected separately and processed for analysis by label-free LC-ESI-MS/MS. RESULTS: Three-hundred sixty three proteins were identified in total, twenty-five being common to all the locations, such as Protein S100-A8, Lysozyme C, Lactoferrin, Statherin, Ig alpha-2, ALB protein, Myeloperoxidase and SMR3B. Many proteins were found exclusively in the AEP collected from one of the regions (46-UAPa, 33-LALi, 59-ULAFa, 31-ULPFa, 44-LPLi and 39-UPPa). CONCLUSIONS: The protein composition of the AEP varied according to its location in the dental arches. These results provide important insights for understanding the differential protective roles of the AEP as a function of its location in the dental arches.

Detecção de alterações na composição da película adquirida em função da sua localização na cavidade bucal: estudo proteômico / Detecção de alterações na composição da película adquirida em função da sua localização na cavidade bucal: estudo proteômicox

A película adquirida do esmalte (PAE) é um filme orgânico, livre de bactérias, formado in vivo como resultado da adsorção seletiva de proteínas salivares sobre a superfície do dente, contendo também glicoproteínas e lipídeos. A presença de proteínas na PAE forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal. O objetivo deste trabalho foi detectar alterações no perfil proteico da PAE formada in vivo de acordo com a sua localização nos arcos dentários. Fizeram parte da pesquisa 9 voluntários, com idade entre 18 e 35 anos, não fumantes, com bom estado de saúde geral e bucal. A película adquirida foi formada no período da manhã, por 120 minutos, após profilaxia com pedra pomes. Películas formadas nas regiões anterior vestibular superior e inferior (ULALa; dentes 13-23 e 33-43), anterior palatina superior (UAPa; dentes 13-23), anterior lingual inferior (LALi; dentes 33-43), posterior vestibular superior e inferior (ULPLa; dentes 14-17, 24-27, 34-37 e 44-47), posterior palatina superior (UPPa; dentes 14-17 e 24-27) e posterior lingual inferior (LPLi; dentes 34-37 e 44-47) foram coletadas separadamente para análise. Após a sua formação, a película foi coletada em papel filtro embebido em ácido cítrico a 3% e processada para análise por LC-ESI-MS/MS. Os espectros MS/MS obtidos foram confrontados com bases de dados de proteínas humanas (SWISS PROT). A quantificação livre de marcadores foi feita utilizando o software PLGS. Um total de 363 proteínas foi encontrado, sendo 252 proteínas únicas de cada grupo e 25 proteínas comuns entre eles (como Protein S100-A8, Lysozyme C, Lactoferrin, Sthatherin, Ig alpha-2 chain C, ALB protein, Myeloperoxidase and Submaxillary gland androgen-regulated protein 3B). Na análise quantitativa, nove comparações foram realizadas e muitas proteínas foram diferentemente expressas entre os grupos, demonstrando assim que a localização na cavidade bucal pode alterar a composição da película adquirida do esmalte. Foram encontradas tanto proteínas típicas da película quanto proteínas não anteriormente descritas na película, cuja função na película foi inferida com base na literatura. Em conclusão houve diferença na composição proteica da película adquirida de acordo com a localização dos arcos dentários. Esses dados devem ser levados em conta quando se pensa no potencial protetor da película adquirida contra a desmineralização dentária, uma vez que esses resultados fornecem informações importantes para a compreensão dos diferentes papeis protetores da AEP dependendo da sua localização nos arcos dentários.(AU)

The acquired enamel pellicle (AEP) is a bacteria-free organic film formed in vivo as a result of selective adsorption of salivary proteins on the surface of the tooth. It also contains glycoproteins and lipids. The presence of proteins in the AEP forms a protective interface on the tooth surface that participates in the interfacial events that occur in the oral cavity. The objective of this study was to detect changes in the protein profile of the AEP formed in vivo according to its location in the dental arches. Nine volunteers, aged 18 to 35 years, non-smokers, with good general and oral health participated in the study. The acquired pellicle was formed in the morning, for 120 minutes, after prophylaxis with pumice. Pellicle formed at upper and lower anterior labial (ULALa; teeth 13-23 and 33-43), upper anterior palatal (UAPa; teeth 13-23), lower anterior lingual (LALi; teeth 33-43), upper and lower posterior labial (ULPLa; teeth 14-17 24 to 27, 34 to 37 and 44 to 47), upper posterior palatal (UPPa; teeth 14 to 17 and 24 to 27) and lower posterior lingual (LPLi; teeth 34 to 37 and 44 to 47) regions was collected separately for analysis. After its formation, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by LC-ESI-MS/MS. The MS/MS spectra obtained were compared with human protein databases (SWISS PROT). Label-free quantification was done using the PLGs software. A total of 363 proteins were found, of which 252 are unique proteins for one of the regions, while 25 proteins care to all of them, including Protein S100-A8, Lysozyme C, Lactoferrin, Sthatherin, Ig alpha-2 chain C, ALB protein, Myeloperoxidase and Submaxillary gland androgen-regulated protein 3B. In the quantitative analysis, 9 comparisons were made and many proteins were differently expressed among the groups, thus demonstrating that the location in the dental arches can change the composition of the AEP. Some proteins not previously found in the AEP were identified and their function in the AEP was inferred from the literature. In conclusion, the composition of the AEP changes as a function of its location in the dental arches. These data should be taken into account when we think about the protective potential of the acquired pellicle against tooth demineralization and provide important insights for understanding the differential protective roles of the AEP as a function of its location in the dental arches.(AU)