Open Access
Proteomic and functional characterisation of extracellular vesicles from collagen VI deficient human fibroblasts reveals a role in cell motility
(Springer, 2023) Badosa, Carmen; Roldán, Mónica; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Jiménez-Mallebrera, Cecilia; Ciencias de la Salud; Osasun Zientziak
Extracellular vesicles (EVs) are key mediators of cell-to-cell communication. Their content reflects the state of diseased cells representing a window into disease progression. Collagen-VI Related Muscular Dystrophy (COL6-RD) is a multi-systemic disease involving different cell types. The role of EVs in this disease has not been explored. We compared by quantitative proteomics the protein cargo of EVs released from fibroblasts from patients with COL6-RD and controls. Isolated EVs contained a significant proportion of the most frequently reported proteins in EVs according to Exocarta and Vesiclepedia. We identified 67 differentially abundant proteins associated with vesicle transport and exocytosis, actin remodelling and the cytoskeleton, hemostasis and oxidative stress. Treatment of control fibroblasts with EVs from either patient or healthy fibroblasts altered significantly the motility of cells on a cell migration assay highlighting the functional relevance of EVs. In parallel, we analysed the secretome from the same cells and found a distinctly different set of 48 differentially abundant proteins related to extracellular matrix organisation and remodelling, growth factor response, RNA metabolism and the proteasome. The EVs and secretome sets of proteins only shared two identifiers indicating that the sorting of proteins towards EVs or the secretory pathway is tightly regulated for different functions. .
Open Access
Effect of immunology biomarkers associated with hip fracture and fracture risk in older adults
(BMC, 2023) Cedeño Veloz, Bernardo Abel; Lozano Vicario, Lucía; Zambom Ferraresi, Fabrício; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Rodríguez-García, Alba; Romero Ortuno, Román; Mondragón Rubio, Jaime; Ruiz-Ruiz, Javier; Ramírez Vélez, Robinson; Izquierdo Redín, Mikel; Martínez Velilla, Nicolás; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Osteoporosis is a skeletal disease that can increase the risk of fractures, leading to adverse health and socioeconomic consequences. However, current clinical methods have limitations in accurately estimating fracture risk, particularly in older adults. Thus, new technologies are necessary to improve the accuracy of fracture risk estimation. In this observational study, we aimed to explore the association between serum cytokines and hip fracture status in older adults, and their associations with fracture risk using the FRAX reference tool. We investigated the use of a proximity extension assay (PEA) with Olink. We compared the characteristics of the population, functional status and detailed body composition (determined using densitometry) between groups. We enrolled 40 participants, including 20 with hip fracture and 20 without fracture, and studied 46 cytokines in their serum. After conducting a score plot and two unpaired t-tests using the Benjamini-Hochberg method, we found that Interleukin 6 (IL-6), Lymphotoxin-alpha (LT-α), Fms-related tyrosine kinase 3 ligand (FLT3LG), Colony stimulating factor 1 (CSF1), and Chemokine (C-C motif) ligand 7 (CCL7) were significantly different between fracture and non-fracture patients (p < 0.05). IL-6 had a moderate correlation with FRAX (R2 = 0.409, p < 0.001), while CSF1 and CCL7 had weak correlations with FRAX. LT-α and FLT3LG exhibited a negative correlation with the risk of fracture. Our results suggest that targeted proteomic tools have the capability to identify differentially regulated proteins and may serve as potential markers for estimating fracture risk. However, longitudinal studies will be necessary to validate these results and determine the temporal patterns of changes in cytokine profiles.
Open Access
Improvement of cognitive function in wild-type and Alzheimer's disease mouse models by the immunomodulatory properties of menthol inhalation or by depletion of T regulatory cells
(Frontiers Media, 2023) Casares, Noelia; Alfaro Larraya, María; Cuadrado-Tejedor, Mar; Lasarte-Cía, Aritz; Navarro Negredo, Flor; Vivas, Isabel; Espelosín, María; Cartas Cejudo, Paz; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; García-Osta, Ana; Lasarte, Juan José; Ciencias de la Salud; Osasun Zientziak
A complex network of interactions exists between the olfactory, immune and central nervous systems. In this work we intend to investigate this connection through the use of an immunostimulatory odorant like menthol, analyzing its impact on the immune system and the cognitive capacity in healthy and Alzheimer’s Disease Mouse Models. We first found that repeated short exposures to menthol odor enhanced the immune response against ovalbumin immunization. Menthol inhalation also improved the cognitive capacity of immunocompetent mice but not in immunodeficient NSG mice, which exhibited very poor fear-conditioning. This improvement was associated with a downregulation of IL-1β and IL-6 mRNA in the brain´s prefrontal cortex, and it was impaired by anosmia induction with methimazole. Exposure to menthol for 6 months (1 week per month) prevented the cognitive impairment observed in the APP/PS1 mouse model of Alzheimer. Besides, this improvement was also observed by the depletion or inhibition of T regulatory cells. Treg depletion also improved the cognitive capacity of the APPNL-G-F/NL-G-F Alzheimer´s mouse model. In all cases, the improvement in learning capacity was associated with a downregulation of IL-1β mRNA. Blockade of the IL-1 receptor with anakinra resulted in a significant increase in cognitive capacity in healthy mice as well as in the APP/PS1 model of Alzheimer´s disease. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals, highlighting the potential of odors and immune modulators as therapeutic agents for CNS-related diseases.
Open Access
Proteostatic modulation in brain aging without associated Alzheimer's disease-and age-related neuropathological changes
(Impact Journals, 2023) Andrés Benito, Pol; Íñigo-Marco, Ignacio; Brullas, Marta; Carmona, Margarita; Río, José Antonio del; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Povedano, Mónica; Ferrer, Isidro; Ciencias de la Salud; Osasun Zientziak
Aims: (Phospho)proteomics of old-aged subjects without cognitive or behavioral symptoms, and without ADneuropathological changes and lacking any other neurodegenerative alteration will increase understanding about the physiological state of human brain aging without associate neurological deficits and neuropathological lesions. Methods: (Phospho)proteomics using conventional label-free- and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) has been assessed in the frontal cortex (FC) of individuals without NFTs, senile plaques (SPs) and age-related co-morbidities classified by age (years) in four groups; group 1 (young, 30–44); group 2 (middle-aged: MA, 45-52); group 3 (early-elderly, 64–70); and group 4 (late-elderly, 75–85). Results: Protein levels and deregulated protein phosphorylation linked to similar biological terms/functions, but involving different individual proteins, are found in FC with age. The modified expression occurs in cytoskeleton proteins, membranes, synapses, vesicles, myelin, membrane transport and ion channels, DNA and RNA metabolism, ubiquitin-proteasome-system (UPS), kinases and phosphatases, fatty acid metabolism, and mitochondria. Dysregulated phosphoproteins are associated with the cytoskeleton, including microfilaments, actin-binding proteins, intermediate filaments of neurons and glial cells, and microtubules; membrane proteins, synapses, and dense core vesicles; kinases and phosphatases; proteins linked to DNA and RNA; members of the UPS; GTPase regulation; inflammation; and lipid metabolism. Noteworthy, protein levels of large clusters of hierarchically-related protein expression levels are stable until 70. However, protein levels of components of cell membranes, vesicles and synapses, RNA modulation, and cellular structures (including tau and tubulin filaments) are markedly altered from the age of 75. Similarly, marked modifications occur in the larger phosphoprotein clusters involving cytoskeleton and neuronal structures, membrane stabilization, and kinase regulation in the late elderly. Conclusions: Present findings may increase understanding of human brain proteostasis modifications in the elderly in the subpopulation of individuals not having AD neuropathological change and any other neurodegenerative change in any telencephalon region.
Open Access
Towards precision prognostication and personalized therapeutics through proteomics
(MDPI, 2023) Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak
Next-generation proteomics has allowed the implementation of biomedical proteome research to uncover disease-affected protein expression profiles. It has also enabled the determination of protein localization, protein interactomes, posttranslational modifications and protein dysfunction in human diseases. Many pillars in personalized medicine, such as diagnostic improvements, drug screening, systems biology or bioinformatics, require the generation of quantitatively consistent proteomics data from translational animal models to human biospecimens to fill the information gap, making omics analysis actionable from a clinical perspective [1-3]. This Special Issue received multiple submissions, of which five original articles were accepted for publication. These contributions cover different phases of precision medicine in the context of proteomics: (i) discovery and quantitation of potential biomarker candidates (three articles), (ii) the proteostatic modulation and mechanisms of action of pharmacological compounds (one article) and (iii) the characterization of posttranslational modifications (one article).
Open Access
Sex-specific role of galectin-3 in aortic stenosis
(BMC, 2023) Matilla Cuenca, Lara; Martín Núñez, Ernesto; Garaikoetxea Zubillaga, Mattie; Navarro, Adela; Tamayo Rodríguez, Ibai; Fernández Celis, Amaya; Gaínza Calleja, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Muntendam, Pieter; Álvarez, Virginia; Sádaba Sagredo, Rafael; Jover, Eva; López Andrés, Natalia; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: Aortic stenosis (AS) is characterized by infammation, fbrosis, osteogenesis and angiogenesis. Men and women develop these mechanisms diferently. Galectin-3 (Gal-3) is a pro-infammatory and pro-osteogenic lectin in AS. In this work, we aim to analyse a potential sex-diferential role of Gal-3 in AS. Methods: 226 patients (61.50% men) with severe AS undergoing surgical aortic valve (AV) replacement were recruited. In AVs, Gal-3 expression and its relationship with infammatory, osteogenic and angiogenic markers was assessed. Valve interstitial cells (VICs) were primary cultured to perform in vitro experiments. Results: Proteomic analysis revealed that intracellular Gal-3 was over-expressed in VICs of male AS patients. Gal-3 secretion was also higher in men’s VICs as compared to women’s. In human AVs, Gal-3 protein levels were signifcantly higher in men, with stronger immunostaining in VICs with myofbroblastic phenotype and valve endothelial cells. Gal-3 levels in AVs were positively correlated with infammatory markers in both sexes. Gal-3 expression was also posi tively correlated with osteogenic markers mainly in men AVs, and with angiogenic molecules only in this sex. In vitro, Gal-3 treatment induced expression of infammatory, osteogenic and angiogenic markers in male’s VICs, while it only upregulated infammatory and osteogenic molecules in women-derived cells. Gal-3 blockade with pharma cological inhibitors (modifed citrus pectin and G3P-01) prevented the upregulation of infammatory, osteogenic and angiogenic molecules. Conclusions: Gal-3 plays a sex-diferential role in the setting of AS, and it could be a new sex-specifc therapeutic target controlling pathological features of AS in VICs.
Open Access
Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer
(Springer Nature, 2023) Macaya, Irati; Roman, Marta; Welch, Connor; Entrialgo-Cadierno, Rodrigo; Salmon, Marina; Santos, Alba; Feliu, Iker; Kovalski, Joanna; López Erdozain, Inés; Rodríguez-Remírez, María; Palomino Echeverría, Sara; Lonfgren, Shane M.; Ferrero, Macarena; Calabuig, Silvia; Ludwig, Iziar A.; Lara-Astiaso, David; Jantus-Lewintre, Eloisa; Guruceaga, Elizabeth; Narayanan, Shruthi; Ponz Sarvisé, Mariano; Pineda Lucena, Antonio; Lecanda, Fernando; Ruggero, Davide; Khatri, Purvesh; Santamaría Martínez, Enrique; Fernández Irigoyen, Joaquín; Ferrer, Irene; Paz-Ares, Luis; Drosten, Matthias; Barbacid, Mariano; Gil-Bazo, Ignacio; Vicent, Silvestre; Ciencias de la Salud; Osasun Zientziak
Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.
Open Access
Docosahexaenoic acid ameliorates contextual fear memory deficits in the Tg2576 Alzheimer´s disease mouse model: cellular and molecular correlates
(MDPI, 2023) Badesso, Sara; Cartas Cejudo, Paz; Espelosín, María; Santamaría Martínez, Enrique; Cuadrado-Tejedor, Mar; García-Osta, Ana; Ciencias de la Salud; Osasun Zientziak
Docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the brain, is essential for successful aging. In fact, epidemiological studies have demonstrated that increased intake of DHA might lower the risk for developing Alzheimer’s disease (AD). These observations are supported by studies in animal models showing that DHA reduces synaptic pathology and memory deficits. Different mechanisms to explain these beneficial effects have been proposed; however, the molecular pathways involved are still unknown. In this study, to unravel the main underlying molecular mechanisms activated upon DHA treatment, the effect of a high dose of DHA on cognitive function and AD pathology was analyzed in aged Tg2576 mice and their wild-type littermates. Transcriptomic analysis of mice hippocampi using RNA sequencing was subsequently performed. Our results revealed that, through an amyloid-independent mechanism, DHA enhanced memory function and increased synapse formation only in the Tg2576 mice. Likewise, the IPA analysis demonstrated that essential neuronal functions related to synaptogenesis, neuritogenesis, the branching of neurites, the density of dendritic spines and the outgrowth of axons were upregulated upon-DHA treatment in Tg2576 mice. Our results suggest that memory function in APP mice is influenced by DHA intake; therefore, a high dose of daily DHA should be tested as a dietary supplement for AD dementia prevention.
Open Access
Metschnikowia pulcherrima as an efficient biocontrol agent of Botrytis cinerea infection in apples: unraveling protection mechanisms through yeast proteomics
(Elsevier, 2023) Fernández San Millán, Alicia; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larraya Reta, Luis María; Farrán Blanch, Inmaculada; Veramendi Charola, Jon; Ciencias de la Salud; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The results obtained in this study show that the Mp-30 strain of Metschnikowia pulcherrima is able to completely prevent Botrytis cinerea infection in apples, which is a major postharvest disease of fruits throughout the world. We have observed that although Mp-30 is able to rapidly colonize wounds, sequestrate iron and secrete antifungal compounds, other unknown mechanisms that occur in the early phase of the yeast-fungal interaction must be implicated in the biocontrol response. The main objective of this study was to identify the pathways involved in the mechanism of action of Mp-30 against B. cinerea in apples. Therefore, differentially accumulated yeast proteins in the presence/absence of B. cinerea on wounded apples were studied to elucidate Mp-30 biocontrol mechanisms and regulation at the protein level. A comparative proteomic analysis showed that 114 yeast proteins were increased and 61 were decreased. The Mp-30 antagonistic response mainly showed the increase of (1) gene expression and protein translation related proteins, (2) trafficking and vesicle-mediated transport related proteins, (3) pyruvate metabolism and mitochondrial proteins related to energy and amino acid production, (4) fatty acid synthesis, and (5) cell envelope related proteins. On the other hand, redox homeostasis, and amino acid and carbon metabolism were downregulated. Since there is no yeast growth enhancement associated with the presence of B. cinerea, such regulation mechanisms may be related to the reprogramming of metabolism, synthesis of new compounds and reorganization of yeast cell structure. Indeed, the results show that several pathways cooperate in restructuring the plasma membrane and cell wall composition, highlighting their major role in the antagonistic interactions for apple protection against gray mold proliferation. These results are of great interest since they provide a clear insight into the yeast mechanisms involved in B. cinerea inactivation during the first hours of contact in the wounded fruit. They shed light on the unknown yeast molecular biocontrol mechanisms.
Open Access
The regulators of peroxisomal acyl-carnitine shuttle CROT and CRAT promote metastasis in melanoma
(Elsevier, 2023) Lasheras Otero, Irene; Feliu, Iker; Maíllo Ruiz de Infante, Alberto; Moreno, Haritz; Redondo Muñoz, Marta; Aldaz Donamaría, Paula; Bocanegra Gondán, Ana Isabel; Olías Arjona, Ana; Lecanda, Fernando; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Larráyoz, Ignacio M.; Gómez-Cabrero, David; Wellbrock, Claudia; Vicent, Silvestre; Arozarena Martinicorena, Imanol; Ciencias; Zientziak; Ciencias de la Salud; Osasun Zientziak
Circulating tumor cells are the key link between a primary tumor and distant metastases, but once in the bloodstream, loss of adhesion induces cell death. To identify the mechanisms relevant for melanoma circulating tumor cell survival, we performed RNA sequencing and discovered that detached melanoma cells and isolated melanoma circulating tumor cells rewire lipid metabolism by upregulating fatty acid (FA) transport and FA betaoxidation‒related genes. In patients with melanoma, high expression of FA transporters and FA beta-oxidation enzymes significantly correlates with reduced progression-free and overall survival. Among the highest expressed regulators in melanoma circulating tumor cells were the carnitine transferases carnitine O-octanoyltransferase and carnitine acetyltransferase, which control the shuttle of peroxisome-derived medium-chain FAs toward mitochondria to fuel mitochondrial FA beta-oxidation. Knockdown of carnitine O-octanoyltransferase or carnitine acetyltransferase and short-term treatment with peroxisomal or mitochondrial FA beta-oxidation inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. Carnitine O-octanoyltransferase and carnitine acetyltransferase depletion could be rescued by medium-chain FA supplementation, indicating that the peroxisomal supply of FAs is crucial for the survival of nonadherent melanoma cells. Our study identifies targeting the FA-based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the antimetastatic activity of the Food and Drug Administration‒approved drug ranolazine carries translational potential.