RESUMEN
The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.
Asunto(s)
Receptor de Colecistoquinina B , Humanos , Receptor de Colecistoquinina B/antagonistas & inhibidores , Receptor de Colecistoquinina B/metabolismo , Línea Celular Tumoral , Trisacáridos/química , Lactosa/análogos & derivados , Lactosa/química , Glucosa/metabolismoRESUMEN
Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
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Hexoquinasa , Neoplasias , Animales , Muerte Celular , Retículo Endoplásmico/metabolismo , Hexoquinasa/genética , Hexoquinasa/metabolismo , Humanos , Ratones , Mitocondrias , Membranas Mitocondriales/metabolismo , Neoplasias/metabolismoRESUMEN
BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
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Retículo Endoplásmico/efectos de los fármacos , Luteolina/farmacología , Mitocondrias/efectos de los fármacos , Neuronas/metabolismo , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Retículo Endoplásmico/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Transducción de SeñalRESUMEN
Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.
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Enfermedad de Alzheimer , Anciano , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Homeostasis , Humanos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMEN
The endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca2+ homeostasis, inflammation, autophagy and apoptosis. The ER-mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER-mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER-mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits.
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Retículo Endoplásmico/fisiología , GTP Fosfohidrolasas/fisiología , Mitocondrias/fisiología , Proteínas Mitocondriales/fisiología , Animales , Humanos , Dinámicas MitocondrialesRESUMEN
The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.
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Calcio/metabolismo , Retículo Endoplásmico/genética , GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Animales , Retículo Endoplásmico/diagnóstico por imagen , GTP Fosfohidrolasas/genética , Células HeLa , Humanos , Transporte Iónico/fisiología , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/ultraestructura , Membranas Mitocondriales , Proteínas Mitocondriales/genética , UltrasonografíaRESUMEN
A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca2+ and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca2+, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca2+ and cAMP (nuclear pores are permeable to solutes up to 50 kDa) and differences in [Ca2+] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca2+ is concerned, in peroxisomes.
Asunto(s)
Señalización del Calcio/fisiología , AMP Cíclico/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Mitocondriales/metabolismo , Membrana Nuclear/metabolismo , Animales , AMP Cíclico/genética , Retículo Endoplásmico/genética , Humanos , Membrana Nuclear/genéticaRESUMEN
Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca(2+) transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid ß-peptide (Aß)-related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca(2+) transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular Aß40 and Aß42 . Analysis of γ-secretase protein expression, maturation and activity revealed that the low Aß concentrations were a result of impaired γ-secretase complex function. Amyloid-ß precursor protein (APP), ß-site APP-cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER-mitochondria contact affects γ-secretase activity and Aß generation. Increased ER-mitochondria contact results in lower γ-secretase activity suggesting a new mechanism by which Aß generation can be controlled.
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Péptidos beta-Amiloides/biosíntesis , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , Técnicas de Silenciamiento del Gen , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Adenosina Trifosfato/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Regulación hacia Abajo , Retículo Endoplásmico/ultraestructura , Células HEK293 , Humanos , Mitocondrias/ultraestructura , ARN Interferente Pequeño/metabolismoRESUMEN
It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and σ1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid ß-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Receptor Cross-Talk , Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Células CHO , Calcio/metabolismo , Línea Celular Tumoral , Cricetinae , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Microdominios de Membrana/metabolismo , Ratones , Mutación , Neuronas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores sigma/metabolismo , Fracciones Subcelulares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Receptor Sigma-1RESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease, caused by poorly known pathogenic mechanisms and aggravated by delayed therapeutic intervention, that still lacks an effective cure. However, it is clear that some important neurophysiological processes are altered years before the onset of clinical symptoms, offering the possibility of identifying biological targets useful for implementation of new therapies. Of note, evidence has been provided suggesting that mitochondria, pivotal organelles in sustaining neuronal energy demand and modulating synaptic activity, are dysfunctional in AD samples. In particular, alterations in mitochondrial Ca2+ signaling have been proposed as causal events for neurodegeneration, although the exact outcomes and molecular mechanisms of these defects, as well as their longitudinal progression, are not always clear. Here, we discuss the importance of a correct mitochondrial Ca2+ handling for neuronal physiology and summarize the latest findings on dysfunctional mitochondrial Ca2+ pathways in AD, analysing possible consequences contributing to the neurodegeneration that characterizes the disease.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Mitocondrias/metabolismo , Orgánulos/metabolismo , Transducción de Señal , Calcio/metabolismoRESUMEN
Rewiring of mitochondrial metabolism has been described in different cancers as a key step for their progression. Calcium (Ca2+) signaling regulates mitochondrial function and is known to be altered in several malignancies, including triple negative breast cancer (TNBC). However, whether and how the alterations in Ca2+ signaling contribute to metabolic changes in TNBC has not been elucidated. Here, we found that TNBC cells display frequent, spontaneous inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations, which are sensed by mitochondria. By combining genetic, pharmacologic and metabolomics approaches, we associated this pathway with the regulation of fatty acid (FA) metabolism. Moreover, we demonstrated that these signaling routes promote TNBC cell migration in vitro, suggesting they might be explored to identify potential therapeutic targets.
RESUMEN
Mitochondria from every tissue are quite similar in their capability to accumulate Ca²âº in a process that depends on the electrical potential across the inner membrane; it is catalyzed by a gated channel (named mitochondrial Ca²âº uniporter), the molecular identity of which has only recently been unraveled. The release of accumulated Ca²âº in mitochondria from different tissues is, on the contrary, quite variable, both in terms of speed and mechanism: a Naâº-dependent efflux in excitable cells (catalyzed by NCLX) and a Hâº/Ca²âº exchanger in other cells. The efficacy of mitochondrial Ca²âº uptake in living cells is strictly dependent on the topological arrangement of the organelles with respect to the source of Ca²âº flowing into the cytoplasm, i.e., plasma membrane or intracellular channels. In turn, the structural and functional relationships between mitochondria and other cellular membranes are dictated by the specific architecture of different cells. Mitochondria not only modulate the amplitude and the kinetics of local and bulk cytoplasmic Ca²âº changes but also depend on the Ca²âº signal for their own functionality, in particular for their capacity to produce ATP. In this review, we summarize the processes involved in mitochondrial Ca²âº handling and its integration in cell physiology, highlighting the main common characteristics as well as key differences, in different tissues.
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Señalización del Calcio , Calcio/metabolismo , Mitocondrias/metabolismo , Animales , Antiportadores/metabolismo , Encéfalo/metabolismo , Canales de Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial , Músculos/metabolismo , Especificidad de Órganos , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
One of the major challenges of modern medicine is to block or prevent the neurodegenerative processes inevitably associated with different pathological conditions [...].
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Enfermedades Neurodegenerativas , Humanos , Transporte de Proteínas , Transducción de SeñalRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.
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Calcio , Receptores Nicotínicos , Calcio/metabolismo , Inositol , Receptores de Inositol 1,4,5-Trifosfato , Músculo Esquelético/metabolismo , Unión NeuromuscularRESUMEN
Mitochondria are autonomous and dynamic cellular organelles orchestrating a diverse range of cellular activities. Numerous cell-signaling pathways target these organelles and Ca2+ is one of the most significant. Mitochondria are able to rapidly and transiently take up Ca2+, thanks to the mitochondrial Ca2+ uniporter complex, as well as to extrude it through the Na+/Ca2+ and H+/Ca2+ exchangers. The transient accumulation of Ca2+ in the mitochondrial matrix impacts on mitochondrial functions and cell pathophysiology. Here we summarize the role of mitochondrial Ca2+ signaling in both physiological (yang) and pathological (yin) processes and the methods that can be used to investigate mitochondrial Ca2+ homeostasis. As an example of the pivotal role of mitochondria in pathology, we described the state of the art of mitochondrial Ca2+ alterations in different pathological conditions, with a special focus on Alzheimer's disease.
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Señalización del Calcio , Células/metabolismo , Células/patología , Mitocondrias/metabolismo , Yin-Yang , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Homeostasis , HumanosRESUMEN
The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.
RESUMEN
The strategic importance for cellular organelles of being in contact with each other, exchanging messenger molecules, is nowadays well established. Different inter-organelle cross-talk pathways finely regulate multiple physiological cellular mechanisms, and their dysregulation has been found to underlie different pathological conditions. In the last years, a great effort has been made to study such organelle interactions, to understand their functional roles within the cell and the molecules involved in their formation and/or modulation. In this contribution, some examples of organelle cross-talk and their contributions in regulating physiological processes are presented. Moreover, the pro and cons of the available methods for a proper, reliable investigation of membrane contact sites are described.
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Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Lípidos de la Membrana/metabolismo , Mitocondrias/metabolismo , Orgánulos/metabolismo , Animales , Autofagia/fisiología , Retículo Endoplásmico/ultraestructura , Humanos , Membranas Intracelulares/ultraestructura , Microscopía Electrónica , Mitocondrias/ultraestructura , Orgánulos/ultraestructuraRESUMEN
Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer's disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca2+ handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER-mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca2+ crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER-mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER-mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling.
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Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Presenilina-2/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Calcio/metabolismo , Línea Celular Tumoral , Citosol/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Gotas Lipídicas/metabolismo , Mutagénesis , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Dominios Proteicos/genéticaRESUMEN
Excitotoxicity is likely to occur in pathological scenarios in which mitochondrial function is already compromised, shaping neuronal responses to glutamate. In fact, mitochondria sustain cell bioenergetics, tune intracellular Ca2+ dynamics, and regulate glutamate availability by using it as metabolic substrate. Here, we suggest the need to explore glutamate toxicity in the context of specific disease models in which it may occur, re-evaluating the impact of mitochondrial dysfunction on glutamate excitotoxicity. Our aim is to signpost new approaches, perhaps combining glutamate and pathways to rescue mitochondrial function, as therapeutic targets in neurological disorders.