Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes'' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.     

Bax dimerizes via a symmetric BH3:groove interface during apoptosis

During apoptotic cell death, Bax and Bak change conformation and homo-oligomerize to permeabilize mitochondria. We recently reported that Bak homodimerizes via an interaction between the BH3 domain and hydrophobic surface groove, that this BH3:groove interaction is symmetric, and that symmetric dimers can be linked via the α6-helices to form the high order oligomers thought responsible for pore formation. We now show that Bax also dimerizes via a BH3:groove interaction after apoptotic signaling in cells and in mitochondrial fractions. BH3:groove dimers of Bax were symmetric as dimers but not higher order oligomers could be linked by cysteine residues placed in both the BH3 and groove. The BH3:groove interaction was evident in the majority of mitochondrial Bax after apoptotic signaling, and correlated strongly with cytochrome c release, supporting its central role in Bax function. A second interface between the Bax α6-helices was implicated by cysteine linkage studies, and could link dimers to higher order oligomers. We also found that a population of Bax:Bak heterodimers generated during apoptosis formed via a BH3:groove interaction, further demonstrating that Bax and Bak oligomerize via similar mechanisms. These findings highlight the importance of BH3:groove interactions in apoptosis regulation by the Bcl-2 protein family.     

Prediction and course of symptoms and lung function around an exacerbation in chronic obstructive pulmonary disease

Background

Frequent exacerbations induce a high burden to Chronic Obstructive Pulmonary Disease (COPD). We investigated the course of exacerbations in the published COSMIC study that investigated the effects of 1-year withdrawal of fluticasone after a 3-month run-in treatment period with salmeterol/fluticasone in patients with COPD.

Methods

In 373 patients, we evaluated diary cards for symptoms, Peak Expiratory Flow (PEF), and salbutamol use and assessed their course during exacerbations.

Results

There were 492 exacerbations in 224 patients. The level of symptoms of cough, sputum, dyspnea and nocturnal awakening steadily increased from 2 weeks prior to exacerbation, with a sharp rise during the last week. Symptoms of cough, sputum, and dyspnea reverted to baseline values at different rates (after 4, 4, and 7 weeks respectively), whereas symptoms of nocturnal awakening were still increased after eight weeks. The course of symptoms was similar around a first and second exacerbation. Increases in symptoms and salbutamol use and decreases in PEF were associated with a higher risk to develop an exacerbation, but with moderate predictive values, the areas under the receiver operating curves ranging from 0.63 to 0.70.

Conclusions

Exacerbations of COPD are associated with increased symptoms that persist for weeks and the course is very similar between a first and second exacerbation. COPD exacerbations are preceded by increased symptoms and salbutamol use and lower PEF, yet predictive values are too low to warrant daily use in clinical practice.     

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis

Background

Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis.

Methods

Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (P_plat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage.

Results

MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo.

Conclusion

MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

Using ontologies to describe mouse phenotypes

The mouse is an important model of human genetic disease. Describing phenotypes of mutant mice in a standard, structured manner that will facilitate data mining is a major challenge for bioinformatics. Here we describe a novel, compositional approach to this problem which combines core ontologies from a variety of sources. This produces a framework with greater flexibility, power and economy than previous approaches. We discuss some of the issues this approach raises.     

Olfactory sensitivity in tsetse flies: a daily rhythm

The diurnal tsetse Glossina morsitans morsitans bites especially in early morning and late afternoon; around midday feeding is at a low. In laboratory apparatus that measures the amount of locomotion under constant conditions over the photophase, the flies display a similar patterning of activity levels. The profile of daily rhythms for G. morsitans reported in the literature includes a number of motor and sensory motor systems that fluctuate cophasically. Lacking is a study on the patterning of the senses' response levels. In this paper we present the first instance of a daily modulation in the sense of smell. We stimulated the antennae with concentration series of host-derived odours and measured the spiking rate of cells at different times during the photophase. The concentration-response curves suggest that the sensitivity of antennal olfactory cells flows in parallel with the other daily rhythms. This was also reflected in electroantennograms (EAGs). The electroantennography was extended to G. fuscipes fuscipes, whose level of spontaneous locomotor activity--instead of following a U- shaped pattern--rises gradually over the photophase. Again, the EAGs appeared to parallel the species' locomotor activity. What we believe happens is that the organism tones down the sensitivity of its odour receptors during periods of anticipated inactivity for reasons of economy.      

Bax targets mitochondria by distinct mechanisms before or during apoptotic cell death: a requirement for VDAC2 or Bak for efficient Bax apoptotic function

In non-apoptotic cells, Bak constitutively resides in the mitochondrial outer membrane. In contrast, Bax is in a dynamic equilibrium between the cytosol and mitochondria, and is commonly predominant in the cytosol. In response to an apoptotic stimulus, Bax and Bak change conformation, leading to Bax accumulation at mitochondria and Bak/Bax oligomerization to form a pore in the mitochondrial outer membrane that is responsible for cell death. Using blue native-PAGE to investigate how Bax oligomerizes in the mitochondrial outer membrane, we observed that, like Bak, a proportion of Bax that constitutively resides at mitochondria associates with voltage-dependent anion channel (VDAC)2 prior to an apoptotic stimulus. During apoptosis, Bax dissociates from VDAC2 and homo-oligomerizes to form high molecular weight oligomers. In cells that lack VDAC2, constitutive mitochondrial localization of Bax and Bak was impaired, suggesting that VDAC2 has a role in Bax and Bak import to, or stability at, the mitochondrial outer membrane. However, following an apoptotic stimulus, Bak and Bax retained the ability to accumulate at VDAC2-deficient mitochondria and to mediate cell death. Silencing of Bak in VDAC2-deficient cells indicated that Bax required either VDAC2 or Bak in order to translocate to and oligomerize at the mitochondrial outer membrane to efficiently mediate apoptosis. In contrast, efficient Bak homo-oligomerization at the mitochondrial outer membrane and its pro-apoptotic function required neither VDAC2 nor Bax. Even a C-terminal mutant of Bax (S184L) that localizes to mitochondria did not constitutively target mitochondria deficient in VDAC2, but was recruited to mitochondria following an apoptotic stimulus dependent on Bak or upon over-expression of Bcl-x_L. Together, our data suggest that Bax localizes to the mitochondrial outer membrane via alternate mechanisms, either constitutively via an interaction with VDAC2 or after activation via interaction with Bcl-2 family proteins.Bax and Bak are the key effectors of the intrinsic apoptotic pathway initiated in response to diverse stimuli including anoikis, DNA damage and growth factor withdrawal.¹ Both proteins are normally dormant in healthy cells, but upon reception of an apoptotic stimulus, they undergo conformation change that allows their self-association to form pores in the mitochondrial outer membrane (MOM).^{2, 3, 4, 5, 6, 7} The consequence of disruption of the MOM is twofold; it impairs the ability of mitochondria to generate ATP by oxidative phosphorylation and it allows the release of intermembrane proteins including cytochrome c that agonizes caspases that dismantle the cell.Bak and Bax share significant structural homology in their inactive states and have conserved mechanism of conformation change and oligomerization.^{3, 8, 9, 10} Further, genetic studies reveal that Bak and Bax perform at least partially overlapping function, with deficiency in both necessary to perturb apoptosis during embryonic development and in response to toxic insult.^{1, 11} However, whether Bak and Bax are regulated similarly is unclear. Whereas Bak is constitutively anchored in the MOM via its hydrophobic C-terminal transmembrane domain, Bax is predominantly cytosolic in the majority of non-apoptotic cells.¹² Recent evidence indicates that Bax is in a dynamic equilibrium between cytosol and mitochondria and is constantly trafficked away from the MOM in non-apoptotic cells.^{13, 14} In response to apoptotic stress this ‘retrotranslocation'' is disrupted causing Bax to accumulate at mitochondria; a hallmark of most apoptotic cells. The mechanism governing the dynamic distribution of Bax in healthy and apoptotic cells is unclear with interactions with pro-survival proteins debated.^{13, 14}Voltage-dependent anion channels (VDACs) are the major channels responsible for ion passage across the MOM. Studies have also implicated an additional role for the VDACs in the regulation of Bak or Bax apoptotic function or potentially even constituting a component of the Bak/Bax apoptotic pore.^{15, 16, 17, 18} However, these studies have provided contrasting findings relating to whether VDACs might positively or negatively regulate Bak/Bax apoptotic function.We used blue native-PAGE (BN-PAGE) to investigate how Bax oligomerizes in the MOM during apoptosis. We observed that VDAC2 is a determinant of the constitutive association of both Bax and Bak with the MOM. The defect in Bax mitochondrial localization can be bypassed by Bak-dependent recruitment during apoptosis. Thus, our data suggest that mitochondrial localization of Bax occurs via distinct mechanisms in healthy and apoptotic cells and that either VDAC2 or Bak is required for the efficient translocation of Bax and hence for the oligomerization at the MOM and Bax apoptotic function.