Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.Regulation of endothelial nitric oxide (NO)² production represents a critical mechanism for the modulation of vascular homeostasis. NO is released by endothelial cells in response to diverse humoral, neural, and mechanical stimuli (1–4). Endothelial cell-derived NO activates guanylate cyclase in vascular smooth muscle cells, leading to increased levels of cGMP and to smooth muscle relaxation. Blood platelets represent another key target for the actions of endothelium-derived NO (5): platelet aggregation is inhibited by NO-induced guanylate cyclase activation. Many other effects of NO have been identified in cultured vascular cells and in vascular tissues, including the regulation of apoptosis, cell adhesion, angiogenesis, thrombosis, vascular smooth muscle proliferation, and atherogenesis, among other cellular responses and (patho)physiological processes.The endothelial isoform of NO synthase (eNOS) is a membrane-associated homodimeric 135-kDa protein that is robustly expressed in endothelial cells (2, 4, 6, 7). Similar to all the mammalian NOS isoforms, eNOS functions as an obligate homodimer that includes a cysteine-complex Zn²⁺ (zinc-tetrathiolate) at the dimer interface (8–10). eNOS is a Ca²⁺/calmodulin-dependent enzyme that is activated in response to the stimulation of a variety of Ca²⁺-mobilizing cell surface receptors in vascular endothelium and in cardiac myocytes. The activity of eNOS is also regulated by phosphorylation at multiple sites (11) that are differentially modulated following the activation of cell surface receptors by agonists such as insulin and vascular endothelial growth factor (VEGF) (12). The phosphorylation of eNOS at Ser-1179 activates eNOS, but phosphorylation at Thr-497 or Ser-116 is associated with inhibition of eNOS activity (13–17). eNOS is reversibly targeted to plasmalemmal caveolae as a consequence of the protein''s N-myristoylation and thiopalmitoylation. The generation of NO by eNOS requires several redox-active cofactors, including nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4), which have key roles in the electron flow required for eNOS catalysis. If the flow of electrons within eNOS is disrupted, the enzyme is uncoupled from NO production and other redox-active products are generated, including hydrogen peroxide and superoxide anion radical (18, 19).In vascular disease states such as diabetes, endothelial dysfunction is characterized by a decrease in NO bioactivity and by a concomitant increase in superoxide formation, while eNOS mRNA and protein levels are maintained or even increased. “Uncoupled” eNOS generates reactive oxygen species (ROS), shifting the nitroso-redox balance and having adverse consequences in the vascular wall (20). Several enzymes expressed in vascular tissues contribute to the production and efficient degradation of ROS, and an enhanced activity of oxidant enzymes and/or reduced activity of antioxidant enzymes may cause oxidative stress. Various agonists, pathological conditions, and therapeutic interventions lead to modulated expression and function of oxidant and antioxidant enzymes. However, the intimate relationship between intracellular redox state, eNOS regulation, and NO bioavailability remains incompletely characterized.BH4 is a key redox-active cofactor for activity of all NOS enzymes (21). The exact role of BH4 in NOS catalysis is not yet completely defined, but this cofactor appears to facilitate electron transfer from the eNOS reductase domain and maintains the heme prosthetic group of the enzyme in its redox-active form (18, 22, 23). Moreover, BH4 promotes formation of active NOS homodimers (24) and inhibits the formation of hydrogen peroxide or superoxide by uncoupled eNOS (18, 19). It has been reported that the endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. Supplementation with BH4 has been shown to improve endothelial function in the models of diabetes and hypertension (25, 26, 27). Moreover, BH4 oxidation is seen in vascular cells in the setting of oxidative stress associated with diabetes (28) and hypertension (29).BH4 can be formed either by a de novo biosynthetic pathway or by a salvage pathway. Guanosine triphosphate cyclohydrolase-1 (GTPCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate. BH4 is generated by further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (30). GTPCH1 appears to be the rate-limiting enzyme in BH4 biosynthesis; overexpression of GTPCH1 is sufficient to augment BH4 levels in cultured endothelial cells (31). On the other hand, dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 from its oxidized form, 7,8-dihydrobiopterin (BH2), in several cell types (30, 32). DHFR is mainly involved in folate metabolism and converts inactive BH2 back to BH4 and plays an important role in the metabolism of exogenously administered BH4. However, the relative contributions of endothelial GTPCH1 and DHFR to the modulation of eNOS-dependent pathways are incompletely understood.In these studies, we have used siRNA-mediated “knockdown” of GTPCH1 and DHFR to explore the relative roles of BH4 synthesis and recycling in the modulation of eNOS bioactivity, as well as in the regulation of NO-dependent signaling pathways in endothelial cells.     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

Conformational Changes in Bcl-2 Pro-survival Proteins Determine Their Capacity to Bind Ligands

Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-x_L, and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-x_L and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-x_L, a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.Apoptosis, or programmed cell death, is a fundamental cellular process required by all multicellular organisms for the elimination of redundant, damaged, or potentially dangerous cells (1). A consequence of dysregulated cell death is the survival of abnormal cells, which in some cases can proliferate uncontrollably and form tumors. Hence, strategies to activate cell death pathways may represent one avenue by which cancer cells can be killed. A major pathway to cell death is regulated by the Bcl-2 family of proteins that consists of both pro-apoptotic and pro-survival members (2). Those family members that promote cell death are divided into two subgroups; that is, the Bax/Bak molecules, which are the essential mediators of apoptosis, and the BH3-only proteins (such as Bim, Bad, Puma, Noxa, and several others), which are the initiators of the apoptotic cascade. Within the pro-survival faction there are five members including Bcl-2 itself, Bcl-x_L, Bcl-w, Mcl-1, and A1. Overexpression of pro-survival proteins can confer a survival advantage to cancer cells. Critically, conventional anti-cancer therapies are often rendered ineffective by this overexpression and other up-stream defects, most prominently mutations in the tumor suppressor p53.One strategy to kill cancer cells is to develop molecules that can mimic the BH3-only proteins (3). These proteins function by engaging the pro-survival proteins, although the downstream consequences of this interaction remain controversial (4). These interactions are mediated by a short sequence motif called the BH3 domain on the BH3-only protein. The structures of a number of BH3 domains in complex with pro-survival proteins have been solved, and all reveal that the BH3 sequence forms an amphipathic helix that inserts into a hydrophobic groove on the surface of the pro-survival proteins (5–10). These structures suggest that it might be possible to develop drugs based on small organic molecules that mimic natural BH3 ligands and activate the cell death pathways.Although a number of “BH3-mimetic” molecules have now been described (11–21), many of these appear to kill cells in a non-mechanism-based manner (22). Only ABT-737, developed by Abbott Laboratories (15, 23), has been shown to be a bona fide BH3-mimetic (22, 24), binding in the same hydrophobic groove of Bcl-x_L as do BH3 ligands (25). ABT-263, a molecule in the same chemical class as ABT-737, has shown efficacy as a single agent in cancer cell lines and animal models of cancer (26–28) and is currently in a phase I/II clinical trial in leukemia and lymphoma patients.One of the important aspects of BH3 peptide interactions with pro-survival proteins is selectivity; some BH3 ligands only bind certain subsets of pro-survival proteins (e.g. Bad only binds Bcl-x_L, Bcl-2, and Bcl-w, whereas Noxa only binds Mcl-1 and A1), but others such as Bim and Puma are more promiscuous and bind all pro-survival proteins tightly (29–31). This selectivity has important implications as it appears that a range of pro-survival proteins needs to be neutralized for cell death to proceed in some cell types (30, 32–34). Selectivity is also an important issue for drug potency. ABT-737 and ABT-263 share the same binding profile as Bad, and consequently, their efficacy seems to be restricted to cells/tumors in which Mcl-1 (to which they do not bind with high affinity) is inactivated or limiting (22, 28, 35–38). Hence, only a small subset of all tumors, in particular some hematological malignancies and small-cell lung cancers, appear to respond to ABT-737/ABT-263 when used as a single agent (15, 28). Therefore, to expand the repertoire of cancers that could be treated with BH3 mimetics, it is likely that the range of binding specificities of such molecules also needs to be expanded, with Mcl-1 being an obvious target candidate. Obatoclax (GX15-070), a small molecule developed by Gemin X Biotechnologies, has been reported to function in this way (14). However, this compound can kill cells deficient in both Bax and Bak, the essential mediators of apoptotic cell death (28); hence, its true mechanism of action is uncertain.Peptide ligands can provide a template for small molecule drug design, particularly when a structure is available for it in complex with its target. For example, highly effective small molecule antagonists of the inhibitors of apoptosis proteins were designed using peptides as a starting point (39, 40). Antagonists of Bcl-x_L based on derivatized terphenyl and terephthalamide scaffolds that apparently mimic the BH3 α-helix have also been reported (17, 18), although in this case no complexes with the protein target have yet been described.Here we use mutagenesis data and structures of BH3 peptides in complex with pro-survival proteins to guide attempts to develop a BH3-mimetic that binds to Mcl-1. Although that goal was not achieved, our results demonstrate extreme conformational flexibility in the pro-survival protein Bcl-x_L and yielded a novel Bcl-x_L-selective antagonist. In contrast, Mcl-1 displays no backbone conformational flexibility when presented with the various ligands we describe.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD: PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION*

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X_L proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (1–3). This form of cellular suicide is widely observed in nature and is not only essential for embryogenesis, immune responses, and tissue homeostasis but is also involved in diseases such as tumor development and progression. Bcl-2 family proteins play a pivotal role in controlling programmed cell death. The major function of these proteins is to directly modulate outer mitochondrial membrane permeability and thereby regulate the release of apoptogenic factors from the intermembrane space into the cytoplasm (for a recent review, see Ref. 4). On the basis of various structural and functional characteristics, the Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-2, Bcl-X_L, or Bcl-w) or promote programmed cell death (e.g. Bax, Bak, or Bok) (5, 6). A second subclass of proapoptotic Bcl-2 family members, the BH3²-only proteins, comprises BAD, Bik, Bmf, Hrk, Noxa, truncated Bid, Bim, and Puma (4). BH3-only proteins share sequence homology only at the BH3 domain. The amphipathic helix formed by the BH3 domain (and neighboring residues) associates with a hydrophobic groove of the antiapoptotic Bcl-2 family members (7, 8). Originally, truncated Bid has been reported to interact with Bax and Bak (9), suggesting that some BH3-only proteins promote apoptosis via at least two different mechanisms: inactivating Bcl-2-like proteins by direct binding and/or by inducing modification of Bax-like molecules. BAD (Bcl-2-associated death promoter, Bcl-2 antagonist of cell death) was described to promote apoptosis by forming heterodimers with the prosurvival proteins Bcl-2 and Bcl-X_L, thus preventing them from binding with Bax (10). More recently, two major models have been suggested for how BH3-only proteins may induce apoptosis. In the direct model, all BH3-only proteins promote cell death by directly binding and inactivating their specific anti-death Bcl-2 protein partner (11, 12). In this model, the relative killing potency of different BH3-only proteins is based on their affinities for antiapoptotic proteins. Thus, the activation of Bax/Bak would be mediated through their release from antiapoptotic counterparts. Contrary to this model, Kim et al. (13) provided support for an alternative hierarchy model, in which BH3-only proteins are divided into two distinct subsets. According to this model, the inactivator BH3-only proteins, like BAD, Noxa, and some others, respond directly to survival factors, resulting in phosphorylation, 14-3-3 binding, and suppression of the proapoptotic function. In the absence of growth factors, these proteins engage specifically their preferred antiapoptotic Bcl-2 proteins. The targeted Bcl-2 proteins then release the other subset of BH3-only proteins designated the activators (truncated Bid, Bim, and Puma) that in turn bind to and activate Bax and Bak.Non-phosphorylated BAD associated with Bcl-2/Bcl-X_L is found at the outer mitochondrial membrane. Phosphorylation of specific serine residues, Ser-112 and Ser-136 of mouse BAD (mBAD) or the corresponding phosphorylation sites Ser-75 and Ser-99 of human BAD (hBAD), results in association with 14-3-3 proteins and subsequent relocation of BAD (14, 15). Phosphorylation of mBAD at Ser-155 (Ser-118 of hBAD) within its BH3 domain disrupts the association with Bcl-2 or Bcl-X_L, promoting cell survival (16). Therefore, the phosphorylation status of BAD at these serine residues reflects a checkpoint for cell death or survival. Although the C-RAF kinase was the first reported BAD kinase (17), its target sites were not clearly defined. However, there is a growing body of evidence for direct participation of RAF in regulation of apoptosis via BAD (18, 19). In addition, Kebache et al. (20) reported recently that the interaction between adaptor protein Grb10 and C-RAF is essential for BAD-mediated cell survival. On the other hand, numerous reports suggest that PKA (21), Akt/PKB (22), PAK (18, 23, 24), Cdc2 (25), RSK (26, 27), CK2 (28), and PIM kinases (29) are involved in BAD phosphorylation as well. The involvement of c-Jun N-terminal kinase in BAD phosphorylation is controversially discussed. Whereas Donovan et al. (30) reported that c-Jun N-terminal kinase phosphorylates mBAD at serine 128, Zhang et al. (31) claimed that c-Jun N-terminal kinase is not a BAD-serine 128 kinase. On the other hand, it has been shown that c-Jun N-terminal kinase is able to suppress IL-3 withdrawal-induced apoptosis via phosphorylation of mBAD at threonine 201 (32). Thus, taken together, with respect to regulation of mBAD by phosphorylation, five serine phosphorylation sites (at positions 112, 128, 136, 155, and 170) and two threonines (117 and 201) have been identified so far. Intriguingly, only little data are available regarding the role of phosphorylation in regulation of hBAD protein, although significant structural differences between these two BAD proteins exist.During apoptosis, some members of the Bcl-2 family of proteins, such as Bax or Bak, have been shown to induce permeabilization of the outer mitochondrial membrane, allowing proteins in the mitochondrial intermembrane space to escape into the cytosol, where they can initiate caspase activation and cell death (for a review, see Refs. 33 and 34). Despite intensive investigation, the mechanism whereby Bax and Bak induce outer membrane permeability remains controversial (34). Based on crystal structure (35), it became evident that Bcl-X_L has a pronounced similarity to the translocation domain of diphtheria toxin (36), a domain that can form pores in artificial lipid bilayers. This discovery provoked the predominant view that upon commitment to apoptosis, the proapoptotic proteins Bax and Bak also form pores in the outer mitochondrial membrane (37). As expected from the structural considerations, Bcl-X_L was found to form channels in synthetic lipid membranes (38). Since then, other Bcl-2 family members like Bcl-2, Bax, and the BH3-only protein Bid have been reported to have channel-forming ability. These pores can be divided into two different types: proteinaceous channels of defined size and ion specificity (38–42) and large lipidic pores that allow free diffusion of 2-megadalton macromolecules (43, 44). With respect to the BH3-only protein BAD, no pore-forming abilities have been reported so far, although human BAD has been found to possess per se high affinity for negatively charged phospholipids and liposomes, mimicking mitochondrial membranes (14).The RAF kinases (A-, B-, and C-RAF) play a central role in the conserved Ras-RAF-MEK-ERK signaling cascade and mediate cellular responses induced by growth factors (45–47). Direct involvement of C-RAF in inhibition of proapoptotic properties of BAD established a link between signal transduction and apoptosis control (48, 49). However, the early works did not identify the exact RAF phosphorylation sites on BAD (17). Here we demonstrate that hBAD serves as a substrate of RAF isoforms. With respect to hBAD phosphorylation by PKA, Akt/PKB, and PAK1 in vivo, we observed different specificity compared with RAF kinases. hBAD phosphorylation by RAF was accompanied by reduced apoptosis in HEK293 cells (transformed human embryonic kidney cells) and NIH 3T3 cells (a mouse embryonic fibroblast cell line). Furthermore, we show that in vitro phosphorylation of hBAD by RAF at serines 75, 99, and 118 regulates the binding of 14-3-3 proteins and association with Bcl-2 and Bcl-X_L. By use of mass spectrometry, we detected several novel in vivo phosphorylation sites of hBAD in addition to the established phosphorylation sites, serines 75, 99, and 118. Finally, we show here that hBAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Mutagenesis of the BH3 Domain of BAX Identifies Residues Critical for Dimerization and Killing

The BCL-2 family of proteins is comprised of proapoptotic as well as antiapoptotic members (S. N. Farrow and R. Brown, Curr. Opin. Genet. Dev. 6:45–49, 1996). A prominent death agonist, BAX, forms homodimers and heterodimerizes with multiple antiapoptotic members. Death agonists have an amphipathic α helix, called BH3; however, the initial assessment of BH3 in BAX has yielded conflicting results. Our BAX deletion constructs and minimal domain constructs indicated that the BH3 domain was required for BAX homodimerization and heterodimerization with BCL-2, BCL-X_L, and MCL-1. An extensive site-directed mutagenesis of BH3 revealed that substitutions along the hydrophobic face of BH3, especially charged substitutions, had the greatest affects on dimerization patterns and death agonist activity. Particularly instructive was the BAX mutant mIII-1 (L63A, G67A, L70A, and M74A), which replaced the hydrophobic face of BH3 with alanines, preserving its amphipathic nature. BAXmIII-1 failed to form heterodimers or homodimers by yeast two-hybrid or immunoprecipitation analysis yet retained proapoptotic activity. This suggests that BAX’s killing function reflects mechanisms beyond its binding to BCL-2 or BCL-X_L to inhibit them or simply displace other protein partners. Notably, BAXmIII-1 was found predominantly in mitochondrial membranes, where it was homodimerized as assessed by homobifunctional cross-linkers. This characteristic of BAXmIII-1 correlates with its capacity to induce mitochondrial dysfunction, caspase activation, and apoptosis. These data are consistent with a model in which BAX death agonist activity may require an intramembranous conformation of this molecule that is not assessed accurately by classic binding assays.

Programmed cell death and its morphologic equivalent, apoptosis, are orchestrated by a distinct genetic pathway that is apparently possessed by all multicellular organisms (22). Moreover, the biochemical details of how encoded proteins function are beginning to emerge. The BCL-2 family of proteins constitutes a central decisional point within the common portion of the apoptotic pathway. This family possesses both proapoptotic (BAX, BAK, BCL-X_S, BAD, BIK, BID, HRK, and BIM) and antiapoptotic (BCL-2, BCL-X_L, MCL-1, and A1) molecules (5, 11). The ratio of antiapoptotic to proapoptotic molecules such as BCL-2/BAX determines the response to a proximal apoptotic signal (14). A striking characteristic of many family members is their propensity to form homo- and heterodimers (16, 19). The BCL-2 family has homology clustered principally within four conserved domains called BH1, BH2, BH3, and BH4 (5, 11). The multidimensional nuclear magnetic resonance (NMR) and X-ray crystallographic structure of a BCL-X_L monomer indicates that the BH1-4 domains correspond to α helices 1 to 7. Notably, the BH1, -2, and -3 domains are in close proximity and create a hydrophobic pocket presumably involved in interactions with other BCL-2 family members (13). The NMR analysis of a BCL-X_L-BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-X_L pocket and a BH3 amphipathic α-helical peptide from BAK (17).Prior mutagenesis studies of BCL-2 and BCL-X_L revealed the importance of BH1 and BH2 domains for both their antiapoptotic function and the capacity to heterodimerize with proapoptotic molecules like BAX or BAK (2, 19, 26). In general, most mutations that disrupt heterodimerization with BAX also lose their death repressor function. However, exceptions do exist; some mutants of BCL-X_L fail to bind BAX or BAK but still repress cell death, suggesting that these functions can be separated for antiapoptotic molecules (2). Moreover, a genetic approach with Bcl-2-deficient and Bax-deficient mice also suggested that BCL-2 and BAX could function independently of one another (10).Deletion studies of the death agonist BAK first implicated the BH3 domain as having the capacity to bind BCL-X_L and promote apoptosis (3). However, the functional significance of BH3 in BAX is uncertain as indicated in the literature. Three deletion analyses indicated the necessity of the BH3 domain in BAX to promote cell death as well as to heterodimerize with BCL-2 (3, 9, 28). Yet, two recent studies reported that BAX functions as a death activator independent of its heterodimerization (21, 27). Moreover, substitution mutants within the BH3 domain showed conflicting specificities of heterodimerization (20, 21, 27).Our initial screen of yeast two-hybrid libraries with BCL-2 as bait yielded multiple clones that possess only the NH₂ terminus of BAX, bearing the BH3 but not the BH1 or the BH2 domains. A similar set of isolates was obtained when BCL-2 (G145A) was used as bait (15). We also noted by deletion analysis and assessment of minimal domains of BAX that the BH3 domain was required for both homodimerization and heterodimerization. Consequently, we undertook an extensive site-directed mutagenesis of the BH3 domain of BAX. These studies demonstrate the importance of the hydrophobic face of the amphipathic α helix of BH3 for the dimerization and cell death activities of BAX. Furthermore, analysis of a BAX mutant indicates that its retained conformation as a cross-linkable dimer at mitochondrial membranes correlates with its intact apoptotic function.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.