Livia Rosa-Fernandes

Technical Challenges of Working with Extracellular Vesicles

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review.

Outside-in, inside-out: Proteomic analysis of endothelial stress mediated by 7-ketocholesterol

Oxysterols are cholesterol oxidation products formed through enzymatic or autoxidation mechanisms. 7-ketocholeterol (7KC) is one of most abundant oxysterols found in atherosclerotic lesions. Its role in atherosclerosis pathogenesis has been broadly studied in a variety of models. The arterial microenvironment is a multicellular dynamic compartment that, among other systemic factors, is continuously stimulated by 7KC. Endothelial cells have a key role on that environment, being in intimate contact with both the blood stream and the vessel wall, the site of disease origin. 7KC has been shown to promote endothelial cell death and/or dysfunction, depending on its concentration. However, its contribution to the cell microenvironment through cell stimulation has not received much attention. Here we applied mass spectrometry-based proteomics followed by bioinformatics workflow to analyze the effect of a non-toxic 7KC concentration on endothelial cell protein expression and secretion in vitro. Trypsin digests were prepared from the secretome of the endothelial cells and from the total cell pellet after 24h exposure to 7KC. All samples were analyzed by high resolution and accurate mass nano-LC MS/MS. After database search and statistical analysis, differentially expressed proteins were selected for further studies. Our workflow identified 1805 secreted proteins and 2203 intracellular proteins, and of these, 48 and 53, respectively, were regulated. Regulated proteins upon 7KC exposure are involved in unfolded protein response, vascular homeostasis, and reduced control of angiogenesis. Moreover, blood coagulation was another main pathway regulated through Tissue Factor Pathway Inhibitor (TFPI), an antithrombotic agent associated with coronary disease that we found to be more than 2 times downregulated. Taken together, these data show differential endothelial protein regulation and secretion upon 7KC exposure for short time periods under non-toxic conditions. Herewith, these data support the role of 7KC in atherosclerosis pathophysiology and thus reinforce the deleterious effect of endothelial cells stress in the arterial microenvironment.

A Perspective on Extracellular Vesicles Proteomics

Increasing attention has been given to secreted extracellular vesicles (EVs) in the past decades, especially in the portrayal of their molecular cargo and role as messengers in both homeostasis and pathophysiological conditions. This review presents the state-of-the-art proteomic technologies to identify and quantify EVs proteins along with their PTMs, interacting partners and structural details. The rapid growth of mass spectrometry-based analytical strategies for protein sequencing, PTMs and structural characterization has improved the level of molecular details that can be achieved from limited amount of EVs isolated from different biological sources. Here we will provide a perspective view on the achievements and challenges on EVs proteome characterization using mass spectrometry. A detailed bioinformatics approach will help us to picture the molecular fingerprint of EVs and understand better their pathophysiological function.

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

Proteins constitute almost 95% of snake venom’s dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50u2009nm and 500u2009nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5′nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Development of a Trypanosoma cruzi strain typing assay using MS2 peptide spectral libraries (Tc-STAMS2)

Background Chagas disease also known as American trypanosomiasis is caused by the protozoan Trypanosoma cruzi. Over the last 30 years, Chagas disease has expanded from a neglected parasitic infection of the rural population to an urbanized chronic disease, becoming a potentially emergent global health problem. T. cruzi strains were assigned to seven genetic groups (TcI-TcVI and TcBat), named discrete typing units (DTUs), which represent a set of isolates that differ in virulence, pathogenicity and immunological features. Indeed, diverse clinical manifestations (from asymptomatic to highly severe disease) have been attempted to be related to T.cruzi genetic variability. Due to that, several DTU typing methods have been introduced. Each method has its own advantages and drawbacks such as high complexity and analysis time and all of them are based on genetic signatures. Recently, a novel method discriminated bacterial strains using a peptide identification-free, genome sequence-independent shotgun proteomics workflow. Here, we aimed to develop a Trypanosoma cruzi Strain Typing Assay using MS/MS peptide spectral libraries, named Tc-STAMS2. Methods/Principal findings The Tc-STAMS2 method uses shotgun proteomics combined with spectral library search to assign and discriminate T. cruzi strains independently on the genome knowledge. The method is based on the construction of a library of MS/MS peptide spectra built using genotyped T. cruzi reference strains. For identification, the MS/MS peptide spectra of unknown T. cruzi cells are identified using the spectral matching algorithm SpectraST. The Tc-STAMS2 method allowed correct identification of all DTUs with high confidence. The method was robust towards different sample preparations, length of chromatographic gradients and fragmentation techniques. Moreover, a pilot inter-laboratory study showed the applicability to different MS platforms. Conclusions and significance This is the first study that develops a MS-based platform for T. cruzi strain typing. Indeed, the Tc-STAMS2 method allows T. cruzi strain typing using MS/MS spectra as discriminatory features and allows the differentiation of TcI-TcVI DTUs. Similar to genomic-based strategies, the Tc-STAMS2 method allows identification of strains within DTUs. Its robustness towards different experimental and biological variables makes it a valuable complementary strategy to the current T. cruzi genotyping assays. Moreover, this method can be used to identify DTU-specific features correlated with the strain phenotype.

NS1 codon usage adaptation to humans in pandemic Zika virus

BACKGROUND Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

Author Correction: Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Distinct urinary glycoprotein signatures in prostate cancer patients

Novel biomarkers are needed to complement prostate specific antigen (PSA) in prostate cancer (PCa) diagnostic screening programs. Glycoproteins represent a hitherto largely untapped resource with a great potential as specific and sensitive tumor biomarkers due to their abundance in bodily fluids and their dynamic and cancer-associated glycosylation. However, quantitative glycoproteomics strategies to detect potential glycoprotein cancer markers from complex biospecimen are only just emerging. Here, we describe a glycoproteomics strategy for deep quantitative mapping of N- and O-glycoproteins in urine with a view to investigate the diagnostic value of the glycoproteome to discriminate PCa from benign prostatic hyperplasia (BPH), two conditions that remain difficult to clinically stratify. Total protein extracts were obtained, concentrated and digested from urine of six PCa patients (Gleason score 7) and six BPH patients. The resulting peptide mixtures were TMT-labeled and mixed prior to a multi-faceted sample processing including hydrophilic interaction liquid chromatography (HILIC) and titanium dioxide SPE based enrichment, endo-/exoglycosidase treatment and HILIC-HPLC pre-fractionation. The isolated N- and O-glycopeptides were detected and quantified using high resolution mass spectrometry. We accurately quantified 729 N-glycoproteins spanning 1,310 unique N-glycosylation sites and observed 954 and 965 unique intact N- and O-glycopeptides, respectively, across the two disease conditions. Importantly, a panel of 56 intact N-glycopeptides perfectly discriminated PCa and BPH (ROC: AUC = 1). This study has generated a panel of intact glycopeptides that has a potential for PCa detection.

Proteome-wide analysis of Trypanosoma cruzi exponential and stationary growth phases reveals a subcellular compartment-specific regulation

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.

MP87-03 URINARY MMP-9 AS CANDIDATE FOR A NON-INVASIVE PROSTATE CANCER BIOMARKER REVEALED BY QUANTITATIVE PROTEOMICS ANALYSIS

INTRODUCTION AND OBJECTIVES: Early studies have found that tumor-associated macrophages (TAMs) promote cancer progression. We previously reported that TAMs promote prostate cancer metastasis via activation of the CCL2-CCR2 axis. Recently, it was reported that the CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was associated with lung metastasis. However, the role of CCR4 and the relationship between CCR2 and CCR4 in prostate cancer is unclear. The aim of this study was to elucidate the role of CCR4 and the relationship between CCL2-CCR2 axis and CCL17/ CCL22-CCR4 axis in prostate cancer progression. METHODS: Human prostate cancer cell line cells and monocyte-lineage cells were used. Transwell migration and invasion assays co-cultured with or without macrophages were performed. Chemokines and their receptors in prostate cancer cells were measured. CCR2 and CCR4 in prostate cancer tissue were immunohistochemically analyzed. RESULTS: Co-culture of macrophages and prostate cancer cells increased prostate cancer cell migration and invasion and induced secretion of CCL2. CCL2 promoted prostate cancer cell migration in an autocrine manner and induced CCR2, CCR4 expressions, and CCL22 secretion of prostate cancer cells. RT-PCR, western blotting, and immunocytochemical staining revealed both CCR2 and CCR4 expressions in prostate cancer cells. CCL22 also promoted prostate cancer cell migration. Blockade of the CCL2-CCR2 or CCL17/22-CCR4 axis with receptor antagonist inhibited the migration of prostate cancer cells. The CCL2-CCR2 and CCL22-CCR4 axes increased phosphorylation of Akt and Erk1/2. Although both CCR2 and CCR4 antagonists could inhibit phosphorylation of Akt and Erk1/2, the CCR4 antagonist, compared with the CCR2 antagonist, strongly inhibited phosphorylation of Akt. CCR4 may have contributed more to prostate cancer cell migration than did CCR2. CCR4 and CCR2 were increased in prostate cancer tissues by IHC staining. Interestingly, the staining intensities of CCR2 and CCR4 in each specimen were significantly correlated. Moreover, the staining intensity of CCR4 was correlated with the progression of TNM stage. CONCLUSIONS: This is the first study to show that CCR4 was expressed in prostate cancer cell lines and human prostate cancer tissues and that the CCL22-CCR4 axis contributed to prostate cancer migration and invasion. Targeting of the CCL22-CCR4 axis, which is activated by TAMs, may be a novel therapeutic target and a potential biomarker for prostate cancer.