Mitochondria-dependent apoptosis in yeast

Mitochondrial involvement in yeast apoptosis is probably the most unifying feature in the field. Reports proposing a role for mitochondria in yeast apoptosis present evidence ranging from the simple observation of ROS accumulation in the cell to the identification of mitochondrial proteins mediating cell death. Although yeast is unarguably a simple model it reveals an elaborate regulation of the death process involving distinct proteins and most likely different pathways, depending on the insult, growth conditions and cell metabolism. This complexity may be due to the interplay between the death pathways and the major signalling routes in the cell, contributing to a whole integrated response. The elucidation of these pathways in yeast has been a valuable help in understanding the intricate mechanisms of cell death in higher eukaryotes, and of severe human diseases associated with mitochondria-dependent apoptosis. In addition, the absence of obvious orthologues of mammalian apoptotic regulators, namely of the Bcl-2 family, favours the use of yeast to assess the function of such proteins. In conclusion, yeast with its distinctive ability to survive without respiration-competent mitochondria is a powerful model to study the involvement of mitochondria and mitochondria interacting proteins in cell death.     

Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Voltage-gated K(+) (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (I(Kv)) revealed the presence of a mitochondria-mediated Mg(2+) and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.     

线粒体与细胞凋亡调控

细胞凋亡是一个受到一系列相关基因严格调控的细胞死亡过程。线粒体是细胞凋亡调控的活动中心。在凋亡因子的刺激下,线粒体释放出不同促凋亡因子如细胞色素C、Smac／Diablo等,激活细胞内凋亡蛋白酶Caspase。我们发现,活化后的Caspase可以反过来作用于线粒体,引发更大量线粒体细胞色素c的释放,构成细胞色素c释放的正反馈调节机制,从而导致电子传递链的中断、膜电势的丧失、胞内ROS的升高以及线粒体产生ATP功能的完全丧失。Bcl-2家族蛋白在细胞色素C释放和细胞凋亡调控中起关键作用。     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Mitochondria-dependent caspase-9 activation is necessary for antigen receptor-mediated effector caspase activation and apoptosis in WEHI 231 lymphoma cells

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.     

Proteases and cellular regulation in plants

Protein degradation is accomplished by a diverse collection of proteases. Recent studies have illustrated the importance of proteolysis in the control of many aspects of cellular regulation from photosynthesis to photomorphogenesis. In addition, new results point to a role for proteolysis in programmed cell death, circadian rhythm, and defense response in plants.     

Calcium binding proteins and cellular regulation

An important feature of cellular regulation is the precise control of intracellular calcium levels. This is accomplished both by dynamic organelle release and sequestration of calcium and by specific calcium active transport mechanisms located in the plasma membrane. The actual calcium signal for mediation of a cellular response is carried out by specific intracellular proteins, the most widely studied examples are calmodulin and troponin C. The recent discovery of phospholipid protein kinase and calcimedins suggests receptor mediation via several independent proteins. The physiological importance of a particular protein as a calcium messenger rests on several features: 1) calcium binding is of the order of 1–10 μm, 2) the protein is known to be localized at the site of proposed action, 3) if translocation occurs upon activation, the time required is consistent with the time course of the physiologic response and 4) substrates or effectors at the next level of action when isolated can be demonstrated to have similar activation kinetics as $\text{in situ}$.     

Short-term pH regulation in plants

Cellular pH regulation consists of two features: (i) Long-term pH homeostasis, which ensures that all H⁺ or OH⁻ produced in excess is ultimately removed from the cell and which requires metabolic energy; (ii) short-term reactions of the cell(s) to sudden shifts in intracellular pH, in order to prevent acute disturbances of metabolism. Recent progress in measuring and understanding of mainly short-term cellular regulation is summarized, including cellular responses to pH loads that arise from different sources such as external pH, weak acids/bases, protonophores, metabolic inhibitors, H⁺/cotransport, light and phytohormones. Whereas the plasma membrane H⁺ pump and metabolic adjustments may serve both long- and short-term pH control, physico-chemical buffering and the translocation of H⁺ from and to cellular compartments render only time-limited capacity for the neutralization of pH loads and seem exhausted within minutes. In spite of the widespread opinion that, because of tight regulation, intracellular pH does not vary with time, there is good evidence for long-lasting pH changes in plant cells, i.e. after hormonal stimulation, light/dark changes or carboxylation during crassulacean acid metabolism (CAM). This emphasizes that cytoplasmic pH, besides being well regulated, is essential not only for the regulation of membrane transport but also as a cellular messenger.     

pH regulation in anoxic plants

BACKGROUND: pH regulation is the result of a complex interaction of ion transport, H+ buffering, H+-consuming and H+-producing reactions. Cells under anoxia experience an energy crisis; an early response thereof (in most tissues) is a rapid cytoplasmic acidification of roughly half a pH unit. Depending on the degree of anoxia tolerance, this pH remains relatively stable for some time, but then drops further due to an energy shortage, which, in concert with a general breakdown of transmembrane gradients, finally leads to cell death unless the plant finds access to an energy source. SCOPE: In this review the much-debated origin of the initial pH change and its regulation under anoxia is discussed, as well as the problem of how tissues deal with the energy crisis and to what extent pH regulation and membrane transport from and into the vacuole and the apoplast is a part thereof. CONCLUSIONS: It is postulated that, because a foremost goal of cells under anoxia must be energy production (having an anaerobic machinery that produces insufficient amounts of ATP), a new pH is set to ensure a proper functioning of the involved enzymes. Thus, the anoxic pH is not experienced as an error signal and is therefore not reversed to the aerobic level. Although acclimated and anoxia-tolerant tissues may display higher cytoplasmic pH than non-acclimated or anoxia-intolerant tissues, evidence for an impeded pH-regulation is missing even in the anoxia-intolerant tissues. For sufficient energy production, residual H+ pumping is vital to cope with anoxia by importing energy-rich compounds; however it is not vital for pH-regulation. Whereas the initial acidification is not due to energy shortage, subsequent uncontrolled acidosis occurring in concert with a general gradient breakdown damages the cell but may not be the primary event.     

Proteolytic regulation of apoptosis

Much of the proteolysis that occurs during apoptosis is directed by caspases, a family of related cysteinyl proteases. A relatively small number of cellular proteins are targeted by caspases, yet their function is dramatically affected and apoptosis is triggered. Other proteases, such as granzymes and calpain, are also involved in the apoptotic signaling process, but in a much more cell type- and/or stimulus type-specific manner. At least three distinct caspase-signaling pathways exist; one activated through ligand-dependent death receptor oligomerization, the second through mitochondrial disruption, and the third through stress-mediated events involving the endoplasmic reticulum. These pathways also appear to interact to amplify weak apoptotic signals and shorten cellular execution time. Finally, defects in caspases contribute to autoimmune disease, cancer and certain neurological disorders.     

Redox regulation of cellular functions

Maintenance of normal intracellular redox status plays an important role in such processes as DNA synthesis, gene expression, enzymatic activity, and others. In addition, it is clear that changes in the redox status of intracellular content and individual molecules, resulting from stress or intrinsic cellular activity, are involved in the regulation of different processes in cells. Small changes in intracellular levels of reactive oxygen species participate in intracellular signaling. Thiol-containing molecules, such as glutathione, thioredoxins, glutaredoxins, and peroxiredoxins, also play an important role in maintaining redox homeostasis and redox regulation. This review attempts to summarize the current knowledge about redox regulation in different cell types.