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During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore.

Bcl-2 family of proteins regulate apoptosis at the level of the mitochondrial outer membrane (MOM) through both protein-protein and protein-membrane interactions. While the role of the membrane as the “locus of action” has been recognized, the detailed molecular mechanisms and the consequences of the interactions of Bcl-2 family members with the membrane are yet to be fully understood. The findings presented here focus on the dynamic interactions of Bcl-2 proteins, most notably tBid with the MOM, and their functional significance on mitochondrial outer membrane permeabilization. We show that the activation of tBid is a multi-step process that is regulated by MOM lipids and proteins. The rate-limiting step in the activation of tBid is an elaborate conformational change that is facilitated by Mtch2, and is required for the activation and recruitment of Bax to the MOM. Furthermore, we demonstrate that binding of both tBid and Bax to the membrane is reversible and is governed by dynamic equilibria that potentially contribute to the propagation of the permeabilization signal within the cell for the regulation of apoptosis. We report that the transfer of tBid between membranes is accelerated by Bax and restricted by Bcl-XL, whereas the transfer of Bax between membranes is slower than and not influenced by tBid. Finally, by studying the effect of varying lipid composition on Bax-mediated permeabilization, we establish that electrostatic interactions mediate the binding of both tBid and Bim to the membrane. We demonstrate that while Bim does not exhibit any preference for a specific anionic lipid, tBid requires cardiolipin in order to undergo its conformational change at the membrane in the absence of Mtch2. Taken together, our work contributes to the growing understanding of the dynamic interactions and changes in conformation of Bcl-2 proteins at the MOM., Doctor of Science (PhD)

In type 1 diabetes (T1D) a combination of genetic predisposition and environmental factors triggers islet inflammation (insulitis) leading to a selective and gradual destruction of the pancreatic beta cells. Beta cells mainly die through apoptosis, triggered at least in part by pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ. Recent findings suggest that the mitochondrial pathway of cell death is involved in this death cascade. Array analysis indicated that TNF-α+IFN-γ induces transcription factors such as NF-ĸB, STAT1, and AP-1 in beta cells. We presently aimed to examine the pathway(s) of apoptosis triggered by TNF-α+IFN-γ in beta cells. TNF-α+IFN-γ induces beta cell apoptosis through the intrinsic pathway of cell death. This involved activation of the BH3 only proteins DP5, PUMA and Bim. Knockdown (KD) of either DP5 or PUMA or both led to a partial protection of INS-1E cells (12-20%), while silencing Bim led to about 60% protection against cytokine-induced apoptosis. Bim is transcriptionally induced by activated STAT1. TNF-α+IFN-γ also induces downregulation of Bcl-XL, an anti-apoptotic Bcl-2 gene which inhibits Bim. Knocking down Bcl-XL alone led to increase in apoptosis, but this was prevented by the parallel KD of Bim.The ultimate goal of our research is to protect beta cells from the autoimmune assault. Previous data revealed that JunB inhibits ER stress and apoptosis in beta cells treated with IL-β+IFN-γ. Here, TNF-α+IFN-γ up-regulated the expression of JunB which was downstream of activated NF-ĸB. JunB KD exacerbated TNF-α+IFN-γ induced beta cell death in primary rat beta cells and INS-1E cells. The gene networks affected by JunB were studied by microarray analysis. JunB regulates 20-25% of the cytokine-modified beta cell genes, including the transcription factor ATF3 and Bcl-XL. ATF3 expression was increased in cytokine-treated human islets and in vitro silencing of JunB led to >60% reduction in ATF3 overexpression. We confirmed direct JunB, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

Apoptosis plays a key role in the regulation and development of healthy multicellular organisms throughout their lifetimes. The mitochondria play a key role in this cellular process, as it contains proapoptotic factors, which once released into the cytosol of the cell, results in the death of the cell. The Bcl-2 family of proteins play a key role in apoptosis, acting as the gateway between life and death of the cell. Proteins such as tBid and Bax act to permeabilize the mitochondrial outer membrane (MOM), releasing the proapoptotic factors into the cell’s cytosol. The interactions between these proteins and the mitochondrial outer membrane have yet to be fully understood. The lipid composition and cholesterol content of the membrane effectively inhibit or promote pore formation by Bax. Specifically, the addition of cholesterol into the membrane inhibits pore formation. This thesis attempts to further understand the effects cholesterol has on the structure of the MOM, and link those changes to the inhibited activity of Bax pore formation. MOM-like lipid bilayers were studied under varying temperatures and with the addition of cholesterol using x-ray reflectivity. Increasing temperatures from 10°C to 30°C resulted in bilayer thinning, as did decreasing cholesterol concentrations below 30%. From 10°C to 20°C, bilayer thickness showed a bell shaped profile, and changed to a linear decrease above about 20°C. This may assist Bax in pore formation, as it has also been observed to cause bilayer thinning. Increasing Cholesterol concentrations up to 30% resulted in little variation in bilayer thickness though hindrance of Bax pore formation is observed at content levels as low as 8%. Thus it is unlikely that bilayer thickening by cholesterol causes the inhibition of Bax pore formation. In addition, cholesterol was observed to increase the electron density of the core of the bilayer at concentration levels above 25%., Master of Science (MSc)

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic beta-cells. Pro-inflammatory cytokines are early mediators of beta-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in beta-cells, but the role for CHOP overexpression in cytokine-induced beta-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-kappa B (NF-kappa B) is a crucial transcription factor regulating beta-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-kappa B activity and expression of key NF-kappa B target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased I kappa B degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting beta-cell death: (1) CHOP directly contributes to cytokine-induced beta-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-kappa B activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis. Cell Death and Differentiation (2012) 19, 1836-1846; doi:10.1038/cdd.2012.67; published online 1 June 2012

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biomedical Genetics, 2010., The imbalance between generation of reactive oxygen species (ROS) and antioxidant can lead to oxidative stress, which has been linked to many diseases. Exposure to hyperoxia has been used as a model for studying oxidative stress, and under this condition, damaged DNA activates cell cycle checkpoints as well as cell death pathways. The concomitant activation of survival-promoting checkpoints and apoptosis seems counter intuitive, therefore it is of great interest to understand how these opposing pathways ultimately dictate cell fate. The cyclin-dependent kinase inhibitor p21 is induced during hyperoxia and causes G1 arrest and promotes cell survival. At the same time, hyperoxia promotes Bak and Bax-dependent cell death. Hence, current study investigates the hypothesis that p21 promotes cell survival through maintaining the balance between Bcl-2 anti- and pro-apoptotic proteins. Here, by using p53-dificient human lung H1299 cells conditionally over-expressing p21, anti-apoptotic Mcl-1 or Bcl-XL, we found that hyperoxia selectively down-regulated anti-apoptotic Mcl-1 and Bcl-XL, while stimulating expression of pro-apoptotic Bak. The loss of Mcl-1 and Bcl-XL were due to increased protein degradation, while up-regulation of Bak was pre-translational. Over-expression of p21 selectively delayed the loss of Mcl-1 and Bcl-XL without blocking the up-regulation of Bak. Both Mcl-1 and Bcl-XL protected against hyperoxia, however, Bcl-XL was found to be the predominant mediator of p21 cytoprotection. By using H1299 cells conditional over-expressing another cell cycle inhibitor p27, the amino, or the carboxyl terminus of p21, or cytoplasm-targeted p21, we found that p21 delays the loss of Mcl-1 and Bcl-XL through G1 arrest-independent mechanism. Altogether, our data suggest hyperoxia induces cell death via altering the balance between Bcl-2 anti- and pro-apoptotic proteins, and p21 protects by selectively delaying the loss of Mcl-1 and more predominantly Bcl-XL. Our study revealed

An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with 15N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled 15N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single 15N methionine within its hydrophobic helix 9 region exhibited 15N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.

Kainic acid induces excitotoxicity and nerve cell degeneration in vulnerable regions of rat brain, most markedly in hippocampus and amygdala. Part of the cell death following kainic acid is apoptotic as shown by caspase 3 activation and chromatin condensation. Here we have studied the regulation of pro- and anti-apoptotic proteins belonging to the Bcl-2 family in rat hippocampus and amygdala by kainic acid in relationship to ensuing neuronal death. The pro-apoptotic protein Bax was up-regulated in hippocampus 6 h after kainic acid administration. The increase in Bax was followed by the appearance of TdT-mediated dUTP nick end label ling-positive cells which were prominent at 24 h. Immunohistochemistry for active Bax revealed a punctated labelling of neurons in the CA3 and hilar regions of hippocampus as well as in amygdala. Double staining for NeuN, a marker for nerve cells, and TdT-mediated dUTP nick end labelling showed that mainly neurons undergo degeneration after kainic acid treatment. In contrast to Bax, the pro-apoptotic BH3-only Bcl-2 proteins Bim and Harakiri/DP5 were down-regulated by kainic acid. This was also observed for the anti-apoptotic proteins Bcl-x and Bcl-w. Immunoreactive Bcl-2 was up-regulated in hippocampus after kainic acid together with an increase in the phosphorylation of serine-87 in Bcl-2, suggesting a post-transcriptional modification of the protein. This was confirmed using immunoprecipitation of total Bcl-2 from hippocampus and amygdala which revealed an increase in serine-87 phospho-Bcl-2 after kainic acid. Inhibition of the c-jun N-terminal protein kinase pathway reduced both serine-87 phosphorylation and cell death after kainic acid. This indicates an important role of Bcl-2 phosphorylation in controlling neuronal death after kainic acid. In contrast to the situation in trophic factor-deprived neurons, no up-regulation of Bim or Harakiri/DP5 proteins occurred after kainic acid, suggesting alternative pathways for regulation of cell