Mass spectrometry and nuclear magnetic resonance based metabolomics applied to the study of polycystic ovary syndrome

Objectives: Three objectives of this thesis have been: (i) Mastering of the main analytical platforms used in metabolomics, (ii) Developing an untargeted metabolomic workflow, involving novel aspects of sample preparation, and data processing for metabolite identification, (iii) Implementing our untargeted metabolomic workflow to the study of human patients with Polycystic Ovary Syndrome (PCOS) and their response to drug treatment Results: In Work 1: Optimization metabolite extraction conditions for NMR analysis, followed by LC/ESI-MS by using the same sample extract with no need for solvent exchange or further pretreatment. In Work 2: Investigate the impact of different aspects of univariate statistical analysis on untargeted LC-MS based metabolomic experiments. In Work 3: Implementation of GC-MS untargeted metabolomic approach to provide new insights on the impact that obesity exerts on the metabolic derangements associated with PCOS. In Work 4: Implementation of multiplatform metabolomics approach based on NMR and LC-MS to provide new insights in PCOS disease in a cohort of young lean PCOS patients. In Work 5: Implementation of multiplatform metabolomics approach based on NMR, GC-MS and LC-MS to provide new insights on the action of drug polytherapy to PCOS disorder. Conclusion: Metabolomics can be consider as a powerful tool for the study of metabolic disorders. Furthermore, metabolite profiling has demonstrated feasibility and flexibility for revealing new mechanistic insights in metabolic disorders that are not been consider when classical analysis is used. Therefore, our metabolomic analysis have demonstrated a great potential as a useful diagnostic technique and can facilitate monitoring of both disease progression and effects of therapeutic treatment.Objetivos: El presente trabajo tiene dos objetivos generalizables que han sido estudiados con más detalle en la presente tesis doctoral. El primero de ellos es mejorar aspectos metodológicos en el ámbito de la metabolómica y el segundo ha sido la aplicación de la metabolómica en el estudio del síndrome del ovario poliquístico (PCOS). Resultados: Del primer objetivo se han realizado dos trabajos: en el primero, la optimización de un método de extracción común para analizar muestras biológicas en dos plataformas analíticas complementarias utilizadas en metabolómica como son la resonancia magnética nuclear y la espectrometría de masas. Del segundo trabajo realizado se han obtenido unas pautas para abordar los retos que surgen del análisis de datos de metabolómica en espectrometría de masas. Del segundo objetivo también han sido realizados dos trabajos: en ambos se ha utilizado la metabolómica no dirigida para abordar el estudio del PCOS. En el primer trabajo, se ha utilizado la metabolómica para conocer el impacto que ejerce la obesidad en los trastornos metabólicos asociados al PCOS. En el segundo trabajo, se ha utilizado la metabolómica no dirigida para evaluar como afecta la aplicación de una politerapia con medicamentos al metabolismo de pacientes con PCOS. Conclusión: La metabolómica puede ser utilizada como una nueva herramienta para estudiar los trastornos metabólicos

Long-term health risks of hyperinsulinaemic androgen excess appear in adolescence with serum markers of oxidative stress and impaired HDL maturation through oxidation of methionine residues in apolipoprotein A1

Curs 2013-2014Director/a: Oscar Yanes Torrad

An endogenous HIV envelope-derived peptide without the terminal NH3+ group anchor is physiologically presented by major histocompatibility complex class I molecules

Cytotoxic T lymphocytes (CTL) recognize viral peptidic antigens presented by major histocompatibility complex (MHC) class I molecules on the surface of infected cells. The CTL response is critical in clearance and prevention of HIV infection. Yet, there are no descriptions of physiological peptides derived from the viral envelope protein. In the few reports on endogenous MHC class I viral peptidic ligands from HIV internal proteins, definitive positive identification by mass spectrometry is lacking. The HIV-1 envelope glycoprotein gp160 induces a strong specific CTL response restricted by several human and murine MHC class I molecules, including H-2Dd. Previous analyses showed that this response can be optimally mimicked with the synthetic decameric peptide 318RGPGRAFVTI327. We aim to identify the endogenous natural peptides mediating the response to this epitope. Our data indicate the presence of, at least, two peptidic species of different length and sharing the same antigenic core, which are associated with the Dd presenting molecule in infected cells. One species is at least, probably, the optimal decapeptide. The second species, identified by mass spectrometry for the first time in HIV, is, unexpectedly, a nonamer, which lacks the correctly positioned N-terminal group to bind to Dd. And yet, it is present in similar amounts and, notably, is equally antigenic. Thus, the physiological set of HIV-derived MHC class I ligands is richer and different than expected from studies with synthetic peptides. This may help raise the plasticity and thus the effectiveness of the immune response against the viral infection. These data have implications for HIV vaccine development.This work was supported by grants from the European Union, Ministerio de Educación y Ciencia, Comisión Interministerial de Ciencia y Tecnología, Comunidad de Madrid, Instituto de Salud Carlos III, and Red Temática de Investigación Cooperativa en SIDA del Fondo de Investigaciones Sanitarias. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

A long N-terminal-extended nested set of abundant and antigenic major histocompatibility complex class I natural ligands from HIV envelope protein

Viral antigens complexed with major histocompatibility complex (MHC) class I molecules are recognized by cytotoxic T lymphocytes on infected cells. Assays with synthetic peptides identify optimal MHC class I ligands often used for vaccines. However, when natural peptides are analyzed, more complex mixtures including long peptides bulging in the middle of the binding site or with carboxyl extensions are found, reflecting lack of exposure to carboxypeptidases in the antigen processing pathway. In contrast, precursor peptides are exposed to extensive cytosolic aminopeptidase activity, and fewer than 1% survive, only to be further trimmed in the endoplasmic reticulum. We show here a striking example of a nested set of at least three highly antigenic and similarly abundant natural MHC class I ligands, 15, 10, and 9 amino acids in length, derived from a single human immunodeficiency virus gp160 epitope. Antigen processing, thus, gives rise to a rich pool of possible ligands from which MHC class I molecules can choose. The natural peptide set includes a 15-residue-long peptide with unprecedented 6 N-terminal residues that most likely extend out of the MHC class I binding groove. This 15-mer is the longest natural peptide known recognized by cytotoxic T lymphocytes and is surprisingly protected from aminopeptidase trimming in living cells.This work was supported by grants from European Union, Ministerio de Educación y Ciencia, Comunidad de Madrid, Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa en Sindrome de Inmunodeficiencia Adquirida (SIDA) del Fondo de Investigaciones Sanitarias (to M. D. V.), Comunidad de Madrid, Instituto de Salud Carlos III, Fundación para la Investigación y la Prevención del Sindrome de Inmunodeficiencia Adquirida en España (to D. L.), and by European Commission Grant QLK2-CT-2001-01167 (to P. M. V. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

Effects of Lifestyle Intervention in Tissue-Specific Lipidomic Profile of Formerly Obese Mice

Lipids are highly diverse in their composition, properties and distribution in different biological entities. We aim to establish the lipidomes of several insulin-sensitive tissues and to test their plasticity when divergent feeding regimens and lifestyles are imposed. Here, we report a proton nuclear magnetic resonance (1H-NMR) study of lipid abundance across 4 tissues of C57Bl6J male mice that includes the changes in the lipid profile after every lifestyle intervention. Every tissue analysed presented a specific lipid profile irrespective of interventions. Glycerolipids and fatty acids were most abundant in epididymal white adipose tissue (eWAT) followed by liver, whereas sterol lipids and phosphoglycerolipids were highly enriched in hypothalamus, and gastrocnemius had the lowest content in all lipid species compared to the other tissues. Both when subjected to a high-fat diet (HFD) and after a subsequent lifestyle intervention (INT), the lipidome of hypothalamus showed no changes. Gastrocnemius and liver revealed a pattern of increase in content in many lipid species after HFD followed by a regression to basal levels after INT, while eWAT lipidome was affected mainly by the fat composition of the administered diets and not their caloric density. Thus, the present study demonstrates a unique lipidome for each tissue modulated by caloric intake and dietary composition. Keywords: lipidomics; tissue-specific; plasticity; energy intake; diet composition; exercise; hypothalamus; gastrocnemius; liver; white adipose tissu

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economia y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y Leon (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigacion Biomedicina 2017 Fundacion BBVA, and Fundacion Ramon Areces. This study was partially supported by the generous donations from Fundacion CRIS Contra el Cancer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.S

Nucleotide depletion reveals the impaired ribosomebiogenesis checkpoint as a barrier against DNA damage

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21‐mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC‐mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability

FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids

Need for tripeptidyl-peptidase II in major histocompatibility complex class I viral antigen processing when proteasomes are detrimental

CD8(+) T lymphocytes recognize infected cells that display virus-derived antigenic peptides complexed with major histocompatibility complex class I molecules. Peptides are mainly byproducts of cellular protein turnover by cytosolic proteasomes. Cytosolic tripeptidyl-peptidase II (TPPII) also participates in protein degradation. Several peptidic epitopes unexpectedly do not require proteasomes, but it is unclear which proteases generate them. We studied antigen processing of influenza virus nucleoprotein epitope NP(147-155), an archetype epitope that is even destroyed by a proteasome-mediated mechanism. TPPII, with the assistance of endoplasmic reticulum trimming metallo-aminopeptidases, probably ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing), was crucial for nucleoprotein epitope generation both in the presence of functional proteasomes and when blocked by lactacystin, as shown with specific chemical inhibitors and gene silencing. Different protein contexts and subcellular targeting all allowed epitope processing by TPPII as well as trimming. The results show the plasticity of the cell's assortment of proteases for providing ligands for recognition by antiviral CD8(+) T cells. Our observations identify for the first time a set of proteases competent for antigen processing of an epitope that is susceptible to destruction by proteasomes.This work was supported in part by grants from Spanish Ministerio de Educación y Ciencia and from Instituto de Salud Carlos III (to M. D. V.), by a grant from Spanish Ministerio de Educación y Ciencia (to L. C. A.), by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa, and by a grant from Comunidad de Madrid (to M. D. V. and L. C. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.S

Metabolomics reveals impaired maturation of HDL particles in adolescents with hyperinsulinaemic androgen excess.

Hyperinsulinaemic androgen excess (HIAE) in prepubertal and pubertal girls usually precedes a broader pathological phenotype in adulthood that is associated with anovulatory infertility, metabolic syndrome and type 2 diabetes. The metabolic derangements that determine these long-term health risks remain to be clarified. Here we use NMR and MS-based metabolomics to show that serum levels of methionine sulfoxide in HIAE girls are an indicator of the degree of oxidation of methionine-148 residue in apolipoprotein-A1. Oxidation of apo-A1 in methionine-148, in turn, leads to an impaired maturation of high-density lipoproteins (HDL) that is reflected in a decline of large HDL particles. Notably, such metabolic alterations occur in the absence of impaired glucose tolerance, hyperglycemia and hypertriglyceridemia, and were partially restored after 18 months of treatment with a low-dose combination of pioglitazone, metformin and flutamide