Open Access
Oncolytic adenovirus Delta-24-RGD induces a widespread glioma proteotype remodeling during autophagy
(Elsevier, 2018) González Morales, Andrea; Zabaleta, Aintzane; García Moure, Marc; Alonso Roldán, Marta; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Adenovirus Delta-24-RGD has shown a remarkable efficacy in a phase I clinical trial for glioblastoma. Delta-24-RGD induces autophagy in glioma cells, however, the molecular derangements associated with Delta-24-RGD infection remains poorly understood. Here, proteomics was applied to characterize the glioma metabolic disturbances soon after Delta-24-RGD internalization and late in infection. Minutes post-infection, a rapid survival reprogramming of glioma cells was evidenced by an early c-Jun activation and a time-dependent dephosphorylation of multiple survival kinases. At 48 h post-infection (hpi), a severe intracellular proteostasis impairment was characterized, detecting differentially expressed proteins related to mRNA splicing, cytoskeletal organization, oxidative response, and inflammation. Specific kinase-regulated protein interactomes for Delta-24-RGD-modulated proteome revealed interferences with the activation dynamics of protein kinases C and A (PKC, PKA), tyrosine-protein kinase Src (c-Src), glycogen synthase kinase-3 (GSK-3) as well as serine/threonine-protein phosphatases 1 and 2A (PP1, PP2A) at 48hpi, in parallel with adenoviral protein overproduction. Moreover, the late activation of the nuclear factor kappa B (NF-κB) correlates with the extracellular increment of specific cytokines involved in migration, and activation of different inflammatory cells. Taken together, our integrative analysis provides further insights into the effects triggered by Delta-24-RGD in the modulation of tumor suppression and immune response against glioma. Significance: The current study provides new insights regarding the molecular mechanisms governing the glioma metabolism during Delta-24-RGD oncolytic adenoviral therapy. The compilation and analysis of intracellular and extracellular proteomics have led us to characterize: i) the cell survival reprogramming during Delta-24-RGD internalization, ii) the proteostatic disarrangement induced by Delta-24-RGD during the autophagic stage, iii) the protein interactomes for Delta-24-RGD-modulated proteome, iv) the regulatory effects on kinase dynamics induced by Delta-24-RGD late in infection, and v) the overproduction of multitasking cytokines upon Delta-24-RGD treatment. We consider that the quantitative molecular maps generated in this study may establish the foundations for the development of complementary adenoviral based-vectors to increase the potency against glioma.
Open Access
Amyloid-driven tau accumulation on mitochondria potentially leads to cognitive deterioration in Alzheimer’s disease
(MDPI, 2021) Cuadrado-Tejedor, Mar; Pérez-González, Marta; Alfaro-Ruiz, Rocío; Badesso, Sara; Sucunza, Diego; Espelosín, María; Ursúa, Susana; Lachén Montes, Mercedes; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Luján, Rafael; García-Osta, Ana; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
Despite the well-accepted role of the two main neuropathological markers (β-amyloid and tau) in the progression of Alzheimer’s disease, the interaction and specific contribution of each of them is not fully elucidated. To address this question, in the present study, an adeno-associated virus (AAV9) carrying the mutant P301L form of human tau, was injected into the dorsal hippocampi of APP/PS1 transgenic mice or wild type mice (WT). Three months after injections, memory tasks, biochemical and immunohistochemical analysis were performed. We found that the overexpression of hTauP301L accelerates memory deficits in APP/PS1 mice, but it did not affect memory function of WT mice. Likewise, biochemical assays showed that only in the case of APP/PS1-hTauP301L injected mice, an important accumulation of tau was observed in the insoluble urea fraction. Similarly, electron microscopy images revealed that numerous clusters of tau immunoparticles appear at the dendrites of APP/PS1 injected mice and not in WT animals, suggesting that the presence of amyloid is necessary to induce tau aggregation. Interestingly, these tau immunoparticles accumulate in dendritic mitochondria in the APP/PS1 mice, whereas most of mitochondria in WT injected mice remain free of tau immunoparticles. Taken together, it seems that amyloid induces tau aggregation and accumulation in the dendritic mitochondria and subsequently may alter synapse function, thus, contributing to accelerate cognitive decline in APP/PS1 mice.
Open Access
Fiber-based label-free D-dimer detection for early diagnosis of venous thromboembolism
(SPIE, 2020) Zubiate Orzanco, Pablo; Urrutia Azcona, Aitor; Ruiz Zamarreño, Carlos; Fernández Irigoyen, Joaquín; Giannetti, Ambra; Baldini, Francesco; Díaz Lucas, Silvia; Matías Maestro, Ignacio; Arregui San Martín, Francisco Javier; Santamaría Martínez, Enrique; Del Villar, Ignacio; Chiavaioli, Francesco; Ingeniaritza Elektrikoa, Elektronikoaren eta Telekomunikazio Ingeniaritzaren; Institute of Smart Cities - ISC; Ingeniería Eléctrica, Electrónica y de Comunicación
D-dimer is a useful diagnostic biomarker for deep vein thrombosis or pulmonary embolism, collectively referred to as venous thromboembolism (VTE). The ability to detect in real-time the amount of D-dimer with a fast and reliable method is a key step to anticipate the appearance of these diseases. The combination of fiber-optic-based platforms for biosensing with the nanotechnologies is opening up the chance for the development of in situ, portable, lightweight, versatile, reliable and high-performance optical sensing devices towards lab-on-fiber technology. The generation of lossy mode resonances (LMRs) by means of the deposition of nm-thick absorbing metal-oxide films on special geometric-modified fibers allows measuring precisely and accurately surface refractive index changes, which are due to the binding interaction between a biological recognition element and the analyte under investigation. This approach enhances the light-matter interaction in a strong way, thus turning out to be more sensitive compared to other optical technology platforms, such as fiber gratings or surface plasmon resonance. Here, the results of a highly specific and sensitive biosensor for the detection of D-dimer based on LMR in fiber-optics are presented by monitoring in real-time the shift of the LMR related to the biomolecule interactions thanks to a conventional wavelength-interrogation system and an ad-hoc developed microfluidics. A detection limit of 100 ng/mL, a value 5-fold below the clinical cutoff value, has been attained for D-dimer spiked in human serum. The comparison of the results achieved with proteomics-based methodologies, which allows for the identification of betaand gamma-chains of fibrinogen, demonstrates the ability of our platform to specifically (>90%) recognize D-dimer.
Open Access
Influence of short-term training on functional capacity and (anti-)inflammatory immune signalling in acute hospitalization
(Wiley, 2020) Ramírez Vélez, Robinson; Martínez Velilla, Nicolás; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Izquierdo Redín, Mikel; Palomino Echeverría, Sara; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
To investigate the infuence of exercise on inflammatory signalling, it was performed cytokine array profiling in human serum to identify inflammatory cytokines produced after a 3 day in-hospital intervention including individualized moderate-intensity resistance, balance, and walking exercises vs. medical usual-care for acute hospitalization in very elderly patients.
Open Access
A proteomic atlas of lineage and cancer-polarized expression modules in myeloid cells modeling immunosuppressive tumor-infiltrating subsets
(MDPI, 2021) Blanco, Ester; Ibañez Vea, María; Hernández, Carlos; Drici, Lylia; Martínez de Morentin Iribarren, Xabier; Gato Cañas, María; Ausín, Karina; Bocanegra Gondán, Ana Isabel; Zuazo Ibarra, Miren; Chocarro de Erauso, Luisa; Arasanz Esteban, Hugo; Fernández Hinojal, Gonzalo; Fernández Irigoyen, Joaquín; Smerdou, Cristian; Garnica, Maider; Echaide Górriz, Míriam; Fernández Rubio, Leticia; Morente Sancho, Pilar; Ramos-Castellanos, Pablo; Llopiz, Diana; Santamaría Martínez, Enrique; Larsen, Martin R.; Escors Murugarren, David; Kochan, Grazyna; Osasun Zientziak; Institute for Multidisciplinary Research in Applied Biology - IMAB; Ciencias de la Salud; Gobierno de Navarra / Nafarroako Gobernua
Monocytic and granulocytic myeloid-derived suppressor cells together with tumor-infiltrating macrophages constitute the main tumor-infiltrating immunosuppressive myeloid populations. Due to the phenotypic resemblance to conventional myeloid cells, their identification and purification from within the tumors is technically difficult and makes their study a challenge. We differentiated myeloid cells modeling the three main tumor-infiltrating types together with uncommitted macrophages, using ex vivo differentiation methods resembling the tumor microenvironment. The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules. The relationships between monocytic MDSCs and TAMs, monocytic MDSCs and granulocytic MDSCs, and hierarchical relationships of expression networks and transcription factors due to lineage and cancer polarization were mapped. Highly purified immunosuppressive myeloid cell populations that model tumor-infiltrating counterparts were systematically analyzed by quantitative proteomics. Full functional interactome maps have been generated to characterize at high resolution the relationships between the three main myeloid tumor-infiltrating cell types. Our data highlights the biological processes related to each cell type, and uncover novel shared and differential molecular targets. Moreover, the high numbers and fidelity of ex vivo-generated subsets to their natu-ral tumor-shaped counterparts enable their use for validation of new treatments in high-throughput experiments.
Open Access
Mitochondrial oxidative stress induces cardiac fibrosis in obese rats through modulation of transthyretin
(MDPI, 2022) Martínez Martínez, Ernesto; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Nieto, María Luisa; Bravo San Pedro, José Manuel; Cachofeiro, Victoria; Ciencias de la Salud; Osasun Zientziak
A proteomic approach was used to characterize potential mediators involved in the improvement in cardiac fibrosis observed with the administration of the mitochondrial antioxidant MitoQ in obese rats. Male Wistar rats were fed a standard diet (3.5% fat; CT) or a high-fat diet (35% fat; HFD) and treated with vehicle or MitoQ (200 ¿M) in drinking water for 7 weeks. Obesity modulated the expression of 33 proteins as compared with controls of the more than 1000 proteins identified. These include proteins related to endoplasmic reticulum (ER) stress and oxidative stress. Proteomic analyses revealed that HFD animals presented with an increase in cardiac transthyretin (TTR) protein levels, an effect that was prevented by MitoQ treatment in obese animals. This was confirmed by plasma levels, which were associated with those of cardiac levels of both binding immunoglobulin protein (BiP), a marker of ER stress, and fibrosis. TTR stimulated collagen I production and BiP in cardiac fibroblasts. This upregulation was prevented by the presence of MitoQ. In summary, the results suggest a role of TTR in cardiac fibrosis development associated with obesity and the beneficial effects of treatment with mitochondrial antioxidants.
Open Access
RTP801 interacts with the tRNA ligase complex and dysregulates its RNA ligase activity in Alzheimer's disease
(Oxford University Press, 2024-09-12) Campoy-Campos, Genís; Solana-Balaguer, Júlia; Guisado-Corcoll, Anna; Chicote-González, Almudena; García-Segura, Pol; Pérez-Sisqués, Leticia; Gabriel Torres, Adrián; Canal, Mercè; Molina-Porcel, Laura; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Pouplana, Lluís Ribas de; Alberch, Jordi; Martí, Eulàlia; Giralt, Albert; Pérez-Navarro, Esther; Malagelada, Cristina; Ciencias de la Salud; Osasun Zientziak
RTP801/REDD1 is a stress-responsive protein overexpressed in neurodegenerative diseases such as Alzheimer's disease (AD) that contributes to cognitive deficits and neuroinflammation. Here, we found that RTP801 interacts with HSPC117, DDX1 and CGI-99, three members of the tRNA ligase complex (tRNA-LC), which ligates the excised exons of intron-containing tRNAs and the mRNA exons of the transcription factor XBP1 during the unfolded protein response (UPR). We also found that RTP801 modulates the mRNA ligase activity of the complex in vitro since RTP801 knockdown promoted XBP1 splicing and the expression of its transcriptional target, SEC24D. Conversely, RTP801 overexpression inhibited the splicing of XBP1. Similarly, in human AD postmortem hippocampal samples, where RTP801 is upregulated, we found that XBP1 splicing was dramatically decreased. In the 5xFAD mouse model of AD, silencing RTP801 expression in hippocampal neurons promoted Xbp1 splicing and prevented the accumulation of intron-containing pre-tRNAs. Finally, the tRNA-enriched fraction obtained from 5xFAD mice promoted abnormal dendritic arborization in cultured hippocampal neurons, and RTP801 silencing in the source neurons prevented this phenotype. Altogether, these results show that elevated RTP801 impairs RNA processing in vitro and in vivo in the context of AD and suggest that RTP801 inhibition could be a promising therapeutic approach.
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
Motor skill learning modulates striatal extracellular vesicles' content in a mouse model of Huntington's disease
(BMC, 2024-06-11) Solana-Balaguer, Júlia; García-Segura, Pol; Campoy-Campos, Genís; Chicote-González, Almudena; Fernández Irigoyen, Joaquín; Santamaría Martínez, Enrique; Pérez-Navarro, Esther; Masana, Mercè; Alberch, Jordi; Malagelada, Cristina; Ciencias de la Salud; Osasun Zientziak
Huntington's disease (HD) is a neurological disorder caused by a CAG expansion in the Huntingtin gene (HTT). HD pathology mostly affects striatal medium-sized spiny neurons and results in an altered cortico-striatal function. Recent studies report that motor skill learning, and cortico-striatal stimulation attenuate the neuropathology in HD, resulting in an amelioration of some motor and cognitive functions. During physical training, extracellular vesicles (EVs) are released in many tissues, including the brain, as a potential means for inter-tissue communication. To investigate how motor skill learning, involving acute physical training, modulates EVs crosstalk between cells in the striatum, we trained wild-type (WT) and R6/1 mice, the latter with motor and cognitive deficits, on the accelerating rotarod test, and we isolated their striatal EVs. EVs from R6/1 mice presented alterations in the small exosome population when compared to WT. Proteomic analyses revealed that striatal R6/1 EVs recapitulated signaling and energy deficiencies present in HD. Motor skill learning in R6/1 mice restored the amount of EVs and their protein content in comparison to naïve R6/1 mice. Furthermore, motor skill learning modulated crucial pathways in metabolism and neurodegeneration. All these data provide new insights into the pathogenesis of HD and put striatal EVs in the spotlight to understand the signaling and metabolic alterations in neurodegenerative diseases. Moreover, our results suggest that motor learning is a crucial modulator of cell-to-cell communication in the striatum.
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.