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.     

Algorithm of OMA for large-scale orthology inference

Since the publication of our article (Roth, Gonnet, and Dessimoz: BMC Bioinformatics 2008 9: 518), we have noticed several errors, which we correct in the following.     

PIDDosome‐induced p53‐dependent ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi‐protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome‐induced p53‐activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro‐tumorigenic effect of PIDDosome‐mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence‐free survival in HCC patients.     

SIMPLE: implementation of recommendations from international evidence-based guidelines on caesarean sections in the Netherlands. Protocol for a controlled before and after study

Background

Caesarean section (CS) rates are rising worldwide. In the Netherlands, the most significant rise is observed in healthy women with a singleton in vertex position between 37 and 42 weeks gestation, whereas it is doubtful whether an improved outcome for the mother or her child was obtained. It can be hypothesized that evidence-based guidelines on CS are not implemented sufficiently. Therefore, the present study has the following objectives: to develop quality indicators on the decision to perform a CS based on key recommendations from national and international guidelines; to use the quality indicators in order to gain insight into actual adherence of Dutch gynaecologists to guideline recommendations on the performance of a CS; to explore barriers and facilitators that have a direct effect on guideline application regarding CS; and to develop, execute, and evaluate a strategy in order to reduce the CS incidence for a similar neonatal outcome (based on the information gathered in the second and third objectives).

Methods

An independent expert panel of Dutch gynaecologists and midwives will develop a set of quality indicators on the decision to perform a CS. These indicators will be used to measure current care in 20 hospitals with a population of 1,000 women who delivered by CS, and a random selection of 1,000 women who delivered vaginally in the same period. Furthermore, by interviewing healthcare professionals and patients, the barriers and facilitators that may influence the decision to perform a CS will be measured. Based on the results, a tailor-made implementation strategy will be developed and tested in a controlled before-and-after study in 12 hospitals (six intervention, six control hospitals) with regard to effectiveness, experiences, and costs.

Discussion

This study will offer insight into the current CS care and into the hindering and facilitating factors influencing obstetrical policy on CS. Furthermore, it will allow definition of patient categories or situations in which a tailor-made implementation strategy will most likely be meaningful and cost effective, without negatively affecting the outcome for mother and child.

Trial registration

http://www.clinicaltrials.gov: NCT01261676     

Aggravation of myocardial dysfunction by injurious mechanical ventilation in LPS-induced pneumonia in rats

Background

Mechanical ventilation (MV) may cause ventilator-induced lung injury (VILI) and may thereby contribute to fatal multiple organ failure. We tested the hypothesis that injurious MV of lipopolysaccharide (LPS) pre-injured lungs induces myocardial inflammation and further dysfunction ex vivo, through calcium (Ca²⁺)-dependent mechanism.

Materials and methods

N = 35 male anesthetized and paralyzed male Wistar rats were randomized to intratracheal instillation of 2 mg/kg LPS or nothing and subsequent MV with lung-protective settings (low tidal volume (V_t) of 6 mL/kg and 5 cmH₂O positive end-expiratory pressure (PEEP)) or injurious ventilation (high V_t of 19 mL/kg and 1 cmH₂O PEEP) for 4 hours. Myocardial function ex vivo was evaluated in a Langendorff setup and Ca²⁺ exposure. Key mediators were determined in lung and heart at the mRNA level.

Results

Instillation of LPS and high V_t MV impaired gas exchange and, particularly when combined, increased pulmonary wet/dry ratio; heat shock protein (HSP)70 mRNA expression also increased by the interaction between LPS and high V_t MV. For the heart, C-X-C motif ligand (CXCL)1 and Toll-like receptor (TLR)2 mRNA expression increased, and ventricular (LV) systolic pressure, LV developed pressure, LV +dP/dt_max and contractile responses to increasing Ca²⁺ exposure ex vivo decreased by LPS. High V_t ventilation aggravated the effects of LPS on myocardial inflammation and dysfunction but not on Ca²⁺ responses.

Conclusions

Injurious MV by high V_t aggravates the effects of intratracheal instillation of LPS on myocardial dysfunction, possibly through enhancing myocardial inflammation via pulmonary release of HSP70 stimulating cardiac TLR2, not involving Ca²⁺ handling and sensitivity.     

Structure of detergent-activated BAK dimers derived from the inert monomer

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Turmeric: old spice,new spice

The history of chemical investigations into the yellow components of turmeric can be traced from 1815. Although the major yellow component of turmeric, curcumin, often is represented as a 1,3-diketone in the solid state and in nonaqueous solution, it exists in the enol form. The struggle to identify the chemical structure of curcumin continued for nearly a century and was complicated by the difficult purification of curcumin and by the presence of two additional yellow components. Food remains the main use for turmeric (old spice); its use as a dye has diminished since the 19th century, but it may have pharmaceutical uses (new spice).     

Refined regional assignment of the human tissue factor pathway inhibitor (TFPI) gene to chromosome band 2q32 by non-isotopic in situ hybridization

Summary The potential usefulness of the tissue factor pathway inhibitor gene for construction of physical and genetic linkage maps of chromosome 2 is reported.     

To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions

The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.