Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat

The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as “ER stress”. Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Regulation of mitochondrial membrane permeabilization by BCL-2 family proteins and caspases

Mitochondria play an important role in the integration and transmission of cell death signals, activating caspases and other cell death execution events by releasing apoptogenic proteins from the intermembrane space. The BCL-2 family of proteins localize (or can be targeted) to mitochondria and regulate the permeability of the mitochondrial outer membrane to these apoptotic factors. Recent evidence suggests that multiple mechanisms may regulate the release of mitochondrial factors, some of which depend on the action of caspases.     

The control of endoplasmic reticulum-initiated apoptosis by the BCL-2 family of proteins

Irreversible perturbations in the homeostasis of the endoplasmic reticulum (ER) are thought to lead to apoptosis and cell loss in a number of important human diseases, including Alzheimer disease, Parkinson disease, and type 2 diabetes. However, the exact mechanisms that lead from ER stress to cell death remain incompletely understood. Recent work has shown that the BCL-2 family of proteins plays a central role in regulating this form of cell death, both locally at the ER and from a distance at the mitochondrial membrane.     

Regulation of apoptosis by Bcl-2 family proteins

For multicellular organisms, the rigorous control of programmed cell death is as important as that of cell proliferation. The mechanisms involved in the regulation of cell death are not yet understood, but a key component is the family of caspases which are activated in a cascade and are responsible for the apoptotic-specific changes and disassembly of the cell. Although the caspases represent a central point in apoptosis, their activation is regulated by a variety of other factors. Among these, Bcl-2 family plays a pivotal role in caspases activation, by this deciding whether a cell will live or die. Bcl-2 family members are known to focus much of their response to the mitochondria level, upstream the irreversible cellular damage, but their functions are not yet well defined. This review summarizes the recent data regarding the Bcl-2 proteins and the ways they regulate the apoptosis.     

Significance of the caspase family in Helicobacter pylori induced gastric epithelial apoptosis

Background and Aims. H. pylori infection results in an increased epithelial apoptosis in gastritis and duodenal ulcer patients. We investigated the role and type of activation of caspases in H. pylori‐induced apoptosis in gastric epithelial cells. Methods. Differentiated human gastric cancer cells (AGS) and human gastric mucous cell primary cultures were incubated with H. pylori for 0.5–24 hours in RPMI 1640 medium, and the effects on cell viability, epithelial apoptosis, and activity of caspases were monitored. Apoptosis was analyzed by detection of DNA‐fragments by Hoechst stain®, DNA‐laddering, and Histone‐ELISA. Activities of caspases were determined in fluorogenic assays and by Western blotting. Cleavage of BID and release of cytochrome c were analyzed by Western blot. Significance of caspase activation was investigated by preincubation of gastric epithelial cells with cell permeable specific caspase inhibitors. Results. Incubation of gastric epithelial cells with H. pylori caused a time and concentration dependent induction of DNA fragmentation (3‐fold increase), cleavage of BID, release of cytochrome c and a concomittant sequential activation of caspase‐9 (4‐fold), caspase‐8 (2‐fold), caspase‐6 (2‐fold), and caspase‐3 (6‐fold). No effects on caspase‐1 and ‐7 were observed. Activation of caspases preceded the induction of DNA fragmentation. Apoptosis could be inhibited by prior incubation with the inhibitors of caspase‐3, ‐8, and ‐9, but not with that of caspase‐1. Conclusions. Activation of certain caspases and activation of the mitochondrial apoptotic pathway are essential for H. pylori induced apoptosis in gastric epithelial cells.     

Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis

During apoptosis, pro‐apoptotic BAX and BAK are activated, causing mitochondrial outer membrane permeabilisation (MOMP), caspase activation and cell death. However, even in the absence of caspase activity, cells usually die following MOMP. Such caspase‐independent cell death is accompanied by inflammation that requires mitochondrial DNA (mtDNA) activation of cGAS‐STING signalling. Because the mitochondrial inner membrane is thought to remain intact during apoptosis, we sought to address how matrix mtDNA could activate the cytosolic cGAS‐STING signalling pathway. Using super‐resolution imaging, we show that mtDNA is efficiently released from mitochondria following MOMP. In a temporal manner, we find that following MOMP, BAX/BAK‐mediated mitochondrial outer membrane pores gradually widen. This allows extrusion of the mitochondrial inner membrane into the cytosol whereupon it permeablises allowing mtDNA release. Our data demonstrate that mitochondrial inner membrane permeabilisation (MIMP) can occur during cell death following BAX/BAK‐dependent MOMP. Importantly, by enabling the cytosolic release of mtDNA, inner membrane permeabilisation underpins the immunogenic effects of caspase‐independent cell death.     

Progress in targeting the BCL-2 family of proteins

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The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.     

ING1 induces apoptosis through direct effects at the mitochondria

The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1, interacts with the proliferating cell nuclear antigen (PCNA) in a UV-inducible manner. ING1 also interacts with members of the 14-3-3 family leading to its cytoplasmic relocalization. Overexpression of ING1 enhances expression of the Bax gene and was reported to alter mitochondrial membrane potential in a p53-dependent manner. Here we show that ING1 translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein. Bioinformatic analysis of the yeast interactome indicates that yeast ING proteins interact with 64 mitochondrial proteins. Also, sequence analysis of ING1 reveals the presence of a BH3-like domain. These data suggest a model in which stress-induced cytoplasmic relocalization of ING1 by 14-3-3 induces ING1-BAX interaction to promote mitochondrial membrane permeability and represent a paradigm shift in our understanding of ING1 function in the cytoplasm and its contribution to apoptosis.     

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok(-/-) mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok(-/-) mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok(-/-) mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eμ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.     

NMR of membrane proteins in micelles and bilayers: the FXYD family proteins

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.     

Use of carbonate extraction in analyzing moderately hydrophobic transmembrane proteins in the mitochondrial inner membrane

Resistance to sodium carbonate extraction is regarded as a canonical way to distinguish integral membrane proteins (MPs) from other membrane‐associated proteins. However, it has been observed that carbonate extraction releases some mitochondrial integral MPs. Here, by analyzing both artificially designed and native mitochondrial inner MPs containing transmembrane domains (TMDs) of different hydrophobicities, we show that carbonate treatment can release moderately hydrophobic TMDs from the mitochondrial inner membrane. These results suggest that resistance and sensitivity to carbonate extraction may be interpreted with caution when analyzing the nature of mitochondrial inner MPs.     

From mosaic to patchwork: Matching lipids and proteins in membrane organization

Abstract

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints – focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization – are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or ‘patchwork membrane'.     

Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX

Interactions between the BCL-2 family proteins determine the cell's fate to live or die. How they interact with each other to regulate apoptosis remains as an unsettled central issue. So far, the antiapoptotic BCL-2 proteins are thought to interact with BAX weakly, but the physiological significance of this interaction has been vague. Herein, we show that recombinant BCL-2 and BCL-w interact potently with a BCL-2 homology (BH) 3 domain-containing peptide derived from BAX, exhibiting the dissociation constants of 15 and 23 nM, respectively. To clarify the basis for this strong interaction, we determined the three-dimensional structure of a complex of BCL-2 with a BAX peptide spanning its BH3 domain. It revealed that their interactions extended beyond the canonical BH3 domain and involved three nonconserved charged residues of BAX. A novel BAX variant, containing the alanine substitution of these three residues, had greatly impaired affinity for BCL-2 and BCL-w, but was otherwise indistinguishable from wild-type BAX. Critically, the apoptotic activity of the BAX variant could not be restrained by BCL-2 and BCL-w, pointing that the observed tight interactions are critical for regulating BAX activation. We also comprehensively quantified the binding affinities between the three BCL-2 subfamily proteins. Collectively, the data show that due to the high affinity of BAX for BCL-2, BCL-w and A1, and of BAK for BCL-X(L), MCL-1 and A1, only a subset of BH3-only proteins, commonly including BIM, BID and PUMA, could be expected to free BAX or BAK from the antiapoptotic BCL-2 proteins to elicit apoptosis.     

Ethyl-nitrosourea transformed astrocytes exhibit mitochondrial membrane hyperpolarization and constrained apoptosis

Recent studies have shown the mitochondria and mitochondrial DNA are altered in gliomas, studied either as primary tissues or in culture. Few studies have been performed which evaluate the mitochondria during the development of glial malignancy. We used an ethyl-nitrosourea (ENU) in vitro model to assess the changes in mitochondrial parameters with progression to astrocyte transformation. When compared to the untreated control cells mitochondrial mass of the ENU treated cells significantly decreased ontologically early with concurrent increase in mitochondrial membrane potential, resulting in hyperpolarization of the mitochondrial membrane. At successive divisions, the degree of spontaneous apoptosis during astrocyte transformation was significantly diminished in the ENU treated cells. With 24 h pre- and co-treatment of ENU cells with citrate, an allosteric inhibitor of phosphofructokinase, the astrocytes still became immortal, but did not manifest any of the mitochondrial changes nor acquire the transformed properties of the ENU treated cells without the inhibition. Indeed, the degree of apoptosis noted in these dually treated cells was increased, associated with a loss of anchorage independence and low density growth. Transformed subclones exposed to citrate after the development of malignant properties also exhibited increased apoptosis, and did not form colonies in low density plating conditions. These results suggest that the development of transformed properties in an ENU model is associated with marked hyperpolarization of mitochondrial membrane potential and diminished spontaneous apoptosis. Exposure to citrate attenuated these mitochondrial changes and in vitro growth properties, with increases in apoptosis. The development of transformed astrocytes involve constraints on apoptosis related to alterations in mitochondrial membrane potential and mass.     

Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis

Endoplasmic reticulum (ER) stress-induced apoptosis may arise from multiple environmental and pharmacological causes, but the precise mechanism(s) involved are not completely known. Members of Bcl-2 protein family are important regulators of apoptosis. In this study, we report that in a process dependent on the proapoptotic Bcl-2 members Bax and Bak, exogenously expressed fluorescent protein localized to the ER lumen is released into the cytosol in cells undergoing ER stress. Upon ER stress induction, endogenous ER luminal proteins are also released into the cytosol in a similar manner accompanied by translocation and anchorage of Bax to the ER membrane. In addition, Bax and truncated-Bid (tBid) mediate a global increase in ER membrane permeability to ER luminal proteins in vitro. Importantly, antiapoptotic Bcl-X_L antagonizes the effects of proapoptotic Bcl-2 proteins on ER membrane permeability. Consistent with Bax translocation to the ER membrane in whole apoptotic cells, there is also increased tight association of Bax with the ER membrane correlated with the increase in ER membrane permeability in vitro. Overall, these data suggest that the regulation of ER membrane permeability by Bcl-2 proteins could be an important molecular mechanism of ER stress-induced apoptosis.     

Bcl-2 family of proteins: life-or-death switch in mitochondria

An increase in the permeability of outer mitochondrial membrane is central to apoptotic cell death, and results in the release of several apoptogenic factors such as cytochrome c into the cytoplasm to activate downstream destructive programs. The voltage-dependent anion channel (VDAC or mitochondrial porin) plays an essential role in disrupting the mitochondrial membrane barrier and is regulated directly by members of the Bcl-2 family proteins. Anti-apoptotic Bcl-2 family members interact with and close the VDAC, whereas some, but not all, proapoptotic members interact with VDAC to open protein-conducting pore through which apoptogenic factors pass. Although the VDAC is involved directly in breaking the mitochondrial membrane barrier and is a known component of the permeability transition pore complex, VDAC-dependent increase in outer membrane permeability can be independent of the permeability transition event such as mitochondrial swelling followed by rupture of the outer mitochondrial membrane. VDAC interacts not only with Bcl-2 family members but also with proteins such as gelsolin, an actin regulatory protein, and appears to be a convergence point for a variety of cell survival and cell death signals.     

Structure prediction of membrane proteins

There is a large gap between the number of membrane protein (MP) sequences and that of their decoded 3D structures, especially high-resolution structures, due to difficulties in crystal preparation of MPs. However, detailed knowledge of the 3D structure is required for the fundamental understanding of the function of an MP and the interactions between the protein and its inhibitors or activators. In this paper, some computational approaches that have been used to predict MP structures are discussed and compared.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.