Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate

Purpose The retinal pigment epithelium (RPE) and underlying Bruch’s membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell–substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch’s membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this “aged” substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. Methods Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. Results Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. Conclusions This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch”s membrane could play a significant role in age-related dysfunction of the RPE.

Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle

Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections.

Effect-based proteomic detection of growth promoter abuse

Unregulated growth promoter use in food-producing animals is an issue of concern both from food safety and animal welfare perspectives. However, the monitoring of such practices is analytically challenging due to the concerted actions of users to evade detection. Techniques based on the monitoring of biological responses to exogenous administrations have been proposed as more sensitive methods to identify treated animals. This study has, for the first time, profiled plasma proteome responses in bovine animals to treatment with nortestosterone decanoate and 17 beta-oestradiol benzoate, followed by dexamethasone administration. Two-dimensional fluorescence differential in-gel electrophoresis analysis revealed a series of hepatic and acute-phase proteins within plasma whose levels were up- or down-regulated within phases of the treatment regime. Surface plasmon resonance (SPR) immuno-assays were developed to quantify responses of identified protein markers during the experimental treatment study with a view to developing methods which can be used as screening tools for growth promoter abuse detection. SPR analysis demonstrated the potential for plasma proteins to be used as indicative measures of growth promoter administrations and concludes that the sensitivity and robustness of any detection approach based on plasma proteome analysis would benefit from examination of a range of proteins representative of diverse biological processes rather being reliant on specific individual markers.

Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions).

In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system.

Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR).

Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions. This article is part of a Special Issue entitled: “Farm animal proteomics”.

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17β-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50 % (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC–MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.

Study of oxidation and non-enzymatic glycosylation posttranslational modifications using a proteomic approach

A glicosilação não-enzimática e o stress oxidativo representam dois processos importantes visto desempenharem um papel importante no que respeita às complicações de vários processos patofisiológicos. No presente, a associação entre a glicosilação não-enzimática e a oxidação de proteínas é reconhecida como sendo um dos principais responsáveis pela acumulação de proteínas não-funcionais que, por sua vez, promove uma contínua sensibilização para um aumento do stress oxidativo ao nível celular. Embora esteja disponível bastante informação no que respeita aos dois processos e suas consequências ao nível estrutural e funcional, permanecem questões por esclarecer acerca do que se desenvolve ao nível molecular. Com o objectivo de contribuir para uma melhor compreensão da relação entre a glicosilação não-enzimática e a oxidação, proteínas modelo (albumina, insulina e histonas H2B e H1) foram submetidas a sistemas in vitro de glicosilação não-enzimática e oxidação em condições controladas e durante um período de tempo específico. A identificação dos locais de glicosilação e oxidação foi realizada através de uma abordagem proteómica, na qual após digestão enzimática se procedeu à análise por cromatografia líquida acoplada a espectrometria de massa tandem (MALDI-TOF/TOF). Esta abordagem permitiu a obtenção de elevadas taxas de cobertura das sequências proteicas, permitindo a identificação dos locais preferenciais de glicosilação e oxidação nas diferentes proteínas estudadas. Como esperado, os resíduos de lisina foram os preferencialmente glicosilados. No que respeita à oxidação, além das modificações envolvendo hidroxilações e adições de oxigénio, foram identificadas deamidações, carbamilações e conversões oxidativas específicas de vários aminoácidos. No geral, os resíduos mais afectados pela oxidação foram os resíduos de cisteína, metionina, triptofano, tirosina, prolina, lisina e fenilalanina. Ao longo do período de tempo estudado, os resultados indicaram que a oxidação teve início em zonas expostas da proteína e/ou localizadas na vizinhança de resíduos de cisteína e metionina, ao invés de exibir um comportamente aleatório, ocorrendo de uma forma nãolinear por sua vez dependente da estabilidade conformacional da proteína. O estudo ao longo do tempo mostrou igualmente que, no caso das proteínas préglicosiladas, a oxidação das mesmas ocorreu de forma mais rápida e acentuada, sugerindo que as alterações estruturais induzidas pela glicosilação promovem um estado pro-oxidativo. No caso das proteínas pré-glicosiladas e oxidadas, foi identificado um maior número de modificações oxidativas assim como de resíduos modificados na vizinhança de resíduos glicosilados. Com esta abordagem é realizada uma importante contribuição na investigação das consequências do dano ‘glico-oxidativo’ em proteínas ao nível molecular através da combinação da espectrometria de massa e da bioinformática.

A proteomic study of the effects of nutrient restriction in utero on postnatal metabolism

It is widely recognized that protein restriction in utero may cause metabolic and endocrine adaptations, which may be of benefit to the neonate on a short-term basis but may cause adverse long-term conditions such as obesity, Type 2 diabetes, metabolic syndrome, hypertension and cardiovascular diseases. Adequate foetal and early post natal nutrient and energy supply is therefore essential for adult animal health, performance and life span. In this project it was investigated the progressive adaptations of the hepatic proteome in male mink offspring exposed to either a low protein (FL) or an adequate protein (FA) diet in utero fed either on a low protein (LP) or on an adequate (AP) diet from weaning until sexual maturity. Specifically, the aim was to determine the metabolic adaptations at selected phases of the animal’s first annual cycle and establish the metabolic priorities occurring during those phases. The three different morphological stages studied during the first year of development included, end of bone growth at 4 months of age, maximal fat accretion at 6 months of age and sexual maturity at 12 months of age. A reference proteome of mink liver coming from these different animal groups were generated using 2D electrophoresis coupled to MALDI-TOF analysis and the way in which dietary treatment affect their proteome was established. Approximately 330 proteins were detected in the mink liver proteome. A total of 27 comparisons were carried out between all different animal groups which resulted in 20 differentially expressed proteins. An extensive survey was conducted towards the characterization of these proteins including their subcellular localization, the biological processes in which they are involved and their molecular functions. This characterization allowed the identification of proteins in various processes including the glycolysis and fatty acid metabolism. The detailed analysis of the different dietary treatment animal groups was indicative of differences in metabolism and also to changes associated with development in mink.

Contamination of farmed gilthead sea bream (Sparus aurata, Linnaeus, 1758) by the parasite Amyloodinium ocellatum: a proteomic approach

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Uniersidade do Algarve, 2015

Proteomic analysis of the influence of the adipocyte secretome on Glioma Gl261 cells

Glioma is the most frequent form of malignant brain tumor in the adults and childhood. There is a global tendency toward a higher incidence of gliomas in highly developed and industrialized countries. Simultaneously obesity is reaching epidemic proportions in such developed countries. It has been highly accepted that obesity may play an important role in the biology of several types of cancer. We have developed an in vitro method for the understanding of the influence of obesity on glioma mouse cells (Gl261). 3T3-L1 mouse pre-adipocytes were induced to the maturity. The conditioned medium was harvested and used into the Gl261 cultures. Using two-dimension electrophoresis it was analyzed the proteome content of Gl261 in the presence of conditioned medium (CGl) and in its absence (NCGl). The differently expressed spots were collected and analyzed by means of mass spectroscopy (MALDI-TOF-MS). Significantly expression pattern changes were observed in eleven proteins and enzymes. RFC1, KIF5C, ANXA2, N-RAP, RACK1 and citrate synthase were overexpressed or only present in the CGl. Contrariwise, STI1, hnRNPs and phosphoglycerate kinase 1 were significantly underexpressed in CGl. Aldose reductase and carbonic anhydrase were expressed only in NCGl. Our results show that obesity remodels the physiological and metabolic behavior of glioma cancer cells. Also, proteins found differently expressed are implicated in several signaling pathways that control matrix remodeling, proliferation, progression, migration and invasion. In general our results support the idea that obesity may increase glioma malignancy, however, some interesting paradox finding were also reported and discussed.

Protein stability in a proteomic perspective

Dissertation presented to obtain the Ph.D degree in Biochemistry

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.