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1.
Biochim Biophys Acta Proteins Proteom ; 1869(8): 140659, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33839314

RESUMO

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.


Assuntos
Saliva/microbiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/microbiologia , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Metagenômica/métodos , Microbiota/genética , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Neoplasias Bucais/microbiologia , Prognóstico , Proteômica/métodos , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo
2.
PLoS One ; 15(12): e0243867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338036

RESUMO

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Proteômica , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Bacterianos , Anotação de Sequência Molecular , Fosfoglucomutase/metabolismo , Reprodutibilidade dos Testes , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
3.
Redox Biol ; 37: 101735, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33011677

RESUMO

The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1. We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cytoF730A) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys824 at the C-terminal of ADAM17cyto with the Cys73 of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.


Assuntos
Proteína ADAM17 , Cisteína , Tiorredoxinas , Cisteína/metabolismo , Células HEK293 , Humanos , Conformação Molecular , Oxirredução , Compostos de Sulfidrila , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
4.
Mol Cell Proteomics ; 20: 100004, 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33578082

RESUMO

Protease activity has been associated with pathological processes that can lead to cancer development and progression. However, understanding the pathological unbalance in proteolysis is challenging because changes can occur simultaneously at protease, their inhibitor, and substrate levels. Here, we present a pipeline that combines peptidomics, proteomics, and peptidase predictions for studying proteolytic events in the saliva of 79 patients and their association with oral squamous cell carcinoma (OSCC) prognosis. Our findings revealed differences in the saliva peptidome of patients with (pN+) or without (pN0) lymph-node metastasis and delivered a panel of ten endogenous peptides correlated with poor prognostic factors plus five molecules able to classify pN0 and pN+ patients (area under the receiver operating characteristic curve > 0.85). In addition, endopeptidases and exopeptidases putatively implicated in the processing of differential peptides were investigated using cancer tissue gene expression data from public repositories, reinforcing their association with poorer survival rates and prognosis in oral cancer. The dynamics of the OSCC-related proteolysis were further explored via the proteomic profiling of saliva. This revealed that peptidase/endopeptidase inhibitors exhibited reduced levels in the saliva of pN+ patients, as confirmed by selected reaction monitoring-mass spectrometry, while minor changes were detected in the level of saliva proteases. Taken together, our results indicated that proteolytic activity is accentuated in the saliva of patients with OSCC and lymph-node metastasis and, at least in part, is modulated by reduced levels of salivary peptidase inhibitors. Therefore, this integrated pipeline provided better comprehension and discovery of molecular features with implications in the oral cancer metastasis prognosis.

5.
Proteomics ; 16(20): 2650-2666, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27493124

RESUMO

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 interacting proteins were predominantly related to "cytoskeleton" and "stress response," whereas p54-S6K2 interactome was more associated to "transcription," "splicing," and "ribosome biogenesis." Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.


Assuntos
Mapas de Interação de Proteínas , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Proteômica , Proteínas Quinases S6 Ribossômicas 70-kDa/análise , Transdução de Sinais
6.
Clin Sci (Lond) ; 130(10): 785-99, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-26823560

RESUMO

EEF1D (eukaryotic translation elongation factor 1δ) is a subunit of the elongation factor 1 complex of proteins that mediates the elongation process during protein synthesis via enzymatic delivery of aminoacyl-tRNAs to the ribosome. Although the functions of EEF1D in the translation process are recognized, EEF1D expression was found to be unbalanced in tumours. In the present study, we demonstrate the overexpression of EEF1D in OSCC (oral squamous cell carcinoma), and revealed that EEF1D and protein interaction partners promote the activation of cyclin D1 and vimentin proteins. EEF1D knockdown in OSCC reduced cell proliferation and induced EMT (epithelial-mesenchymal transition) phenotypes, including cell invasion. Taken together, these results define EEF1D as a critical inducer of OSCC proliferation and EMT.


Assuntos
Carcinoma de Células Escamosas/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias Bucais/genética , Fator 1 de Elongação de Peptídeos/genética , Carcinoma de Células Escamosas/diagnóstico , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias de Cabeça e Pescoço/diagnóstico , Humanos , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Fenótipo , Carcinoma de Células Escamosas de Cabeça e Pescoço
7.
Tumour Biol ; 37(7): 9045-57, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26762409

RESUMO

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-ß1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Neoplasias Bucais/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Intervalo Livre de Doença , Matriz Extracelular/patologia , Feminino , Fibroblastos/patologia , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Neoplasias Bucais/patologia , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Pró-Colágeno/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral/fisiologia , Regulação para Cima/fisiologia
8.
Biochim Biophys Acta ; 1854(1): 46-54, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25448015

RESUMO

Proteomics experiments often generate a vast amount of data. However, the simple identification and quantification of proteins from a cell proteome or subproteome is not sufficient for the full understanding of complex mechanisms occurring in the biological systems. Therefore, the functional annotation analysis of protein datasets using bioinformatics tools is essential for interpreting the results of high-throughput proteomics. Although large-scale proteomics data have rapidly increased, the biological interpretation of these results remains as a challenging task. Here we reviewed basic concepts and different programs that are commonly used in proteomics data functional annotation, emphasizing the main strategies focused in the use of gene ontology annotations. Furthermore, we explored the characteristics of some tools developed for functional annotation analysis, concerning the ease of use and typical caveats on ontology annotations. The utility and variations between different tools were assessed through the comparison of the resulting outputs generated for an example of proteomics dataset.


Assuntos
Biologia Computacional/métodos , Ontologia Genética , Proteoma/metabolismo , Proteômica/métodos , Animais , Bases de Dados de Proteínas , Humanos , Ligação Proteica , Mapas de Interação de Proteínas , Proteoma/genética , Transdução de Sinais
9.
PLoS One ; 9(12): e115004, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25506919

RESUMO

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.


Assuntos
Agrina/fisiologia , Carcinoma de Células Escamosas/fisiopatologia , Proteoglicanas de Heparan Sulfato/fisiologia , Neoplasias Bucais/fisiopatologia , Agrina/genética , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteoglicanas de Heparan Sulfato/genética , Humanos , Neoplasias Bucais/genética
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