Human Bcl-2 Protein Research Articles

Human anti-apoptotic Bcl-2 and Bcl-xL proteins protect yeast cells from aging induced oxidative stress

Aging is a degenerative, biological, and time-dependent process that affects all organisms. Yeast aging is a physiological phenomenon characterized by the progressive transformation of yeast cells, resulting in modifications to their viability and vitality. Aging in yeast cells is comparable to that in higher organisms in some respects; however, due to their straightforward and well-characterized genetic makeup, these cells present unique advantages when it comes to researching the aging process. Here, we assessed the impact of human anti-apoptotic Bcl-2 and Bcl-xL proteins on aging using a yeast model. The findings clearly showed that these proteins exhibited remarkable anti-aging properties in yeast cells. Our data indicate that the presence of both proteins enhanced the reproductive survival of aging cells, likely by effecting the components functioning as both pro- and anti-oxidants, depending on the stage of yeast cell lifespan. Both proteins partially protected yeast cells from aging-related morphological deformations and cellular damage during the aging period. In particular, Bcl-xL expressing yeast cells reached the maximum activity levels for almost all of the major antioxidant enzymes and the total antioxidant status on the 8th day of lifespan and could provide effective protection at the latest stage of the investigated aging period. The chemometric data analysis of IR spectra confirmed the findings of the morphological and biochemical analyses. In this regard, specifically, understanding the mechanism of action on the cellular redox state of Bcl-xL in yeast may facilitate comprehension of its indirect antioxidant function in higher eukaryotes.

Monkeypox virus C7L is a Bcl-2-fold protein that binds human pro-apoptotic Bcl-2 proteins

Monkeypox virus C7L is a Bcl-2-fold protein that binds human pro-apoptotic Bcl-2 proteins

Anticancer Activities of Re(I) Tricarbonyl and Its Imidazole-Based Ligands: Insight from a Theoretical Approach.

Rhenium complexes have been observed experimentally to exhibit good inhibitory activity against malignant cells. Hence, our motivation is to explore this activity from a theoretical perspective. In the present study, density functional theory (DFT) and in silico molecular docking approaches were utilized to unravel the unique properties of metal-based rhenium tricarbonyl complexes as effective anticancer drugs. All DFT calculations and geometric optimizations were conducted using the well-established hybrid functional B3LYP-GD(BJ)/Gen/6-311++G(d,p)/LanL2DZ computational method. The FT-IR spectroscopic characterization of the complexes: fac-[Re(Pico)(CO)3(Pz)] (R1), fac-[Re(Pico)(CO)3(Py)] (R2), fac-[Re(Dfpc)(CO)3(H2O)] (R3), fac-[Re(Dfpc)(CO)3(Pz)] (R4), fac-[Re(Dfpc)(CO)3(Py)] (R5), fac-[Re(Tfpc)(CO)3(H2O)] (R6), fac-[Re(Tfpc)(CO)3(Py)] (R7), and fac-[Re(Tfpc)(CO)3(Im)] (R8) was explored. To gain insights into the electronic structural properties, bioactivity, and stability of these complexes, the highest occupied molecular orbital-lowest unoccupied molecular orbital analysis, binding energy, and topological analysis based on quantum theory of atoms-in-molecules were considered. The anticancer activities of the complexes were measured via in silico molecular docking against human BCL-2 protein (IG5M) and proapoptotic (agonist) BAX 1 protein (450O). The results showed that the studied complexes exhibited good binding affinity (-3.25 to -10.16 kcal/mol) and could cause significant disruption of the normal physiological functions of the studied proteins. The results of DFT calculations also showed that the studied complexes exhibited good stability and are suitable candidates for the development of anticancer agents.

Insight into Bcl-2 proteins' functioning at mitochondrial membrane level.

Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes. 1. A. Ul Mushtaq, J. Åden, L.A. Clifton, H. Wacklin-Knecht, M. Campana, A.P.G. Dingeldein, C. Persson, T. Sparrman, G. Gröbner. Neutron reflectometry and NMR spectroscopy of full length Bcl-2 protein reveal its membrane localization and conformation. Communications Biology 4 (2021) 507.

Domain-specific insight into the recognition of BH3-death motifs by the pro-survival Bcl-2 protein

Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lymphoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, cancers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molecular basis of Bcl-2’s ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane-mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2’s functional domains engage in recognizing and inhibiting apoptotic proteins via their prominent BH3 motifs. This way, we provide a unique residue- and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

In vivo, in vitro and Molecular Modelling Analysis of Isoquercetin, Roseoside, Coreximine, Anonaine, and Arianacin Molecules.

Annona muricata is a member of the Annonaceae family. This plant has a high concentration of acetogenin, which gives it excellent therapeutic property. Researchers have tested this miraculous herb in breast cancer cells treatment and observed that it could be a source of anti-cancer agents. The proposed study focused on screening the anticancer biological activity of Annona muricata plant by using the in vitro, in vivo, and in silico methods. In in vitro analysis, the IC50 was determined on two-dimensional and three-dimensional breast cancer cells. 2D cells were cultured on flat dishes typically made of plastic, while 3D cells were cultured using the hanging drop method. In in vivo analysis, Drosophila melanogaster was preferred, and the LC50 was determined. In in silico analysis, molecular docking studies have been carried out on the different classes of Annona muricata acetogenins against the target proteins. Nearly, five acetogenins were selected from the literature, and docking was performed against human Bcl-2, Bad and Akt-1 proteins. In vitro and in vivo results revealed the IC50 value of 2D MDA-MB-231 cells as 330 μg.mℓ-1, of 2D MCF-7 cells as290 μg.mℓ-1, and of 3D MCF-7 and MDA-MB-231 cells about 0.005 g.mℓ-1; the LC50 value of Drosophila melanogaster was determined as 0.1 g.mℓ-1. In silico results revealed that the docked complex formed by Isoquercetin showed better binding affinity towards target proteins. As a result of the analysis, the Annona muricata plant has been observed to be effective against cancer and likely to be a potential drug.

Neutron reflectometry and NMR spectroscopy of full-length Bcl-2 protein reveal its membrane localization and conformation

B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis. Anti-apoptotic Bcl-2 proteins possess a hydrophobic tail-anchor enabling them to translocate to their target membrane and to shift into an active conformation where they inhibit pro-apoptotic Bcl-2 proteins to ensure cell survival. To address the unknown molecular basis of their cell-protecting functionality, we used intact human Bcl-2 protein natively residing at the mitochondrial outer membrane and applied neutron reflectometry and NMR spectroscopy. Here we show that the active full-length protein is entirely buried into its target membrane except for the regulatory flexible loop domain (FLD), which stretches into the aqueous exterior. The membrane location of Bcl-2 and its conformational state seems to be important for its cell-protecting activity, often infamously upregulated in cancers. Most likely, this situation enables the Bcl-2 protein to sequester pro-apoptotic Bcl-2 proteins at the membrane level while sensing cytosolic regulative signals via its FLD region.

Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promising Cancer Drug Target.

Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

Deleterious Missense Single Nucleotide Polymorphism Alters the Structural Conformation of Human Anti-Apoptotic Bcl-2 Protein

Deleterious Missense Single Nucleotide Polymorphism Alters the Structural Conformation of Human Anti-Apoptotic Bcl-2 Protein

Crystal structures of ORFV125 provide insight into orf virus-mediated inhibition of apoptosis.

Premature apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Orf virus (ORFV) is a large double-stranded DNA virus belonging to the poxviridae. ORFV encodes for an apoptosis inhibitory protein ORFV125 homologous to B-cell lymphoma 2 or Bcl-2 family proteins, which has been shown to inhibit host cell encoded pro-apoptotic Bcl-2 proteins. However, the structural basis of apoptosis inhibition by ORFV125 remains to be clarified. We show that ORFV125 is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins including Bax, Bak, Puma and Hrk with modest to weak affinity. We then determined the crystal structures of ORFV125 alone as well as bound to the highest affinity ligand Bax BH3 motif. ORFV125 adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. In contrast with a previously predicted structure, ORFV125 adopts a domain-swapped dimeric topology, where the α1 helix from one protomer is swapped into a neighbouring unit. Furthermore, ORFV125 differs from the conserved architecture of the Bcl-2 binding groove and instead of α3 helix forming one of the binding groove walls, ORFV125 utilizes an extended α2 helix that comprises the equivalent region of helix α3. This results in a subtle variation of previously observed dimeric Bcl-2 architectures in other poxvirus and human encoded Bcl-2 proteins. Overall, our results provide a structural and mechanistic basis for orf virus-mediated inhibition of host cell apoptosis.

The structural basis of Bcl-2 mediated cell death regulation in hydra.

Apoptosis is regulated by evolutionarily conserved signaling pathways to remove damaged, diseased or unwanted cells. Proteins homologous to the B-cell lymphoma 2 (Bcl-2) family of proteins, the primary arbiters of mitochondrially mediated apoptosis, are encoded by the cnidarian Hydra vulgaris. We mapped interactions between pro-survival and pro-apoptotic Bcl-2 proteins of H. vulgaris by affinity measurements between Hy-Bcl-2-4, the sole confirmed pro-survival Bcl-2 protein, with BH3 motif peptides of two Bcl-2 proteins from hydra that displayed pro-apoptotic activity, Hy-Bak1 and Hy-BH3-only-2, and the BH3 motif peptide of the predicted pro-apoptotic protein Hy-Bax. In addition to peptides from hydra encoded pro-apoptotic proteins, Hy-Bcl-2-4 also engaged BH3 motif peptides from multiple human pro-apoptotic Bcl-2 proteins. Reciprocally, human pro-survival Bcl-2 proteins Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1 bound to BH3 spanning peptides from hydra encoded pro-apoptotic Hy-Bak1, Hy-BH3-only and Hy-Bax. The molecular details of the interactions were determined from crystal structures of Hy-Bcl-2-4 complexes with BH3 motif peptides of Hy-Bak1 and Hy-Bax. Our findings suggest that the Bcl-2 family in hydra may function in a manner analogous to the Bcl-2 family in humans, and less like the worm Caenorhabditis elegans where evolutionary gene deletion has simplified the apoptotic program. Combined, our results demonstrate the powerful conservation of the interaction pattern between hydra and human Bcl-2 family members. Furthermore, our data reveal mechanistic differences in the mode of binding between hydra and sponges such as Geodia cydonium, with hydra encoded Bcl-2 resembling the more promiscuous pro-apoptotic Bcl-2 members found in mammals compared with its sponge counterpart.

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies.

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

Analysis of Mutant Constructs of Human BIK and their Interaction with KSHV vBCL‐2

Kaposi's sarcoma‐associated herpesvirus (KSHV) is a skin cancer caused by a gamma‐ herpesvirus.The virus is a “tumor” transforming virus and thus often leads to cancer in the infected patient. Its association with AIDS over the past two decades has brought increased attention to the roll viral infection plays in the evolution of cancer. KSHV has been shown to interfere in one major pathway that controls cell growth, the apoptotic pathway, important for the maintenance of normal growth and development. Viruses require a live cell for survival and have evolved a mechanism to interfere with apoptosis to create immortal cell hosts. The virus expresses a protein, vBCL‐2 that interferes with pro‐apoptotic proteins and promotes tumor growth. This viral protein is a functional homology of the human Bcl2 protein and has been shown to interact with several BH3 domain containing family members. Previously we had identified an interaction between the KSHV vBcl2 protein and the human BH3 domain containing protein, BIK using the Yeast Two Hybrid System. Current studies have demonstrated critical residues in the BH3 domain of the host Bcl2 family member, BAK, that are critical for vBcl2 interaction. We generated mutants with amino acid substitutions in homologous residues in the BH3 domain of the BIK protein to elucidate the binding interface in this interaction. Using the yeast two hybrid (Y2H) assay and biochemical methods, we analyzed the effect the mutations had on the vBcl2‐hBIK interaction. Identification of the critical amino acid residues in the BH3 domain of BIK will provide valuable insight into the mechanism of viral invasion of the cell and aid in the development of drug targets to prevent tumor development.

Abstract LB-72: An immunoassay detecting Bcl-2:Bim and Mcl-1:Bim complexes in the phase 1 monotherapy study of the Bcl-2 inhibitor, ABT-199/GDC-0199, in chronic lymphocytic leukemia patients

Abstract Introduction Proteins in the Bcl-2 family are key regulators of the apoptotic process. This family comprises of pro- and anti-apoptotic proteins. Overexpression of anti-apoptotic proteins such as Bcl-2 and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bim, can inhibit pro-apoptotic proteins’ functions. Bcl-2 and Bim are highly expressed in CLL patients and a Bcl-2:Bim complex is formed at mitochondria in these patients. ABT-199 (GDC-0199) is a potent, selective, small molecule inhibitor of Bcl-2. Preclinical data shows Bcl-2:Bim disruption can serve as a proximal PD biomarker for this treatment. Conversely, an increase of Mcl-1:Bim complex could indicate an inhibition of Mcl-1's anti-apoptotic function thereby blocking a mechanism of escape from Bcl-2 inhibitor treatment. Here we describe the development of an immunoassay for detecting Bcl-2:Bim and Mcl-1:Bim complexes in CLL patient blood. This assay is used to evaluate these two complexes in patient PBMC samples from the Phase 1 monotherapy study of GDC-0199 in CLL/SLL (M12-175). Method Bcl-2:Bim or Mcl-1:Bim complexes from PBMC lysates were captured in solution using anti-Bcl-2 or anti-Mcl-1 antibody, followed by incubation with anti-Bim antibody, which was immobilized on a MSD plate. Bcl-2:Bim or Mcl-1:Bim concentration was calculated using a standard curve. The standard curve was generated using Bcl-2:Bim or Mcl-1:Bim complexes formed from recombinant human Bcl-2 or Mcl-1 protein mixed with recombinant human Bim protein at equal molar concentration. In pre-clinical studies, PBMCs from healthy donor treated with ABT-0199 ex-vivo, or from the SUDHL4 tumor xenograft model was run. In the M12-175 clinical study, PBMCs were collected from patients once prior to GDC-0199 treatment, 6 hour and 24 hours post first dose. Results Our data from pre-clinical studies showed a dose dependent decrease of Bcl-2:Bim association in PBMC lysates. In the clinical study M12-175, we had data from 26 patients at 6h timepoint and 27 patients at 24h timepoint. Data was limited to patients with sufficient amounts of total protein (>7 ug) in their PBMC lysates which was the minimum required for this immunoassay. Since post- treatment samples were only taken after the first dose, this limited the evaluation of most patient samples to the starting dose of ABT-199, 20 mg in 3 patients, 50 mg in 23 patients and 200 mg in 1 patient evaluated, with most patients subsequently receiving intra-patient dose escalation to the target doses, the highest tolerable dose tested so far is 600 mg. Strongest decrease in Bcl-2:Bim complex was observed at 200 mg which was the highest dose tested with this assay. Variable decrease in the Bcl-2:Bim complex was observed at the 20 mg and 50 mg doses, range from 1-3 in fold change and 22 % of them with a 2 fold decrease at 24h. A concomitant increase in Mcl-1:Bim was observed in a subset of patients. 14% of them with a 2 fold increase at 24h. Conclusions ABT-199 treatment resulted in a decrease in Bcl-2:Bim complexes measured using our MSD immunoassay run on PBMC lysates from patients on the M12-175 monotherapy study in CLL/SLL patients. Measurable decreases were observed in the majority of patients (although to variable extents) even at the lowest doses of 20 mg and 50 mg, with stronger decreases in the patient at 200 mg. Citation Format: Jessica Li, Jun Chen, Ward Kadel, Brenda Chyla, evelyn McKeegan, Elizabeth Punnoose, Walter Darbonne. An immunoassay detecting Bcl-2:Bim and Mcl-1:Bim complexes in the phase 1 monotherapy study of the Bcl-2 inhibitor, ABT-199/GDC-0199, in chronic lymphocytic leukemia patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-72. doi:10.1158/1538-7445.AM2014-LB-72

A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.

Antiapoptotic potency of Bcl-2 proteins primarily relies on their stability, not binding selectivity

AbstractAll 6 human prosurvival Bcl-2 proteins can drive cancer development and contribute to therapy resistance. However, their relative abilities to protect cells against cancer therapy were not examined previously. We report that Bcl-2, Bcl-xL, or Bcl-w consistently protected leukemic cells better than Bcl-B, Bfl-1, or Mcl-1 against a wide variety of anticancer regimens. Current thinking would attribute this to differences in their ability to bind to BH3-only proteins, Bax, and Bak. To address this, we established the first complete, quantitative cellular interaction profile of all human prosurvival Bcl-2 proteins with all their proapoptotic relatives. Binding was unexpectedly promiscuous, except for Bad and Noxa, and did not explain the differential antiapoptotic capacity of the Bcl-2 proteins. Rather, Bcl-B, Bfl-1, or Mcl-1 proved less potent due to steady-state or drug-induced proteasomal degradation. All 6 Bcl-2 proteins similarly protected against the diverse anticancer regimens when expressed at equal protein levels, in agreement with their broad interaction profile. Therefore, clinical diagnostics should include all family members and should be performed at the protein rather than at the messenger RNA level. In drug development, targeting the ubiquitination machinery of prosurvival Bcl-2 proteins will complement and potentially improve on targeting Bcl-2 protein interactions with BH3 mimetics.

The BCL-2 database, Act 2: moving beyond dualism to diversity and pleiotropy

Dear Editor, BCL2 was discovered almost 30 years ago as part of a translocation (t14;18) in human B-cell lymphoma.1 Unique to all other oncogenes known to that date, the product of this gene turned out to act as a cytoprotective factor rather than as a cell growth promoter,2 providing a remarkable first in biology. The human BCL-2 protein was also the first identified component of the cellular machinery of apoptosis. Evolutionary conservation of this machinery became evident when it was realized that Caenorhabditis elegans was containing an antiapoptotic Bcl-2-like protein, CED-9.3 Soon thereafter, it also became clear that BCL-2 was belonging to a large group of proteins that can either repress (such as BCL-xL or MCL-1) or promote (e.g. BAX or BAK) apoptosis. The concept (first issued by Stanley Korsmeyer4) that the ratio between BCL-2 and BAX, and more generally between proapoptotic members and prosurvival members (the so-called ‘rheostat' hypothesis), can determine cell survival or death after an apoptotic stimulus had a deep and sustained influence on the field. Most of the works performed on BCL-2 family proteins and most of the reviews written on the subject so far are rooted in this epistemological and ontological dualism, which has proven immensely useful in understanding the basic aspects of apoptosis regulation by BCL-2 family members. Yet, a number of studies have highlighted the need to transcend this simplistic dualism. These studies may be grouped into four main branches: (a) the demonstration that a distinct subclass of proapoptotic proteins sharing a single region of sequence similarity with BCL-2, termed the BH3 motif, act upstream of the various ‘multi-BH' BCL-2 homologs; (b) evidence showing that at least certain BCL-2 proteins could be bi-functional, shifting from a cytoprotective to a cytotoxic activity and vice versa, depending on changes in the expression level, cell types, alternative splicing and post-translational modifications; (c) the findings that BCL-2 proteins, which act at the mitochondrial level to regulate apoptosis, could also exert functions at non-mitochondrial sites and, more importantly, could play physiological (non-apoptotic) roles in cells uncommitted to death, such as regulation of mitochondrial dynamics, metabolism and calcium homeostasis, or during development;5 (d) phylogenomics and bioinformatics studies documenting the important degree of divergence between BCL-2 homologous sequences,6 and the potential huge number of BH3-containing proteins (due to the poor definition of the BH3 signature).7 Collectively, these different points suggest that a major shift of paradigm is taking place in our understanding of BCL-2 proteins, moving from a notion of opposition (anti- versus proapoptotic members) to a notion of diversity (in sequence, structure, function, evolution) and pleiotropy (i.e., vital roles unrelated to cell death regulation).8 Taking this paradigm shift from a phylogenomics perspective, we have previously reported that duplications played a prominent role in the divergent evolution of BCL-2 homologous genes and contributed to the generation of substantial lineage-specific diversity in the BCL-2 complement of extant metazoans.6 Contrary to cellular BCL-2 homologous proteins, BH3-only proteins are evolutionarily unrelated to each other (i.e., they have not arisen from a common ancestor by duplication). Intriguingly, more than 30 cellular and viral proteins have recently been claimed to contain ‘BH3-like' motifs,7 suggesting that the distribution of BH3-containing proteins might be broader than initially conceived. The large and heterogeneous mixture of proteins forming the contemporary BCL-2 group poses major challenges to researchers trying to investigate their structure–function relationships. As an attempt to help address this issue, we have built a novel version of the BCL-2 Database (http://bcl2db.ibcp.fr/). This novel version of BCL2DB represents an effort toward providing a more global picture of BCL-2 proteins, with the objective of facilitating the reframe from dualism to multiplicity.

Content Determination of Active Component in Huangqi Yinyanghuo Group and Its Effects on hTERT and Bcl-2 Protein in Osteosarcoma.

To screen the optimal extraction process and content determination of active component of Huangqi Yinyanghuo group (HYG) and to study the effects of HYG on human telomerase reverse transcriptase (hTERT) and Bcl-2 protein in osteosarcoma (HOS) cells, providing the theoretical basis for clinical application of HYG in treatment of osteosarcoma, orthogonal design table L9(43) was used to design the extraction process of HYG, and icariin was taken as the investigation index to optimize the extraction process of HYG. 0.125, 0.25, 0.5, 1, 2, 4, and 8 μmol/L HYG were taken to act separately on logarithmic growth phase osteosarcoma HOS cells, CCK-8 assay was used to determine cell viability, and immunohistochemical SP assay was used to determine the expression of hTERT and Bcl-2 protein. Apoptosis rate was positively correlated with the dose of HYG, and the expressions of hTERT and Bcl-2 protein were significantly decreased with the prolonged duration of action. Under the effect of HYG, dose was negatively correlated with osteosarcoma cell survival fraction; osteosarcoma cell survival fraction was positively correlated with hTERT and Bcl-2 protein; duration of action was negatively correlated with hTERT and Bcl-2 protein; and hTERT and Bcl-2 protein were in a synchronous relationship.

Extracellular BCL2 Proteins Are Danger-Associated Molecular Patterns That Reduce Tissue Damage in Murine Models of Ischemia-Reperfusion Injury

BackgroundIschemia-reperfusion (I/R) injury contributes to organ dysfunction in a variety of clinical disorders, including myocardial infarction, stroke, organ transplantation, and hemorrhagic shock. Recent investigations have demonstrated that apoptosis as an important mechanism of cell death leading to organ dysfunction following I/R. Intracellular danger-associated molecular patterns (DAMPs) released during cell death can activate cytoprotective responses by engaging receptors of the innate immune system.Methodology/Principal FindingsIschemia was induced in the mouse hind limb by tourniquet or in the heart by coronary artery ligation. Reperfusion injury of skeletal or cardiac muscle was markedly reduced by intraperitoneal or subcutaneous injection of recombinant human (rh)BCL2 protein or rhBCL2-related protein A1 (BCL2A1) (50 ng/g) given prior to ischemia or at the time of reperfusion. The cytoprotective activity of extracellular rhBCL2 or rhBCL2A1 protein was mapped to the BH4 domain, as treatment with a mutant BCL2 protein lacking the BH4 domain was not protective, whereas peptides derived from the BH4 domain of BCL2 or the BH4-like domain of BCL2A1 were. Protection by extracellular rhBCL2 or rhBCL2A1 was associated with a reduction in apoptosis in skeletal and cardiac muscle following I/R, concomitant with increased expression of endogenous mouse BCL2 (mBCL2) protein. Notably, treatment with rhBCL2A1 protein did not protect mice deficient in toll-like receptor-2 (TLR2) or the adaptor protein, myeloid differentiation factor-88 (MyD88).Conclusions/SignificanceTreatment with cytokine-like doses of rhBCL2 or rhBCL2A1 protein or BH4-domain peptides reduces apoptosis and tissue injury following I/R by a TLR2-MyD88-dependent mechanism. These findings establish a novel extracellular cytoprotective activity of BCL2 BH4-domain proteins as potent cytoprotective DAMPs.