1,720,972 research outputs found
The role of mitochondria in shaping the oncogenic signaling
Full text linkCancer cells are endowed with the capability to reprogram cell metabolism in order to support neoplastic growth and cell death escape. A key role in this process is played by signaling pathways, mainly cascades regulated by kinases. Integration of survival and death stimuli occurs on mitochondria, where many of these signals converge to the regulation of a channel, the permeability transition pore (PTP), that triggers mitochondrial depolarization and release of pro-apoptotic factors from mitochondria. PTP opening commits cells to death, and it is regulated by a variety of factors. Here I have studied how signal transduction pathways affect cell metabolism, particularly mitochondrial bioenergetics, which could eventually lead to modulation of PTP opening.
In the first part of my work, I have explored the presence of the PTP in cells depleted of mitochondrial DNA (ρ0 cells) which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions, among which cancer. I have observed that these cells are indeed equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells. In detail, inhibition of PTP opening is a survival mechanism achieved by two different mechanisms: the first one is the mitochondrial binding of the glycolytic enzyme hexokinase (HK) II, which is up-regulated in ρ0 cells; the second one relies on the hyper-activation of the ERK-GSK3 signaling pathway that converge on mitochondria where it maintains the PTP regulator cyclophilin D (CyP-D) in the dephosphorylated form. I observed that mitochondria of ρ0 cells maintain a membrane potential which is readily dissipated after displacement of HK II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by keeping ρ0 cells in serum and glucose starvation. The PTP inhibitor cyclosporin A (CsA) is able to decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. Moreover, glucose and serum deprivation causes concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of CyP-D and PTP opening. Indeed, GSK3α/β inhibition with indirubin-3′-oxime decreases PTP-induced cell death in ρ0 cells following nutrient ablation.
In the second part of my work, I have focused my attention on a tumor predisposition syndrome, called Neurofibromatosis type 1 (NF1), caused by loss of function of neurofibromin, which acts as a negative regulator of the Ras signaling cascades. Here I have investigated whether the Ras hyper-activation induced by neurofibromin ablation can affect mitochondria bioenergetics, thus contributing to the metabolic rewiring of NF1 tumors. I observed that the absence of neurofibromin confers to mouse embryonic fibroblasts (MEFs) the capability to form colonies in an in vitro tumorigenesis assay, and that the use of an ERK inhibitor completely abrogates colony formation. Moreover, I injected wild-type and Nf1-/- MEFs subcutaneously into nude mice in order to test the capability of the cells to form tumors in vivo. I observed that the absence of Nf1 leads to the growth of a tumor mass within a month, whereas no tumor can be formed in the same time frame by wild-type MEFs. I started the analysis of mitochondrial bioenergetics by measurements of oxygen consumption rate (OCR) performed with an extracellular flux analyzer on adherent cell monolayers. Nf1-/- MEFs display a lower OCR than wild-type MEFs both in basal condition and upon treatment with a low concentration of an uncoupler, which stimulates respiration maximally. Moreover, I found that the fraction of oxygen consumption coupled to ATP production by mitochondrial ATP synthase is lower in cells without Nf1, suggesting that ATP requirements are mainly supplied by glycolysis. Accordingly, upon glucose starvation or inhibition of glycolysis with 5-thioglucose Nf1-/- cells display a stronger decrease in ATP levels compared to wild-type cells. The different OCRs between wild-type and Nf1-/- cells are not related to differences in mitochondrial mass or membrane potential.
In order to establish whether a modulation of respiratory chain activity could account for the observed OCR differences, I analyzed the expression level of the respiratory chain complexes. Complex I (NADH dehydrogenase) is down-regulated by Nf1 ablation, as a lower level of some of its subunits and of the assembled complex I was detected. Furthermore, the enzymatic activity of complex I is expectedly lower in Nf1-/- cells. Interestingly, administration of an ERK inhibitor increases the expression of complex I subunits resulting in its augmented assembly and activity; remarkably, this effect is more pronounced in Nf1-/- MEFs than in wild-type cells.
I also observed down-regulation of complex II (succinate dehydrogenase) activity in knock-out cells compared to wild-type MEFs. Interestingly, I found that complex II interacts with the mitochondrial chaperone TRAP1 and the kinase ERK, a fraction of which is mitochondrial; these interactions are stronger in Nf1-/- than in wild-type cells, as assessed by blue native gel analysis. However, silencing of TRAP1 does not change the activity of complex II; in accordance to this, the OCR of cells in which TRAP1 has been down-regulated does not vary, yet modulation of TRAP1 levels affects maximal respiration of Nf1-/- cells. Interestingly, silencing of TRAP1 in Nf1-deficient MEFs compromises the tumorigenic properties of these cells.
Taken together, these observations suggest that the absence of neurofibromin leads to a more glycolytic metabolism by down-modulating the activity of respiratory chain complexes. The metabolic switch observed upon Nf1 deletion is a typical marker of cancer cells that favors tumor progression. We hypothesize that Ras-ERK signaling is upstream to the regulation of mitochondrial bioenergetics, and that the metabolic changes prompted by Ras-ERK activation can contribute to the transformed phenotype that we observe in Nf1-/- MEFs
Induction of the permeability transition pore in cells depleted of mitochondrial DNA
No full textAbstractRespiratory complexes are believed to play a role in the function of the mitochondrial permeability transition pore (PTP), whose dysregulation affects the process of cell death and is involved in a variety of diseases, including cancer and degenerative disorders. We investigated here the PTP in cells devoid of mitochondrial DNA (ρ0 cells), which lack respiration and constitute a model for the analysis of mitochondrial involvement in several pathological conditions. We observed that mitochondria of ρ0 cells maintain a membrane potential and that this is readily dissipated after displacement of hexokinase (HK) II from the mitochondrial surface by treatment with either the drug clotrimazole or with a cell-permeant HK II peptide, or by placing ρ0 cells in a medium without serum and glucose. The PTP inhibitor cyclosporin A (CsA) could decrease the mitochondrial depolarization induced by either HK II displacement or by nutrient depletion. We also found that a fraction of the kinases ERK1/2 and GSK3α/β is located in the mitochondrial matrix of ρ0 cells, and that glucose and serum deprivation caused concomitant ERK1/2 inhibition and GSK3α/β activation with the ensuing phosphorylation of cyclophilin D, the mitochondrial target of CsA. GSK3α/β inhibition with indirubin-3′-oxime decreased PTP-induced cell death in ρ0 cells following nutrient ablation. These findings indicate that ρ0 cells are equipped with a functioning PTP, whose regulatory mechanisms are similar to those observed in cancer cells, and suggest that escape from PTP opening is a survival factor in this model of mitochondrial diseases. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)
The Chaperone TRAP1 As a Modulator of the Mitochondrial Adaptations in Cancer Cells
Full text linkMitochondria can receive, integrate, and transmit a variety of signals to shape many biochemical activities of the cell. In the process of tumor onset and growth, mitochondria contribute to the capability of cells of escaping death insults, handling changes in ROS levels, rewiring metabolism, and reprograming gene expression. Therefore, mitochondria can tune the bioenergetic and anabolic needs of neoplastic cells in a rapid and flexible way, and these adaptations are required for cell survival and proliferation in the fluctuating environment of a rapidly growing tumor mass. The molecular bases of pro-neoplastic mitochondrial adaptations are complex and only partially understood. Recently, the mitochondrial molecular chaperone TRAP1 (tumor necrosis factor receptor associated protein 1) was identified as a key regulator of mitochondrial bioenergetics in tumor cells, with a profound impact on neoplastic growth. In this review, we analyze these findings and discuss the possibility that targeting TRAP1 constitutes a new antitumor approach
Design and Test of Molecules that Interfere with the Recognition Mechanisms between the SARS-CoV-2 Spike Protein and Its Host Cell Receptors
No full text: The disruptive impact of the COVID-19 pandemic has led the scientific community to undertake an unprecedented effort to characterize viral infection mechanisms. Among these, interactions between the viral glycosylated Spike and the human receptors ACE2 and TMPRSS2 are key to allowing virus invasion. Here, we report and test a fully rational methodology to design molecules that are capable of perturbing the interactions between these critical players in SARS-CoV-2 pathogenicity. To this end, we computationally identify substructures on the fully glycosylated Spike protein that are not intramolecularly optimized and are thus prone to being stabilized by forming complexes with ACE2 and TMPRSS2. With the aim of competing with the Spike-mediated cell entry mechanisms, we have engineered the predicted putative interaction regions in the form of peptide mimics that could compete with Spike for interaction with ACE2 and/or TMPRSS2. Experimental models of viral entry demonstrate that the designed molecules are able to interfere with viral entry into ACE2/TMPRSS2 expressing cells, while they have no effects on the entry of control viral particles that do not harbor the Spike protein or on the entry of Spike-presenting viral particles into cells that do not display its receptors on their surface
Metabolic plasticity of tumor cell mitochondria
Full text linkMitochondria are dynamic organelles that exchange a multiplicity of signals with other cell compartments, in order to finely adjust key biological routines to the fluctuating metabolic needs of the cell. During neoplastic transformation, cells must provide an adequate supply of the anabolic building blocks required to meet a relentless proliferation pressure. This can occur in conditions of inconstant blood perfusion leading to variations in oxygen and nutrient levels. Mitochondria afford the bioenergetic plasticity that allows tumor cells to adapt and thrive in this ever changing and often unfavorable environment. Here we analyse how mitochondria orchestrate the profound metabolic rewiring required for neoplastic growth
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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