A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis.

MAC inhibitors antagonize the pro-apoptotic effects of tBid and disassemble Bax / Bak oligomers.

Mitochondrial Apoptotic Channel inhibitors or iMACs are di-bromocarbazole derivatives with anti-apoptotic function which have been tested and validated in several mouse models of brain injury and neurodegeneration. Owing to the increased therapeutic potential of these compounds, we sought to expand our knowledge of their mechanism of action. We investigated the kinetics of MAC inhibition in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and semiquantitative western blot analyses.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content.

BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP).

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC-MS.

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases.

Regulation of Bax mitochondrial localization by Bcl-2 and Bcl-x(L): keep your friends close but your enemies closer.

Bax-induced mitochondrial outer membrane permeabilization (MOMP) is considered as one of the key control switches of apoptosis. MOMP requires Bax relocation to and insertion into the outer mitochondrial membrane to oligomerize and form pores allowing the release of apoptogenic factors such as cytochrome c. Even if these essential steps are now well-defined, it is necessary to better understand the molecular changes underlying the switch between inactive Bax and active (pore-forming) Bax.

Lysosome-mediated apoptosis is associated with cathepsin D-specific processing of bid at Phe24, Trp48, and Phe183.

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts.

Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.

Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity.

In vivo processing of CXCL12α/SDF-1α after intravenous and subcutaneous administration to mice.

CXCL12α has been shown to be selectively processed at the N- and C-termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions.

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions.

BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation.

BAX cooperates with truncated BID (tBID) and Ca(2+) in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization.

MAC inhibitors suppress mitochondrial apoptosis.

MAC (mitochondrial apoptosis-induced channel) forms in the mitochondrial outer membrane and unleashes cytochrome c to orchestrate the execution of the cell. MAC opening is the commitment step of intrinsic apoptosis. Hence closure of MAC may prevent apoptosis.

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID.

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX(oligo)) and by a combination of monomeric BAX (BAX(mono)) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t(c)BID). We also examined whether the effects induced by BAX(oligo) or by BAX(mono) activated with t(c)BID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t(c)BID plus BAX(mono) produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology.

Bad targets the permeability transition pore independent of Bax or Bak to switch between Ca2+-dependent cell survival and death.

Calcium oscillations exert physiological control on mitochondrial energy metabolism and can also lead to mitochondrial membrane permeabilization and cell death. The outcome of the mitochondrial calcium signaling is altered by stress factors such as ceramide or staurosporine. However, the mechanism of this proapoptotic switch remains unclear.

Endophilin B1/Bif-1 stimulates BAX activation independently from its capacity to produce large scale membrane morphological rearrangements.

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes.

Oligomeric BAX induces mitochondrial permeability transition and complete cytochrome c release without oxidative stress.

In the present study, we investigated the mechanism of cytochrome c release from isolated brain mitochondria induced by recombinant oligomeric BAX (BAX(oligo)). We found that BAX(oligo) caused a complete release of cytochrome c in a concentration- and time-dependent manner. The release was similar to those induced by alamethicin, which causes maximal mitochondrial swelling and eliminates barrier properties of the OMM.

Regulation of antiapoptotic MCL-1 function by gossypol: mechanistic insights from in vitro reconstituted systems.

Small-molecule drugs that induce apoptosis in tumor cells by activation of the BCL-2-regulated mitochondrial outer membrane permeabilization (MOMP) pathway hold promise for rational anticancer therapies. Accumulating evidence indicates that the natural product gossypol and its derivatives can kill tumor cells by targeting antiapoptotic BCL-2 family members in such a manner as to trigger MOMP. However, due to the inherent complexity of the cellular apoptotic network, the precise mechanisms by which interactions between gossypol and individual BCL-2 family members lead to MOMP remain poorly understood.

Mitochondrial cholesterol contributes to chemotherapy resistance in hepatocellular carcinoma.

Cholesterol metabolism is deregulated in carcinogenesis, and cancer cells exhibit enhanced mitochondrial cholesterol content whose role in cell death susceptibility and cancer therapy has not been investigated. Here, we describe that mitochondria from rat or human hepatocellular carcinoma (HC) cells (HCC) or primary tumors from patients with HC exhibit increased mitochondrial cholesterol levels. HCC sensitivity to chemotherapy acting via mitochondria is enhanced upon cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase or squalene synthase (SS), which catalyzes the first committed step in cholesterol biosynthesis.

Mechanism of mitochondrial glutathione-dependent hepatocellular susceptibility to TNF despite NF-kappaB activation.

Background & Aims: Nuclear factor kappaB (NF-kappaB) is the master regulator of tumor necrosis factor (TNF) susceptibility. Although mitochondrial glutathione (mGSH) depletion was shown to sensitize hepatocytes to TNF despite NF-kappaB activation, the mechanisms involved, particularly the role of Bax oligomerization and mitochondrial outer membrane (MOM) permeabilization, 2 critical steps in cell death, remained unexplored.

Methods: TNF signaling at the premitochondrial and mitochondrial levels was analyzed in primary mouse hepatocytes with or without mGSH depletion.

GlialCAM, an immunoglobulin-like cell adhesion molecule is expressed in glial cells of the central nervous system.

Using structure based genome mining targeting vascular endothelial and platelet derived growth factor immunoglobulin (Ig) like folds, we have identified a sequence corresponding to a single transmembrane protein with two Ig domains, which we cloned from a human brain cDNA library. The cDNA is identical to hepatocyte cell adhesion molecule (hepaCAM), which was originally described as a tumor suppressor gene in liver. Here, we show that the protein is predominantly expressed in the mouse and human nervous system.

Detection and identification of plasma proteins that bind GlialCAM using ProteinChip arrays, SELDI-TOF MS, and nano-LC MS/MS.

In order to fully understand biological processes it is essential to identify interactions in protein complexes. There are several techniques available to study this type of interactions, such as yeast two-hybrid screens, affinity chromatography, and coimmunoprecipitation. We propose a novel strategy to identify protein-protein interactions, comprised of first detecting the interactions using ProteinChips and SELDI-TOF MS, followed by the isolation of the interacting proteins through affinity beads and RP-HPLC and finally identifying the proteins using nano-LC MS/MS.

BIM and tBID are not mechanistically equivalent when assisting BAX to permeabilize bilayer membranes.

BIM and tBID are two BCL-2 homology 3 (BH3)-only proteins with a particularly strong capacity to trigger BAX-driven mitochondrial outer membrane permeabilization, a crucial event in mammalian apoptosis. However, the means whereby BIM and tBID fulfill this task is controversial. Here, we used a reconstituted liposomal system bearing physiological relevance to explore systematically how the BAX-permeabilizing function is influenced by interactions of BIM/BID-derived proteins and BH3 motifs with multidomain BCL-2 family members and with membrane lipids.

Purification of the extracellular domain of the membrane protein GlialCAM expressed in HEK and CHO cells and comparison of the glycosylation.

Adhesion molecules are essential for a wide range of biological and physiological functions, including cell-cell interactions, cell interactions with the extracellular matrix, cell migration, proliferation and survival. Defects in cell adhesion have been associated with pathological conditions such as neoplasia, and neurodegenerative diseases. We have identified a new adhesion molecule of the immunoglobulin family, GlialCAM.

A tale of two mitochondrial channels, MAC and PTP, in apoptosis.

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial.

A high-throughput mammalian protein expression, purification, aliquoting and storage pipeline to assemble a library of the human secretome.

In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, we established a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP was based on a transient expression system in HEK293-EBNA cells at 100 to 500 mL culture scale, combined with an automated affinity purification procedure targeting >75% purity.