Despite the early characterisation of Ca 2+ fluxes in isolated mitochondria and the ability of this ion to up‐regulate dehydrogenases of the Krebs cycle, the low affinity of the organelle uptake ...pathways was a long‐standing obstacle to the recognition of a physiological role for mitochondrial Ca 2+ uptake. This review begins with a historical account of the main results that proved the contrary and provides a brief description of mitochondrial Ca 2+ signals. Then, it discusses the characteristics of Ca 2+ regulation of mitochondrial function. Finally, it summarizes recent discoveries on structural aspects of mitochondrial reticulum and its connections to signalling partners such as the endoplasmic reticulum or the plasma membrane.
Specific targeting of the recombinant, Ca2+-sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied ...to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of Ca2+ with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix Ca2+ (Ca2+m) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of Ca2+m using the fluorescent indicator, rhod2. Comparison of Ca2+m with the activity of the Ca2+-sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix Ca2+ changes. Peak Ca2+m values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) Ca2+ oscillations can be decoded by mitochondria.
Regulation of autophagy by cytoplasmic p53 Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo ...
Nature cell biology,
06/2008, Letnik:
10, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, ...mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca 2+ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca 2+ , ...the possibility that physiological stimuli that raise the Ca 2+ of the cytoplasm could trigger major mitochondrial Ca 2+ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude
of the cytoplasmic Ca 2+ rises. The direct measurement of mitochondrial Ca 2+ with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation,
the cytoplasmic Ca 2+ signal is always paralleled by a much larger rise in Ca 2+ in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca 2+ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus
on four main topics: (i) the current knowledge of the functional properties of the Ca 2+ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca 2+ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca 2+ sources; (iii) the physiological consequences of Ca 2+ transport for both mitochondrial function and the modulation of the cytoplasmic Ca 2+ signal; and (iv) evidence that alterations of mitochondrial Ca 2+ signalling may occur in pathophysiological conditions.
Background: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human ...disease models with variable results. Objectives: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full‐length protein in patients could ameliorate hemorrhagic phenotypes. Results: A FVII–green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII–GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X–FVII and p364X–FVII) were transfected and found to secrete low amounts of FVII (∼1% of Wt–FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose‐dependent increase of secreted FVII molecules (p316X–FVII, 24 ± 12 ng mL−1, 3.6 ± 0.8% of Wt–FVII activity; p364X–FVII, 26 ± 10 ng mL−1, 3.7±0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. Conclusions: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation‐specific therapeutic approach in FVII deficiency.
In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through ...glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.
ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly ...luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100-200 microM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.
Over the past few years, extensive progress has been made in elucidating the role of calcium in the signaling of apoptosis. This has led to the characterization of calcium's role in the induction of ...apoptosis and in the regulation of effector proteases. In this review, we attempt to summarize the current knowledge regarding a segment of these studies, the interaction between the endoplasmic reticulum (ER) and mitochondria. This interface has been shown to play a crucial role in transferring agonist induced Ca
2+ signals to mitochondria during physiological processes. Recent evidence, however, extended the role of this Ca
2+ transfer to apoptotic pathways, showing that modulation of mitochondrial Ca
2+ uptake from the ER side has a prominent role in modulating cellular fate.
Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been ...extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca2+ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca2+ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of 'apoptotic' Ca2+ signals, with special emphasis on the mitochondria and cytosolic Ca2+-dependent enzymes.
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