DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.     

Control of Cellular Bcl-xL Levels by Deamidation-Regulated Degradation

The cellular concentration of Bcl-x_L is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-x_L undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-x_L is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-x_L for degradation. Additionally, we show that degradation of deamidated Bcl-x_L is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-x_L, underscoring its importance in Bcl-x_L regulation. Our findings strongly suggest that deamidation-regulated Bcl-x_L degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.     

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.     

Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage

The therapeutic value of DNA-damaging antineoplastic agents is dependent upon their ability to induce tumor cell apoptosis while sparing most normal tissues. Here, we show that a component of the apoptotic response to these agents in several different types of tumor cells is the deamidation of two asparagines in the unstructured loop of Bcl-xL, and we demonstrate that deamidation of these asparagines imports susceptibility to apoptosis by disrupting the ability of Bcl-xL to block the proapoptotic activity of BH3 domain-only proteins. Conversely, Bcl-xL deamidation is actively suppressed in fibroblasts, and suppression of deamidation is an essential component of their resistance to DNA damage-induced apoptosis. Our results suggest that the regulation of Bcl-xL deamidation has a critical role in the tumor-specific activity of DNA-damaging antineoplastic agents.     

Structure assembly of Bcl-xL through α5-α5 and α6-α6 interhelix interactions in lipid membranes

Lipid bilayer membrane is the main site where Bcl-x_L executes its anti-apoptotic function. Here we used site-directed mutagenesis and cysteine-directed cross-linking to trap the structure of Bcl-x_L upon membrane insertion. Cys151 on α5-helix and Asn185 on α6-helix of two neighboring Bcl-x_L are found in close positions, respectively. The FRET based binding assay indicated that the BH3-peptide binding pocket in Bcl-x_L is disrupted after its membrane insertion. Co-immunoprecipitation experiments showed that the membrane-bound Bcl-x_L sequestered tBid by direct interaction at physiological pH. If Bcl-x_L behaves similarly at low pH as it does at physiological pH, the membrane-bound Bcl-x_L should bind to tBid through protein regions other than the BH3 domain of tBid and the hydrophobic pocket of Bcl-x_L. Previously, a crystallography study demonstrated that Bcl-x_L formed homodimers through domain swapping in water, where Cys151 and Asn185 of two monomeric subunits are far apart from each other and the BH3-peptide binding pocket is intact. Our results indicated that Bcl-x_L dimer trapped by cross-linking in lipids is distinct from the domain swapped dimer, suggesting that Bcl-x_L transits through a structural change from the water-soluble state to the membrane-bound state and there are multiple possibilities for structural reorganization of Bcl-x_L protein.     

An Interaction between Bcl-xL and the Voltage-dependent Anion Channel (VDAC) Promotes Mitochondrial Ca2+ Uptake

The role of the antiapoptotic protein Bcl-x_L in regulating mitochondrial Ca²⁺ ([Ca²⁺]_mito) handling was examined in wild-type (WT) and Bcl-x_L knock-out (Bcl-x_L-KO) mouse embryonic fibroblast cells. Inositol 1,4,5-trisphosphate-generating agonist evoked cytosolic Ca²⁺ transients that produced a larger [Ca²⁺]_mito uptake in WT cells compared with Bcl-x_L-KO. In permeabilized cells, stepping external [Ca²⁺] from 0 to 3 μm also produced a larger [Ca²⁺]_mito uptake in WT; moreover, the [Ca²⁺]_mito uptake capacity of Bcl-x_L-KO cells was restored by re-expression of mitochondrially targeted Bcl-x_L. Bcl-x_L enhancement of [Ca²⁺]_mito uptake persisted after dissipation of the mitochondrial membrane potential but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane-localized voltage-dependent anion channel (VDAC) is a known Ca²⁺ permeability pathway that directly interacts with Bcl-x_L. Bcl-x_L interacted with VDAC1 and -3 isoforms, and peptides based on the VDAC sequence disrupted Bcl-x_L binding. Peptides reduced [Ca²⁺]_mito uptake in WT but were without effect in Bcl-x_L-KO cells. In addition, peptides reduced [Ca²⁺]_mito uptake in VDAC1 and VDAC3 knock-out but not VDAC1 and -3 double knock-out mouse embryonic fibroblast cells, confirming that Bcl-x_L interacts functionally with VDAC1 and -3 but not VDAC2. Thus, an interaction between Bcl-x_L and VDAC promotes matrix Ca²⁺ accumulation by increasing Ca²⁺ transfer across the outer mitochondrial membrane.     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Biophysical changes reduce energetic demand in growth factor–deprived lymphocytes

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-x_L expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-x_L cells depleted of IL-3 and primary CD8⁺ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.     

BimL displacing Bcl-xL promotes Bax translocation during TNFα-induced apoptosis

Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFα)-induced apoptosis. Bim_L, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim_L activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the role of Bim_L in Bax activation during TNFα-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x_L and Bax decreased after TNFα treatment, while that between Bcl-x_L and Bim_L increased. Bax did not co-immunoprecipitate with Bim_L before or after the TNFα treatment. In addition, the increased interaction between Bim_L and Bcl-x_L was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim_L redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNFα-induced apoptosis.     

1H, 13C and 15N chemical shift assignments of unliganded Bcl-xL and its complex with a photoresponsive Bak-derived peptide

Here we report the ¹H, ¹³C and ¹⁵N resonance assignments of free Bcl-x_L and of Bcl-x_L in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding.     

Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein-protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72-87) peptide and the anti-apoptotic protein Bcl-x_L, the effect of both increasing and decreasing Bak peptide length on Bcl-x_L binding was investigated. Previous work demonstrated that the Bak(72-87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-x_L. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-x_L and Bcl-2 equally well. Approximate binding affinity calculations on these peptide-protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72-87) variants for improved affinity to Bcl-x_L. The best variants obtained from this library exhibit an apparent K_d to Bcl-x_L 4-fold lower than that of wild-type Bak(72-87).     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x_L, which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x_L was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x_L regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (ρ⁰) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x_L against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x_L.     

Deficiency for the Cysteine Protease Cathepsin L Impairs Myc-Induced Tumorigenesis in a Mouse Model of Pancreatic Neuroendocrine Cancer

Motivated by the recent implication of cysteine protease cathepsin L as a potential target for anti-cancer drug development, we used a conditional MycER^TAM;Bcl-x_L model of pancreatic neuroendocrine tumorigenesis (PNET) to assess the role of cathepsin L in Myc-induced tumor progression. By employing a cysteine cathepsin activity probe in vivo and in vitro, we first established that cathepsin activity increases during the initial stages of MycER^TAM;Bcl-x_L tumor development. Among the cathepsin family members investigated, only cathepsin L was predominately produced by beta-tumor cells in neoplastic pancreata and, consistent with this, cathepsin L mRNA expression was rapidly upregulated following Myc activation in the beta cell compartment. By contrast, cathepsins B, S and C were highly enriched in tumor-infiltrating leukocytes. Genetic deletion of cathepsin L had no discernible effect on the initiation of neoplastic growth or concordant angiogenesis. However, the tumors that developed in the cathepsin L-deficient background were markedly reduced in size relative to their typical wild-type counterparts, indicative of a role for cathepsin L in enabling expansive tumor growth. Thus, genetic blockade of cathepsin L activity is inferred to retard Myc-driven tumor growth, encouraging the potential utility of pharmacological inhibitors of cysteine cathepsins in treating late stage tumors.     

Helix orientations in membrane-associated Bcl-X<Subscript>L</Subscript> determined by <Superscript>15</Superscript>N-solid-state NMR spectroscopy

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x_L is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x_L associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x_L its membrane interactions are weak. In the presence of membranes Bcl-x_L can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x_L hydrophobic binding pocket becomes accessible for protein–protein interactions whilst the structure of the N-terminal region remains intact. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Engineering deamidation-susceptible asparagines leads to improved stability to thermal cycling in a lipase

At high temperatures, protein stability is influenced by chemical alterations; most important among them is deamidation of asparagines. Deamidation kinetics of asparagines depends on the local sequence, solvent, pH, temperature, and the tertiary structure. Suitable replacement of deamidated asparagines could be a viable strategy to improve deamidation-mediated loss in protein properties, specifically protein thermostability. In this study, we have used nano RP-HPLC coupled ESI MS/MS approach to identify residues susceptible to deamidation in a lipase (6B) on heat treatment. Out of 15 asparagines and six glutamines in 6B, only five asparagines were susceptible to deamidation at temperatures higher than 75°C. These five positions were subjected to site saturation mutagenesis followed by activity screen to identify the most suitable substitutions. Only three of the five asparagines were found to be tolerant to substitutions. Best substitutions at these positions were combined into a mutant. The resultant lipase (mutC) has near identical secondary structure and improved thermal tolerance as compared to its parent. The triple mutant has shown almost two-fold higher residual activity compared to 6B after four cycles at 90°C. MutC has retained more than 50% activity even after incubation at 100°C. Engineering asparagines susceptible to deamidation would be a potential strategy to improve proteins to withstand very high temperatures.     

N-acetylphytosphingosine-induced apoptosis of Jurkat cells is mediated by the conformational change in Bak

N-acetylphytosphingosine (NAPS), a sphingolipid derivative, is one of the well-known signal molecules that mediates various cellular functions, including cell growth, differentiation, and apoptosis. In this study, we demonstrated that NAPS induces apoptosis of Jurkat cells by activating Bak, but not Bax, which are both members of a proapoptotic subfamily of the Bcl-2 proteins. NAPS activated caspase-8 in a FADD-independent manner, but the lack of caspase-8 did not suppress the activation of caspase-3 and -9 and cell death, indicating that caspase-8 activation does not play an important role in NAPS-induced cell death. The overexpression of Bcl-x_L, an anti-apoptotic protein, completely inhibited the activation of the caspases and apoptosis, assuming that NAPS-induced apoptosis was initiated by the mitochondria. The expression levels of pro- and anti-apoptotic Bcl-2 family members were not changed by the NAPS treatment. However, Bad was translocated from the cytosol into the mitochondria, where it bound to Bcl-x_L, and Bak was dissociated from Bcl-x_L and conformationally changed. Taken together, these findings indicate that NAPS induced apoptosis of Jurkat cells in a mitochondria-dependent manner that was controlled by the translocation of Bad and the conformational change in Bak. These authors contributed equally to this paper     

Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c

The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-x_L were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-x_L. In contrast, hILP, but not Bcl-x_L, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-x_L may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.