Selected Reaction Monitoring (SRM) frente a Western Blot

We propose the SRM technology as a complementary method to the Western Blot for the detection and quantification of proteins in a sample. The technique Western Blot has its own limitations: i) only a protein-of-choice is detected, ignoring any non-relevant proteins, ii) the sensitivity of the technique depends on the specificity of the antibody and iii) Western Blot is expensive and time-consuming. The advantages of SRM with respect Western Blot are remarkable: i) you can detect up to hundreds of different proteins in a sample, ii) SRM is more sensitive, because just 50 copies of the target protein per cell are enough for the detection and iii) once it has been made an investment in the necessary machinery to develop this technique, the detection of proteins in a sample turns into a cheaper, faster, more specific and full-quantitative procedure, without the need of using antibodies. First of all, SRM requires the identification of little peptides, obtained by tryptic digestion, whose sequence must be unique for a single protein or isoform. There is software for that aim. Then, it’s necessary to create isotope-labeled peptides of that identified for acting as internal standards. That sample is introduced in a triple quadrupole mass spectrometer: it passes through a first quadrupole, which functions as a filter, where the fragments are selected, previously ionized, attending to the mass/charge (m/z) relation that correspond to that unique fragments of the protein of interest. In this first selection may be other peptides from other proteins, with the same m/z but with different sequence. To select those that are exclusive from the target protein, the fragments are moved to a second quadrupole, where they are fragmented again with a physical method, and so new smaller fragments are generated. All the new fragments are conduced to the third quadrupole, where just those which come from the protein of interest are selected, attending at their m/z again. The target peptide concentration is determined by measuring the observed signal response for the target peptide relative to that of the isotopic-labeled peptide, the concentration of which is calculated from a pre-determined calibration-response curve. Calibration curves have to be generated for each target peptide in the sample. Because SRM technology is increasing its use, there have been developed databases where the scientific community upload information about protocols and standards for each protein with the aim to facilitate the work to other researchers.

Nutritional and biochemical profiling of Leucopaxillus candidus (Bres.) singer wild mushroom

The wild mushroom Leucopaxillus candidus (Bres.) Singer was studied for the first time to obtain information about its chemical composition, nutritional value and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. candidus methanolic extract was tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). L. candidus was shown to be an interesting species in terms of nutritional value, with high content in proteins and carbohydrates, but low fat levels, with the prevalence of polyunsaturated fatty acids. Mannitol was the most abundant free sugar and β-tocopherol was the main tocopherol isoform. Other compounds detected were oxalic and fumaric acids, p-hydroxybenzoic and cinnamic acids. The methanolic extract revealed antioxidant activity and did not show hepatoxicity in porcine liver primary cells. The present study provides new information about L. candidus.

Papel de C3G y p38α en la regulación de la migración, invasión tumoral en carcinoma de colon. Función de fibulina 3

Colorectal cancer (CRC) represents the third most common cancer type and the second leading cause of cancer-related death in the western world. CRC results from the accumulation of both acquired genetic and epigenetic changes that transform normal glandular epithelium into adenocarcinoma (Lao and Grady 2011), affecting several genes such as Apc, K-ras, dcc/Smad4 and p53 or DNA mismatch repair genes (Pancione et al. 2012). p38 MAPKs are a subfamily of Serine-Threonine kinases activated by different stimuli that control fundamental cellular processes such as cell growth, proliferation, differentiation, migration and apoptosis (Dhillon et al. 2007, Nebreda and Porras 2000, Wagner and Nebreda 2009). There are four p38 MAPKs isoforms in mammals: α, β, δ and γ. p38α MAPK is ubiquitously expressed and is the most abundant isoform (Cuenda and Rousseau 2007). p38α is involved in the regulation of many cellular functions, among them, cell migration and invasion. In cancer, it can act as either a promoter or a suppressor of tumor growth, playing different roles during tumor progression (del Barco Barrantes and Nebreda 2012). C3G is a guanine nucleotide exchange factor (GEF) mainly for the Ras family members: Rap1 (Gotoh et al. 1995) and R-Ras (Gotoh et al. 1997), but it can also act through GEF independent mechanisms. C3G regulates several cellular functions such as cell death, adhesion, migration and invasion (Radha et al. 2011). In collaboration with Dr. Carmen Guerrero’s group (Centro del Investigación del Cáncer de Salamanca), our group has found a new functional relationship between C3G and p38α MAPK involved in the regulation of cell death in MEFs (Gutierrez-Uzquiza et al. 2010) and in the chronic myeloid leukemia (CML) K562 cell line (Maia et al. 2009). Moreover, C3G and p38α act through a common regulatory pathway to control cell adhesion in K562 cells regulating focal adhesion proteins (Maia et al. 2013)...

CYP3A5 genetic polymorphism in HIV-patients

Poster presented at the From Basic Sciences to Clinical Research - First International Congress of CiiEM. Egas Moniz, Caparica, Portugal, 27-28 November 2015

Mcardle disease: Update of reported mutations and polymorphisms in the Pygm gene

McArdle disease is an autosomal recessive disorder caused by inherited deficiency of the muscle isoform of glycogen phosphorylase (or ‘myophosphorylase´), which catalyzes the first step of glycogen catabolism, releasing glucose-1-phosphate from glycogen deposits. As a result, muscle metabolism is impaired, leading to different degrees of exercise intolerance. Patients range from asymptomatic to severely affected, including in some cases limitations in activities of daily living. The PYGM gene codifies myophosphoylase and to date 147 pathogenic mutations and 39 polymorphisms have been reported. Exon 1 and 17 are mutational hot-spots in PYGM and 50% of the described mutations are missense.

The role of Phospholipases as novel potential modulators of aggressiveness in Glioblastoma

Glioblastoma is the most malignant brain tumor in adults. The standard care of treatment is tumor resection, radiotherapy, and chemotherapy. Despite these invasive therapeutic approaches, glioblastoma prognosis remains unchanged. Therefore, a better understanding of the molecular mechanisms driving tumor transformation is needed to uncover novel therapeutic strategies. Several studies have shown the significance of lipid signaling and phospholipases (PLCs) in the regulation of different mechanisms in the central nervous system as well as in glioblastoma pathogenesis. This work suggests a potential role of PLCβ1 in the maintenance of a less aggressive phenotype of the tumor. Indeed, it was demonstrated that PLCβ1 gene was relatively less expressed in glioblastoma patients compared to their healthy/low-grade counterparts. Moreover, PLCβ1 silencing, in both immortalized and primary cell lines, led to increased cell migration, invasion, proliferation, cell survival and induced the upregulation of mesenchymal markers and metalloproteinases. Moreover, PLCγ1, another abundant PLC isoform in the brain, has been identified as a key element for the aggressiveness of glioblastoma. Data collected on patients’ biopsies and engineered cell models, suggested a strong correlation between PLCγ1 expression level and the acquisition of a more aggressive tumor phenotype. Finally, this trend was further probed using patient-derived glioblastoma stem cells (GSCs), which are a specific tumor population that drives aggressiveness, resistance, and recurrence in glioblastoma. GSCs analysis on the transcriptomic profiles confirmed that PLCγ1 downregulation modulated positively the activation of pathways that negatively regulate cell motility and migration and led to a decreased expression of genes involved in cancer development and progression. Taken together, these data highlight the importance of further investigating phospholipases as potential prognostic biomarkers and targets in the development of new therapeutic strategies for glioblastoma.

Ruolo di Glutaredossina-2 nella trasformazione tumorale oncocitaria

Gli oncocitomi sono tumori epiteliali caratterizzati da un accumulo di mitocondri strutturalmente e funzionalmente compromessi, a prognosi generalmente benigna. Le cause genetiche della trasformazione oncocitaria sono tuttora sconosciute; pertanto, lo studio di oncocitomi in contesti familiari sindromici è utile nella ricerca dei determinanti genetici predisponenti il fenotipo. Diversi membri di una famiglia affetta da sindrome dell’iperparatiroidismo con tumore della mandibola (HPT-JT), dovuta ad un'ampia delezione in CDC73, hanno mostrato recidiva di tumori paratiroidei oncocitari. Il sequenziamento dell’esoma ha escluso mutazioni private della famiglia; all'interno della delezione ereditata, tuttavia, sono stati individuati elementi regolatori del gene glutaredossina 2 (GLRX2), codificante un'isoforma mitocondriale deputata alla deglutationilazione proteica reversibile -modificazione modulante l’attività di numerosi target- il cui ruolo nel cancro non è noto. La proteina è risultata assente in tutti i tumori e dimezzata nei tessuti sani dei soggetti. Per indagare se la sua assenza alteri la deglutationilazione proteica predisponendo al fenotipo oncocitario, sono stati generati modelli cellulari TPC1 e HCT116 GLRX2 KO in cui sono stati riscontrati un ridotto tasso proliferativo ed un'alterata glutationilazione proteica, particolarmente in seguito a stress ossidativo. Un esperimento pilota in vivo ha mostrato cellule KO oncocitoidi, con mitocondri morfologicamente alterati, suggerendo che l’alterazione redox innescata dall’assenza di GLRX2 possa indurre una disfunzione metabolica mitocondriale tale da mimare quelle osservate negli oncocitomi. L’analisi proteomica ha individuato diversi target di glutationilazione nei campioni KO identificando proteine del ciclo di Krebs e della catena respiratoria mitocondriale. In particolare, una marcata glutationilazione del complesso della piruvato deidrogenasi (PDHc) è stata correlata ad una ridotta sintesi di ATP dipendente da piruvato. Considerando l'importanza dello stress ossidativo nella fisiopatologia del cancro ed il ruolo del glutatione nella risposta antiossidante, GLRX2 rappresenta un potenziale candidato nella regolazione del metabolismo ossidativo nelle cellule tumorali esposte allo stress e nella modulazione del fenotipo tumorale.