High variability of perezone content in rhizomes of Acourtia cordata wild plants, environmental factors related, and proteomic analysis.

With the aim of exploring the source of the high variability observed in the production of perezone, in Acourtia cordata wild plants, we analyze the influence of soil parameters and phenotypic characteristics on its perezone content. Perezone is a sesquiterpene quinone responsible for several pharmacological effects and the A. cordata plants are the natural source of this metabolite. The chemistry of perezone has been widely studied, however, no studies exist related to its production under natural conditions, nor to its biosynthesis and the environmental factors that affect the yield of this compound in wild plants. We also used a proteomic approach to detect differentially expressed proteins in wild plant rhizomes and compare the profiles of high vs. low perezone-producing plants. Our results show that in perezone-producing rhizomes, the presence of high concentrations of this compound could result from a positive response to the effects of some edaphic factors, such as total phosphorus (Pt), total nitrogen (Nt), ammonium (NH4), and organic matter (O. M.), but could also be due to a negative response to the soil pH value. Additionally, we identified 616 differentially expressed proteins between high and low perezone producers. According to the functional annotation of this comparison, the upregulated proteins were grouped in valine biosynthesis, breakdown of leucine and isoleucine, and secondary metabolism such as terpenoid biosynthesis. Downregulated proteins were grouped in basal metabolism processes, such as pyruvate and purine metabolism and glycolysis/gluconeogenesis. Our results suggest that soil parameters can impact the content of perezone in wild plants. Furthermore, we used proteomic resources to obtain data on the pathways expressed when A. cordata plants produce high and low concentrations of perezone. These data may be useful to further explore the possible relationship between perezone production and abiotic or biotic factors and the molecular mechanisms related to high and low perezone production.

Rhizobium etli CFN42 proteomes showed isoenzymes in free-living and symbiosis with a different transcriptional regulation inferred from a transcriptional regulatory network.

A comparative proteomic study at 6 h of growth in minimal medium (MM) and bacteroids at 18 days of symbiosis of Rhizobium etli CFN42 with the Phaseolus vulgaris leguminous plant was performed. A gene ontology classification of proteins in MM and bacteroid, showed 31 and 10 pathways with higher or equal than 30 and 20% of proteins with respect to genome content per pathway, respectively. These pathways were for energy and environmental compound metabolism, contributing to understand how Rhizobium is adapted to the different conditions. Metabolic maps based on orthology of the protein profiles, showed 101 and 74 functional homologous proteins in the MM and bacteroid profiles, respectively, which were grouped in 34 different isoenzymes showing a great impact in metabolism by covering 60 metabolic pathways in MM and symbiosis. Taking advantage of co-expression of transcriptional regulators (TF's) in the profiles, by selection of genes whose matrices were clustered with matrices of TF's, Transcriptional Regulatory networks (TRN´s) were deduced by the first time for these metabolic stages. In these clustered TF-MM and clustered TF-bacteroid networks, containing 654 and 246 proteins, including 93 and 46 TFs, respectively, showing valuable information of the TF's and their regulated genes with high stringency. Isoenzymes were specific for adaptation to the different conditions and a different transcriptional regulation for MM and bacteroid was deduced. The parameters of the TRNs of these expected biological networks and biological networks of E. coli and B. subtilis segregate from the random theoretical networks. These are useful data to design experiments on TF gene-target relationships for bases to construct a TRN.

Expression Profiles of Kidney Mitochondrial Proteome during the Progression of the Unilateral Ureteral Obstruction: Focus on Energy Metabolism Adaptions.

Kidney diseases encompass many pathologies, including obstructive nephropathy (ON), a common clinical condition caused by different etiologies such as urolithiasis, prostatic hyperplasia in males, tumors, congenital stenosis, and others. Unilateral ureteral obstruction (UUO) in rodents is an experimental model widely used to explore the pathophysiology of ON, replicating vascular alterations, tubular atrophy, inflammation, and fibrosis development. In addition, due to the kidney's high energetic demand, mitochondrial function has gained great attention, as morphological and functional alterations have been demonstrated in kidney diseases. Here we explore the kidney mitochondrial proteome differences during a time course of 7, 14, and 21 days after the UUO in rats, revealing changes in proteins involved in three main metabolic pathways, oxidative phosphorylation (OXPHOS), the tricarboxylic acid cycle (TCA), and the fatty acid (FA) metabolism, all of them related to bioenergetics. Our results provide new insight into the mechanisms involved in metabolic adaptations triggered by the alterations in kidney mitochondrial proteome during the ON.

Characterization of Mitochondrial Proteome and Function in Luminal A and Basal-like Breast Cancer Subtypes Reveals Alteration in Mitochondrial Dynamics and Bioenergetics Relevant to Their Diagnosis.

Breast cancer (BC) is the most prevalent cancer and the one with the highest mortality among women worldwide. Although the molecular classification of BC has been a helpful tool for diagnosing and predicting the treatment of BC, developments are still being made to improve the diagnosis and find new therapeutic targets. Mitochondrial dysfunction is a crucial feature of cancer, which can be associated with cancer aggressiveness. Although the importance of mitochondrial dynamics in cancer is well recognized, its involvement in the mitochondrial function and bioenergetics context in BC molecular subtypes has been scantly explored. In this study, we combined mitochondrial function and bioenergetics experiments in MCF7 and MDA-MB-231 cell lines with statistical and bioinformatics analyses of the mitochondrial proteome of luminal A and basal-like tumors. We demonstrate that basal-like tumors exhibit a vicious cycle between mitochondrial fusion and fission; impaired but not completely inactive mitochondrial function; and the Warburg effect, associated with decreased oxidative phosphorylation (OXPHOS) complexes I and III. Together with the results obtained in the cell lines and the mitochondrial proteome analysis, two mitochondrial signatures were proposed: one signature reflecting alterations in mitochondrial functions and a second signature exclusively of OXPHOS, which allow us to distinguish between luminal A and basal-like tumors.

Quantitative Proteomic Analysis of Cervical Cancer Tissues Identifies Proteins Associated With Cancer Progression.

BACKGROUND/AIM: To date, several proteomics studies in cervical cancer (CC) have focused mainly on squamous cervical cancer (SCC). Our study aimed to discover and clarify differences in SCC and CAD that may provide valuable information for the identification of proteins involved in tumor progression, in CC as a whole, or specific for SCC or CAD. MATERIALS AND METHODS: Total protein extracts from 15 individual samples corresponding to 5 different CC tissue types were compared with a non-cancerous control group using bidimensional liquid chromatography-mass spectrometry (2D LC-MS/MS), isobaric tags for relative and absolute quantitation (ITRAQ), principal component analysis (PCA) and gene set enrichment analysis (GSEA). RESULTS: A total of 622 statistically significant different proteins were detected. Exocytosis-related proteins were the most over-represented, accounting for 25% of the identified and quantified proteins. Based on the experimental results, reticulocalbin 3 (RCN3) and Ras-related protein Rab-14 (RAB14) were chosen for further downstream in vitro and vivo analyses. RCN3 was overexpressed in all CC tissues compared to the control and RAB14 was overexpressed in squamous cervical cancer (SCC) compared to invasive cervical adenocarcinoma (CAD). In the tumor xenograft experiment, RAB14 protein expression was positively correlated with increased tumor size. In addition, RCN3-expressing HeLa cells induced a discrete size increment compared to control, at day 47 after inoculation. CONCLUSION: RAB14 and RCN3 are suggested as potential biomarkers and therapeutic targets in the treatment of CC.

Principal component analysis on LC­MS/MS and 2DE­MALDI­TOF in glioblastoma cell lines reveals that mitochondria act as organelle sensors of the metabolic state in glioblastoma.

Glioblastoma is a difficult disease to diagnose. Proteomic techniques are commonly applied in biomedical research, and can be useful for early detection, making an accurate diagnosis and reducing mortality. The relevance of mitochondria in brain development and function is well known; therefore, mitochondria may influence the development of glioblastoma. The T98G (with oxidative metabolism) and U87MG (with glycolytic metabolism) cell lines are considered to be useful glioblastoma models for studying these tumors and the role of mitochondria in key aspects of this disease, such as prognosis, metastasis and apoptosis. In the present study, principal component analysis of protein abundance data identified by liquid chromatography coupled to tandem mass spectrometry (LC­MS/MS) and matrix­assisted laser desorption/ionization­time of flight mass spectrometry (MALDI­TOF) from 2D gels indicated that representative mitochondrial proteins were associated with glioblastoma. The selected proteins were organized into T98G­ and U87MG­specific protein­protein interaction networks to demonstrate the representativeness of both proteomic techniques. Gene Ontology overrepresentation analysis based on the relevant proteins revealed that mitochondrial processes were associated with metabolic changes, invasion and metastasis in glioblastoma, along with other non­mitochondrial processes, such as DNA translation, chaperone responses and autophagy. Despite the lower resolution of 2D electrophoresis, principal component analysis yielded information of comparable quality to that of LC­MS/MS. The present analysis pipeline described a specific and more complete metabolic status for each cell line, defined a clear mitochondrial performance for distinct glioblastoma tumors, and introduced a useful strategy to understand the heterogeneity of glioblastoma.

Mitochondrial proteomic profile of complex IV deficiency fibroblasts: rearrangement of oxidative phosphorylation complex/supercomplex and other metabolic pathways / Perfil proteómico mitocondrial de los fibroblastos con deficiencia del complejo IV: reordenamiento del complejo/supercomplejo de la fosforilación oxidativa y otras vías metabólicas

Abstract: Background: Mitochondriopathies are multisystem diseases affecting the oxidative phosphorylation (OXPHOS) system. Skin fibroblasts are a good model for the study of these diseases. Fibroblasts with a complex IV mitochondriopathy were used to determine the molecular mechanism and the main affected functions in this disease. Methods: Skin fibroblast were grown to assure disease phenotype. Mitochondria were isolated from these cells and their proteome extracted for protein identification. Identified proteins were validated with the MitoMiner database. Results: Disease phenotype was corroborated on skin fibroblasts, which presented a complex IV defect. The mitochondrial proteome of these cells showed that the most affected proteins belonged to the OXPHOS system, mainly to the complexes that form supercomplexes or respirosomes (I, III, IV, and V). Defects in complex IV seemed to be due to assembly issues, which might prevent supercomplexes formation and efficient substrate channeling. It was also found that this mitochondriopathy affects other processes that are related to DNA genetic information flow (replication, transcription, and translation) as well as beta oxidation and tricarboxylic acid cycle. Conclusions: These data, as a whole, could be used for the better stratification of these diseases, as well as to optimize management and treatment options.

Resumen: Introducción: Las mitocondriopatías son enfermedades multisistémicas que afectan el funcionamiento de la fosforilación oxidativa (OXPHOS). Un buen modelo de estudio para estas enfermedades es el cultivo primario de fibroblastos. En este trabajo se utilizaron fibroblastos con mitocondriopatía del complejo IV para determinar cuáles son las principales funciones afectadas en esta enfermedad. Métodos: Se realizaron cultivos primarios de fibroblastos para corroborar el fenotipo de la enfermedad. Las mitocondrias se aislaron de estas células y se extrajo su proteoma para su identificación. Las proteínas identificadas se validaron con la base de datos de MitoMiner. Resultados: Los fibroblastos conservaron el fenotipo de la enfermedad que incluye un defecto del complejo IV. El proteoma mitocondrial de estas células mostró que las proteínas más afectadas pertenecen al sistema de OXPHOS, principalmente los complejos que forman supercomplejos o respirosomas (I, III, IV y V). El defecto en el complejo IV al parecer se debió a problemas de ensamblaje que pueden evitar la formación de los supercomplejos y la eficiente canalización de sustratos. También se observó que esta mitocondriopatía afecta otros procesos relacionados con el flujo de información genética del DNA (replicación, transcripción y traducción), así como con la beta oxidación y el ciclo de los ácidos tricarboxílicos (TCA). Conclusiones: En conjunto, estos datos podrían utilizarse para una mejor clasificación de estas enfermedades, así como para la optimización de las opciones de manejo y tratamiento.

Mitochondrial proteomic profile of complex IV deficiency fibroblasts: rearrangement of oxidative phosphorylation complex/supercomplex and other metabolic pathways.

BACKGROUND: Mitochondriopathies are multisystem diseases affecting the oxidative phosphorylation (OXPHOS) system. Skin fibroblasts are a good model for the study of these diseases. Fibroblasts with a complex IV mitochondriopathy were used to determine the molecular mechanism and the main affected functions in this disease. METHODS: Skin fibroblast were grown to assure disease phenotype. Mitochondria were isolated from these cells and their proteome extracted for protein identification. Identified proteins were validated with the MitoMiner database. RESULTS: Disease phenotype was corroborated on skin fibroblasts, which presented a complex IV defect. The mitochondrial proteome of these cells showed that the most affected proteins belonged to the OXPHOS system, mainly to the complexes that form supercomplexes or respirosomes (I, III, IV, and V). Defects in complex IV seemed to be due to assembly issues, which might prevent supercomplexes formation and efficient substrate channeling. It was also found that this mitochondriopathy affects other processes that are related to DNA genetic information flow (replication, transcription, and translation) as well as beta oxidation and tricarboxylic acid cycle. CONCLUSIONS: These data, as a whole, could be used for the better stratification of these diseases, as well as to optimize management and treatment options.