Using mass spectrometry-based proteomics to discover cancer candidate biomarkers and to identify novel ADAM17 substrates

Orientador: Adriana Franco Paes LemeTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A espectrometria de massas aplicada à análise proteômica permite a caracterização e quantificação em larga-escala de proteínas expressas em uma determinada condição ou sistema biológico. Na presente tese, mostramos por meio de diferentes abordagens a utilidade da espectrometria de massas (1) no estudo de proteínas secretadas ou clivadas da superfície (secretoma) por células tumorigênicas como fonte de biomarcadores em câncer (2) na validação de candidatos biomarcadores em saliva humana de pacientes com câncer oral e (3) no estudo de alvos de uma metaloprotease envolvida na progressão de câncer, a ADAM17 (A disintegrin and metalloproteinase 17). Os resultados gerados pela espectrometria de massas juntamente com ferramentas estatísticas e de bioinformática para visualização de dados em agrupamento, heatmaps, rede de interação proteína-proteína, enriquecimento de vias e processos biológicos indicaram proteínas específicas direcionadas à hipótese que puderam ser validadas por métodos complementares quantitativos e funcionais. No contexto de biomarcadores em câncer, um painel de marcadores foi indicado a partir da análise do secretoma de células de carcinoma, melanoma e não cancerosas. Dentre os candidatos em carcinoma, proteínas envolvidas no sistema complemento foram validadas com expressão aumentada tanto em tecidos (proteínas C3 e CFB) quanto em saliva de pacientes com carcinoma oral de células escamosas (proteínas C3, CFB, C4B e SERPINA1). Agrina e perlecan, também observados com expressão alterada no secretoma de células de carcinoma e validados com expressão aumentada em tecidos de paciente com câncer oral, foram estudados quanto à função em processos tumorigênicos: migração, adesão, proliferação e sensibilidade à cisplatina. No estudo do degradoma da ADAM17, mimecan, perlecan e glypican-1 foram revelados como novos alvos dessa protease, e os sítios de clivagem determinados por espectrometria de massas. Além disso, funções e processos biológicos associados à clivagem de glypican-1 também foram estudados por meio da análise de seus parceiros de interação no meio extracelular seguido de ensaios funcionais. Em resumo, a espectrometria de massas esteve presente em todos os passos do método científico, desde a geração de hipóteses por meio da proteômica baseada em descoberta, até o teste e confirmação de hipóteses por meio da proteômica baseada em alvosAbstract: Mass spectrometry applied to proteomic analysis allows large-scale characterization and quantification of proteins in specific conditions or biological systems. In this thesis, we showed different mass spectrometry-based approaches for (1) the study of cell secreted or released from the cell-surface proteins (secretome) as a source of candidate biomarkers in cancer (2) validation of candidate biomarkers in human saliva of patients with oral cancer and (3) the study of the target substrates of an important metalloprotease involved in cancer progression, the ADAM17 (A disintegrin and metalloproteinase 17). Together with statistical and bioinformatics tools, such as clustering, heatmaps, network analysis and enrichment analysis, we found specific hyphothesis-driven proteins that were further validated using complementary quantitative and functional methods. In the context of cancer biomarker, a panel of candidate biomarkers was revealed from the secretome analysis of carcinoma, melanoma and non-tumorigenic cell lines. Among the carcinoma candidates, proteins involved in complement system were validated with higher expression in tissues (C3 and CFB) and saliva (C3, CFB, C4B and SERPINA1) from patients with oral squamous cell carcinoma. The function of agrin and perlecan, which were also found with increased expression in squamous cell carcinoma tissue, were studied in tumorigenic processes: migration, adhesion, proliferation and sensibility to cisplatin. In the context of ADAM17 degradome, mimecan, perlecan and glypican-1 (GPC1) were revealed as novel substrates and their cleavage sites were determined using mass spectrometry. Besides, the function and biological processes associated with GPC1 shedding were studied by identifying the binding partners of GPC1 in the extracellular media and by performing functional assays. In summary, mass spectrometry was present in all part of the scientific method, from hyphothesis generation using discovery proteomics to hyphothesis testing using targeted proteomicsDoutoradoBioquimicaDoutora em Biologia Funcional e Molecula

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow–derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors

Integrated proteomics identified up-regulated focal adhesion-mediated proteins in human squamous cell carcinoma in an orthotopic murine model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by co95e98208FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/54067-3; 2010/19278-0; 2011/22421-2; 2009/53839-2; 2011/02267-9470567/2009-0; 470549/2011-4; 301702/2011-0; 470268/2013-1; 505413/2013-

Hyper-truncated Asn355- And Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity

Integrative analysis to select cancer candidate biomarkers to targeted validation

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOTargeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS.Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS6414363543652FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2009/54067-3; 2010/19278-0; 2011/22421-2; 2009/53839-2470567/2009-0; 470549/2011-4; 301702/2011-0; 470268/2013-

Zika Virus Impairs Neurogenesis and Synaptogenesis Pathways in Human Neural Stem Cells and Neurons

Growing evidences have associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly. Nonetheless, signaling mechanisms that promote the disease outcome are far from being understood, affecting the development of suitable therapeutics. In this study, we applied shotgun mass spectrometry (MS)-based proteomics combined with cell biology approaches to characterize altered molecular pathways on human neuroprogenitor cells (NPC) and neurons derived from induced pluripotent stem cells infected by ZIKV-BR strain, obtained from the 2015 Brazilian outbreak. Furthermore, ZIKV-BR infected NPCs showed unique alteration of pathways involved in neurological diseases, cell death, survival and embryonic development compared to ZIKV-AF, showing a human adaptation of the Brazilian viral strain. Besides, infected neurons differentiated from NPC presented an impairment of neurogenesis and synaptogenesis processes. Taken together, these data explain that CNS developmental arrest observed in Congenital Zika Syndrome is beyond neuronal cell death

Quantitative proteomic analysis of amastigotes from <i>Leishmania (L</i>.<i>) amazonensis</i> LV79 and PH8 strains reveals molecular traits associated with the virulence phenotype

<div><p>Background</p><p>Leishmaniasis is an antropozoonosis caused by <i>Leishmania</i> parasites that affects around 12 million people in 98 different countries. The disease has different clinical forms, which depend mainly on the parasite genetics and on the immunologic status of the host. The promastigote form of the parasite is transmitted by an infected female phlebotomine sand fly, is internalized by phagocytic cells, mainly macrophages, and converts into amastigotes which replicate inside these cells. Macrophages are important cells of the immune system, capable of efficiently killing intracellular pathogens. However, <i>Leishmania</i> can evade these mechanisms due to expression of virulence factors. Different strains of the same <i>Leishmania</i> species may have different infectivity and metastatic phenotypes <i>in vivo</i>, and <i>w</i>e have previously shown that analysis of amastigote proteome can give important information on parasite infectivity. Differential abundance of virulence factors probably accounts for the higher virulence of PH8 strain parasites shown in this work. In order to test this hypothesis, we have quantitatively compared the proteomes of PH8 and LV79 lesion-derived amastigotes using a label-free proteomic approach.</p><p>Methodology/Principal findings</p><p>In the present work, we have compared lesion development by <i>L</i>. <i>(L</i>.<i>) amazonensis</i> PH8 and LV79 strains in mice, showing that they have different virulence <i>in vivo</i>. Viability and numbers of lesion-derived amastigotes were accordingly significantly different. Proteome profiles can discriminate parasites from the two strains and several proteins were differentially expressed.</p><p>Conclusions/Significance</p><p>This work shows that PH8 strain is more virulent in mice, and that lesion-derived parasites from this strain are more viable and more infective <i>in vitro</i>. Amastigote proteome comparison identified GP63 as highly expressed in PH8 strain, and Superoxide Dismutase, Tryparedoxin Peroxidase and Heat Shock Protein 70 as more abundant in LV79 strain. The expression profile of all proteins and of the differential ones precisely classified PH8 and LV79 samples, indicating that the two strains have proteins with different abundances and that proteome profiles correlate with their phenotypes.</p></div

Serum N-Glycomics Stratifies Bacteremic Patients Infected with Different Pathogens

Bacteremia—i.e., the presence of pathogens in the blood stream—is associated with long-term morbidity and is a potential precursor condition to life-threatening sepsis. Timely detection of bacteremia is therefore critical to reduce patient mortality, but existing methods lack precision, speed, and sensitivity to effectively stratify bacteremic patients. Herein, we tested the potential of quantitative serum N-glycomics performed using porous graphitized carbon liquid chromatography tandem mass spectrometry to stratify bacteremic patients infected with Escherichia coli (n = 11), Staphylococcus aureus (n = 11), Pseudomonas aeruginosa (n = 5), and Streptococcus viridans (n = 5) from healthy donors (n = 39). In total, 62 N-glycan isomers spanning 41 glycan compositions primarily comprising complex-type core fucosylated, bisecting N-acetylglucosamine (GlcNAc), and α2,3-/α2,6-sialylated structures were profiled across all samples using label-free quantitation. Excitingly, unsupervised hierarchical clustering and principal component analysis of the serum N-glycome data accurately separated the patient groups. P. aeruginosa-infected patients displayed prominent N-glycome aberrations involving elevated levels of fucosylation and bisecting GlcNAcylation and reduced sialylation relative to other bacteremic patients. Notably, receiver operating characteristic analyses demonstrated that a single N-glycan isomer could effectively stratify each of the four bacteremic patient groups from the healthy donors (area under the curve 0.93–1.00). Thus, the serum N-glycome represents a new hitherto unexplored class of potential diagnostic markers for bloodstream infections