A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.

https://science.sciencemag.org/content/sci/281/5381/1309.full.pdf

Mitochondria and apoptosis

Bcl-2 and related cytoplasmic proteins are key regulators of apoptosis, the cell suicide program critical for development, tissue homeostasis, and protection against pathogens. Those most similar to Bcl-2 promote cell survival by inhibiting adapters needed for activation of the proteases (caspases) that dismantle the cell. More distant relatives instead promote apoptosis, apparently through mechanisms that include displacing the adapters from the pro-survival proteins. Thus, for many but not all apoptotic signals, the balance between these competing activities determines cell fate. Bcl-2 family members are essential for maintenance of major organ systems, and mutations affecting them are implicated in cancer.

https://science.sciencemag.org/content/sci/281/5381/1322.full.pdf

The Bcl-2 Protein Family: Arbiters of Cell Survival

BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have been studied intensively for the past decade owing to their importance in the regulation of apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the point of no return for clonogenic cell survival and thereby affect tumorigenesis and host-pathogen interactions and regulate animal development. Recent structural, phylogenetic and biological analyses, however, suggest the need for some reconsideration of the accepted organizational principles of the family and how the family members interact with one another during programmed cell death. Although these insights into interactions among BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the mechanisms they use to activate caspases remains elusive.

http://dipbsf.uninsubria.it/monti/BFPN%202009/bcl2-1008.pdf

The BCL-2 protein family: opposing activities that mediate cell death

Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors

A variety of physiological death signals, as well as pathological cellular insults, trigger the genetically programmed pathway of apoptosis (Vaux and Korsmeyer 1999). Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction, of which mitochondrial dysfunction is the best characterized (for reviews, see Green and Reed 1998; Thornberry and Lazebnik 1998). As the BCL-2 family members reside upstream of irreversible cellular damage and focus much of their efforts at the level of mitochondria, they play a pivotal role in deciding whether a cell will live or die (Fig. 1). The founder of this family, the BCL-2 proto-oncogene, was discovered at the chromosomal breakpoint of t(14;18) bearing human B-cell lymphomas. The BCL-2 family of proteins has expanded significantly and includes both proas well as anti-apoptotic molecules. Indeed, the ratio between these two subsets helps determine, in part, the susceptibility of cells to a death signal (Oltvai et al. 1993) (Fig. 1). An additional characteristic of the members of this family is their frequent ability to form homoas well as heterodimers, suggesting neutralizing competition between these proteins. A further characteristic of probable functional significance is their ability to become integral membrane proteins. BCL-2 family members possess up to four conserved BCL-2 homology (BH) domains designated BH1, BH2, BH3, and BH4, which correspond to a-helical segments (Adams and Cory 1998; Kelekar and Thompson 1998; Reed 1998) (Fig. 2). Many of the anti-apoptotic members display sequence conservation in all four domains. The pro-apoptotic molecules frequently display less sequence conservation of the first a-helical segment, BH4. Deletion and mutagenesis studies argue that the amphipathic a-helical BH3 domain serves as a critical death domain in the pro-apoptotic members. This concept is supported by an emerging subset of “BH3-domain-only” members who display sequence homology only within the BH3 domain and to date are all pro-apoptotic. However, the three-dimensional structure of at least one BH3-domainonly molecule, BID, demonstrates a very similar overall a-helical content to the anti-apoptotic molecule BCL-XL (Chou et al. 1999; McDonnell et al. 1999). Many BCL-2 family members also contain a carboxy-terminal hydrophobic domain, which in the case of BCL-2 is essential for its targeting to membranes such as the mitochondrial outer membrane (Nguyen et al. 1993).

/pdf/bcl-2-family-members-and-the-mitochondria-in-apoptosis-45he6ri17y.pdf

BCL-2 family members and the mitochondria in apoptosis

Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes. Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive. The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins. A key feature of these pore-forming domains is the ability to form ion channels in biological membranes. Here we demonstrate that Bcl-xL shares this functional feature. Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel. This channel is pH-sensitive and becomes cation-selective at physiological pH. The ion-conducting channel(s) formed by Bcl-xL display multiple conductance states that have identical ion selectivity. Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed.

Bcl-x(L) forms an ion channel in synthetic lipid membranes.

We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50–200 μM), short duration (<100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2894%2900931-7

Cyclic adp-ribose does not affect cardiac or skeletal muscle ryanodine receptors

Bay K 8644, an L-type Ca2+ channel agonist, was shown previously to increase resting sarcoplasmic reticulum (SR) Ca2+ loss and convert post-rest potentiation to decay in dog and ferret ventricular muscle. Here, the effects of Bay K 8644 on local SR Ca2+ release events (Ca2+ sparks) were measured in isolated ferret ventricular myocytes, using laser scanning confocal microscopy and the fluorescent Ca2+ indicator fluo-3. The spark frequency under control conditions was fairly constant during 20 s of rest after interruption of electrical stimulation. Bay K 8644 (100 nmol/L) increased the spark frequency by 466+/-90% of control at constant SR Ca2+ load but did not change the spatial and temporal characteristics of individual sparks. The increase in spark frequency was maintained throughout the period of rest. The increase in Ca2+ spark frequency induced by Bay K 8644 was not affected by superfusion with Ca2+-free solution (with 10 mmol/L EGTA) but was suppressed by the addition of 10 micromol/L nifedipine (which by itself did not alter resting Ca2+ spark frequency). This suggests that the effect of Bay K 8644 on Ca2+ sparks is mediated by the sarcolemmal dihydropyridine receptor but is also independent of Ca2+ influx. Low concentrations of caffeine (0.5 mmol/L) increased both the average frequency and duration of sparks. Ryanodine (50 nmol/L) increased the spark frequency and also induced long-lasting Ca2+ signals. This may indicate long-lasting openings of SR Ca2+ release channels and a lack of local SR Ca2+ depletion. In lipid bilayers, Bay K 8644 had no effect on either single-channel current amplitude or open probability of the cardiac ryanodine receptor. It is concluded that Bay K 8644 activates SR Ca2+ release at rest, independent of Ca2+ influx and perhaps through a functional linkage between the sarcolemmal dihydropyridine receptor and the SR ryanodine receptor. In contrast, caffeine and ryanodine modulate Ca2+ sparks by a direct action on the SR Ca2+ release channels.

Patricio Velez

Papers

Bcl-x(L) forms an ion channel in synthetic lipid membranes.

Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2

Cyclic adp-ribose does not affect cardiac or skeletal muscle ryanodine receptors

Bay K 8644 Increases Resting Ca2+ Spark Frequency in Ferret Ventricular Myocytes Independent of Ca Influx Contrast With Caffeine and Ryanodine Effects