Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.The extensive industrial use of polychlorinated biphenyls (PCBs) during the 20th century has resulted in the release of an estimated several million pounds of PCBs into the environment (2). Due to the hydrophobicity and chemical stability of these compounds, PCBs ultimately accumulate in subsurface anaerobic sediments, where reductive dechlorination by anaerobic microorganisms is proposed to be an essential step in PCB degradation and detoxification (6). Although anaerobic reductive dechlorination has been documented in the environment and in the laboratory, attempts to identify and isolate anaerobic PCB-dechlorinating microbes by classical enrichment and isolation techniques have been unsuccessful (for a review, see reference 2). Isolation of anaerobic PCB-dechlorinating microbes has been hindered in part by the inability to maintain and sequentially transfer dechlorinating consortia in defined medium. May et al. (24) were the first to demonstrate that single colonies could be obtained by plating highly enriched PCB-dechlorinating enrichment cultures on agar-solidified media. Although two of the colonies exhibited para dechlorination activity when transferred back to liquid enrichment medium, the colonies contained a mixed community of microorganisms and dechlorination required the addition of sediment to the medium. More recently, highly enriched PCB-ortho-dechlorinating enrichment cultures were developed from Baltimore Harbor sediments in minimal media that contained sediments and a single congener (3) or Aroclor 1260 (37). These were the first confirmed reports of sustained ortho dechlorination of PCBs throughout sequential transfers in medium with estuarine sediments. Finally, Cutter et al. demonstrated that a consortium of PCB-ortho-dechlorinating anaerobes from Baltimore Harbor could be sequentially transferred and maintained in minimal medium without the addition of sterile sediment (9). With the ability to maintain PCB dechlorination in a completely defined medium, highly enriched PCB-dechlorinating consortia could be developed by sequential transfers in medium that contained the minimal growth requirements for dechlorinating species.The current study identifies putative PCB-dechlorinating anaerobes in ortho-dechlorinating enrichment cultures by a comprehensive approach that combines traditional selective enrichment techniques with molecular monitoring (SEMM). Microbial consortia enriched for PCB ortho dechlorination in minimal medium were analyzed by comparative sequence analysis of genes coding for 16S rRNA (16S rDNA) amplified from total community DNAs. Protocols were developed for chromosomal DNA extraction from sediment, 16S rDNA amplification by PCR, cloning of partial 16S rDNA PCR fragments, screening by restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing for comparative sequence analysis. By utilizing these techniques, shifts in the microbial community were monitored as the cultures were further enriched for PCB-dechlorinating anaerobes by elimination of undefined medium components (i.e., sediment), changes in carbon source, and addition of selective physiological inhibitors. The results presented herein demonstrate the applicability of the SEMM approach for the selection and monitoring of highly defined PCB-dechlorinating microbial consortia.     

Physiological Characterization of a Bacterial Consortium Reductively Dechlorinating 1,2,3- and 1,2,4-Trichlorobenzene

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO₂, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.Chlorobenzenes are widespread pollutants and accumulate in the food chain due to their hydrophobicity and strong persistence against chemical and microbial degradation (34). Anaerobic reductive dechlorination of chlorinated benzenes was demonstrated for enrichment cultures from biofilm reactors, sewage sludge, river sediment, and soil (3, 4, 15, 16, 22, 31, 37). Dechlorination pathways for all multiply chlorinated benzenes were elucidated (4, 15). Some dechlorination patterns can be rationalized by thermodynamic considerations (3, 13), but little is known about the microorganisms participating in chlorobenzene dechlorination.Anaerobic bacteria transforming chlorobenzoates and/or chlorophenols have been isolated in pure cultures (5, 7, 18, 27, 39, 40, 45, 48). Desulfomonile tiedjei (12), strain 2CP-1 (7), Desulfitobacterium chlororespirans (39), and Desulfitobacterium sp. strain PCE1 (18) grow anaerobically by chlororespiration. So far, it has not been possible to evaluate whether the anaerobic dechlorination of chlorobenzenes proceeds via a similar mechanism, since pure cultures are not available.While the effect of oxygen and nitrate on the dechlorination of chloroaromatics is reported to be negative for most cultures (32), the effect of sulfur oxyanions is controversial. Some reports stated an inhibitory role of sulfate in the reductive dehalogenation of various chlorinated or fluorinated aromatics (17, 19, 25, 26); other studies found only slight inhibition (24), no inhibition (14), or even a stimulated rate of dechlorination (17, 23). For one mixed culture, the mineralization of chlorophenols was concomitantly coupled to the reduction of sulfur oxyanions (20, 21). With pure cultures of D. tiedjei, it could be shown that sulfite and thiosulfate inhibited the dechlorination of 3-chlorobenzoate in growing cells, nongrowing cells, and cell extracts, while sulfate inhibited dechlorination only in growing cells (46).The high toxicity (22) and the low solubility of chlorobenzenes in water prevented the successful isolation of bacteria with chlorobenzenes as electron acceptors. It is therefore essential to study alternative electron acceptors that could be used by chlorobenzene-dechlorinating bacteria and that could substitute for chlorobenzenes during enrichment and isolation. Information about reductive dechlorination of chlorobenzenes in the presence of other electron acceptors is also needed for the evaluation of dechlorination processes at natural sites and for in situ remediation projects. To our knowledge, detailed studies of the effects of alternative electron acceptors on the dechlorination of chlorobenzenes have not been reported so far.The aim of the present study was to describe the physiological properties of a mixed culture effectively dechlorinating trichlorobenzenes and to determine the effects of various specific inhibitors and alternative electron acceptors. For these experiments, we used a stable, sediment-free mixed consortium growing in a defined, synthetic mineral medium. This consortium has been established in our laboratory from a fluidized bed bioreactor (1, 33) and reductively dechlorinates 1,2,3-trichlorobenzene to 1,3-dichlorobenzene and 1,2,4-trichlorobenzene to 1,4- and 1,3-dichlorobenzene. By inhibiting the activity of methanogenic bacteria using the specific inhibitor bromoethanesulfonate (BES), we showed that dechlorination occurs independently from methanogenic bacteria (1), as has also been shown for other enrichment cultures dechlorinating chlorobenzenes (22, 31).     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

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.     

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.     

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.     

Size-Selective Predation on Groundwater Bacteria by Nanoflagellates in an Organic-Contaminated Aquifer

Time series incubations were conducted to provide estimates for the size selectivities and rates of protistan grazing that may be occurring in a sandy, contaminated aquifer. The experiments involved four size classes of fluorescently labeled groundwater bacteria (FLB) and 2- to 3-μm-long nanoflagellates, primarily Spumella guttula (Ehrenberg) Kent, that were isolated from contaminated aquifer sediments (Cape Cod, Mass.). The greatest uptake and clearance rates (0.77 bacteria · flagellate⁻¹ · h⁻¹ and 1.4 nl · flagellate⁻¹ · h⁻¹, respectively) were observed for 0.8- to 1.5-μm-long FLB (0.21-μm³ average cell volume), which represent the fastest growing bacteria within the pore fluids of the contaminated aquifer sediments. The 19:1 to 67:1 volume ratios of nanoflagellate predators to preferred bacterial prey were in the lower end of the range commonly reported for other aquatic habitats. The grazing data suggest that the aquifer nanoflagellates can consume as much as 12 to 74% of the unattached bacterial community in 1 day and are likely to have a substantive effect upon bacterial degradation of organic groundwater contaminants.While heterotrophic protists have been found in pristine and contaminated aquifers (3, 29, 34, 37, 54–57), very little research has been performed to elucidate their role in the subsurface. In other environments (e.g., surface and marine waters, topsoil, and wastewater treatment plants), it is well documented that they typically consume bacteria (2, 11, 15, 41, 42, 47), although some have been observed to consume high-molecular-weight organics (48, 59) and even viruses (20, 39). Protists typically graze selectively, depending upon the size (1, 9, 17, 25, 52), growth condition (18, 53), species (16, 17, 35), and motility (18) of their prey. In carbon-limited environments, protists decrease bacterial competition, resulting in a greater bacterial uptake rate for organic substrate per unit of bacterial biomass (27). Based upon indirect field observations, it is also hypothesized that this may be the role they play in organically contaminated aquifers (31). In nutrient-limited environments, protists may release nitrogen or phosphorus needed by bacteria (10, 28, 44, 61).Studies at the U.S. Geological Survey’s (USGS) Toxic Substances Hydrology Program research site at the Massachusetts Military Reservation (MMR) on Cape Cod, Mass., have shown that sandy aquifer sediments can harbor large protistan populations even at relatively low levels (≤2 mg/liter) of dissolved organic matter (30). Protistan abundances in the MMR aquifer plume range from 1 × 10⁴ to 7 × 10⁴ g (dry weight)⁻¹ (30) and consist primarily of nanoflagellates (2 to 3 μm in length) (29) that belong to the genera Bodo, Cercomonas, Cryptaulax, Cyanthomonas, Goniomonas, and Spumella, along with some undescribed species (37). A few amoebae (63) and no ciliates (29) have been observed.Results of a principal-component factor analysis of protistan and bacterial abundances and chemical constituents in the MMR plume suggested that the flagellates were preying upon unattached bacteria (30). Additional evidence of predation was obtained from flowthrough columns of aquifer sediment from which fluorescently labeled unattached bacteria eluted at much lower rates than they did from sterile (protist-free) controls (31). However, these results provide only indirect evidence of predation because no enumerations of the bacteria within the flagellates were performed.The purpose of the research reported in this paper was to directly determine whether the MMR nanoflagellates can consume unattached bacteria in the plume and the extent to which they engage in size-selective grazing. Rates of bacterivory (grazing and clearance rates) were estimated in the laboratory by using fluorescently labeled, monodispersed bacteria (FLB) and nanoflagellates that had been isolated from the MMR aquifer plume. Although other methods exist (19, 26, 38, 43, 45, 62, 64–66), we chose to use fluorescent labeling to study flagellate bacterivory because this procedure requires shorter incubation times and relies upon direct visual observation of the prey within the predator. In addition, experiments could be designed with different sizes of FLB to determine if the nanoflagellates can discriminate between prey. This involved using FLB with cell lengths of 0.1 to 0.5, 0.5 to 0.8, 0.8 to 1.5, and >1.5 μm (average cell volumes of 0.06, 0.14, 0.21, and 0.87 μm³, respectively) in the grazing experiments.     

Identification and Characterization of a Novel CprA Reductive Dehalogenase Specific to Highly Chlorinated Phenols from Desulfitobacterium hafniense Strain PCP-1

Desulfitobacterium hafniense strain PCP-1 reductively dechlorinates pentachlorophenol (PCP) to 3-chlorophenol and a variety of halogenated aromatic compounds at the ortho, meta, and para positions. Several reductive dehalogenases (RDases) are thought to be involved in this cascade of dehalogenation. We partially purified a novel RDase involved in the dechlorination of highly chlorinated phenols from strain PCP-1 cultivated in the presence of 2,4,6-trichlorophenol. The RDase was membrane associated, and the activity was sensitive to oxygen, with a half-life of 128 min upon exposure to air. The pH and temperature optima were 7.0 and 55°C, respectively. Several highly chlorinated phenols were dechlorinated at the ortho positions. The highest dechlorinating activity levels were observed with PCP, 2,3,4,5-tetrachlorophenol, and 2,3,4-trichlorophenol. 3-Chloro-4-hydroxyphenylacetate, 3-chloro-4-hydroxybenzoate, dichlorophenols, and monochlorophenols were not dechlorinated. The apparent K_m value for PCP was 46.7 μM at a methyl viologen concentration of 2 mM. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activity, suggesting the involvement of a corrinoid cofactor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation revealed 2 bands with apparent molecular masses of 42 and 47 kDa. Mass spectrometry analysis using Mascot to search the genome sequence of D. hafniense strain DCB-2 identified the 42-kDa band as NADH-quinone oxidoreductase, subunit D, and the 47-kDa band as the putative chlorophenol RDase CprA3. This is the first report of an RDase with high affinity and high dechlorinating activity toward PCP.Halogenated compounds are generally known as toxic environmental pollutants. Hydrogenolytic reductive dehalogenation, a reaction involving the replacement of one halogen atom with one hydrogen atom, is the predominant mechanism for their transformation in anaerobic environments. This process can sustain microbial growth via electron transport-coupled phosphorylation (10, 26, 31). The majority of the known reductive dehalogenases (RDases) belong to the CprA/PceA family. These are single-polypeptide membrane-associated anaerobic enzymes that are synthesized as preproteins with a cleavable twin arginine translocation (TAT) peptide signal. They contain one corrinoid and two iron-sulfur clusters as cofactors.CprA enzymes catalyzing the reductive dechlorination of chloroaromatics have been purified from Desulfitobacterium hafniense strain DCB-2 (6), Desulfitobacterium dehalogenans (30), Desulfitobacterium chlororespirans strain Co23 (12, 14), Desulfitobacterium sp. strain PCE1 (29), and D. hafniense strain PCP-1 (28) and characterized, and PceA enzymes have been purified from Sulfurospirillum multivorans (22, 23), Desulfitobacterium sp. strain PCE-S (18, 19), D. hafniense strain TCE1 (29), Dehalococcoides ethenogenes 195 (15, 16), Desulfitobacterium sp. strain PCE1 (29), Dehalobacter restrictus (17, 25), Desulfitobacterium sp. strain Y51 (27), and Dehalococcoides sp. strain VS (20) and characterized. However, none of these enzymes showed high dechlorinating activity toward highly chlorinated phenols such as pentachlorophenol (PCP).D. hafniense strain PCP-1 is the only known strict anaerobic bacterium which reductively dechlorinates PCP to 3-chlorophenol (3-CP) and a variety of halogenated aromatic compounds at the ortho, meta, and para positions (2, 7). It dechlorinates PCP at the ortho, ortho, para, and meta positions in the following order: PCP → 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP) → 3,4,5-trichlorophenol (3,4,5-TCP) → 3,5-dichlorophenol (3,5-DCP) → 3-CP (7). Several RDases are thought to operate during this sequence of dechlorinations. Two RDases have already been purified from strain PCP-1. The first one, CrdA, is a membrane-associated enzyme, not related to CprA/PceA-type RDases, that mediates ortho dechlorination of 2,4,6-TCP and several chlorophenols (3). The second enzyme, CprA5, catalyzes the meta and para dechlorination of 3,5-DCP and several chlorophenols (28). Three other putative cprA genes were identified in strain PCP-1 (cprA2, cprA3, and cprA4), which suggests that other RDases with different specificities toward halogenated compounds exist in this strain (8, 31, 32). In this study, we have partially purified and characterized a new CprA-type RDase (CprA3) from strain PCP-1. CprA3 is the first reported RDase with high affinity toward PCP and with high ortho-dechlorinating activity toward PCP and other highly chlorinated phenols.     

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling: RELATIVE IMPORTANCE OF THE DE NOVO BIOPTERIN SYNTHESIS VERSUS SALVAGE PATHWAYS*

Tetrahyrobiopterin (BH4) is a required cofactor for the synthesis of nitric oxide by endothelial nitric-oxide synthase (eNOS), and BH4 bioavailability within the endothelium is a critical factor in regulating the balance between NO and superoxide production by eNOS (eNOS coupling). BH4 levels are determined by the activity of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in de novo BH4 biosynthesis. However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but the functional importance of DHFR in maintaining eNOS coupling remains unclear. We investigated the role of DHFR in regulating BH4 versus BH2 levels in endothelial cells and in cell lines expressing eNOS combined with tet-regulated GTPCH expression in order to compare the effects of low or high levels of de novo BH4 biosynthesis. Pharmacological inhibition of DHFR activity by methotrexate or genetic knockdown of DHFR protein by RNA interference reduced intracellular BH4 and increased BH2 levels resulting in enzymatic uncoupling of eNOS, as indicated by increased eNOS-dependent superoxide but reduced NO production. In contrast to the decreased BH4:BH2 ratio induced by DHFR knockdown, GTPCH knockdown greatly reduced total biopterin levels but with no change in BH4:BH2 ratio. In cells expressing eNOS with low biopterin levels, DHFR inhibition or knockdown further diminished the BH4:BH2 ratio and exacerbated eNOS uncoupling. Taken together, these data reveal a key role for DHFR in eNOS coupling by maintaining the BH4:BH2 ratio, particularly in conditions of low total biopterin availability.In vascular disease states such as atherosclerosis and diabetes, endothelial nitric oxide (NO) bioactivity is reduced, and oxidative stress is increased, resulting in endothelial dysfunction. It has become apparent that enzymatic “coupling” of endothelial NO synthase by its cofactor tetrahydrobiopterin (BH4)² plays a key role in maintaining endothelial function. Indeed, the balance between NO and superoxide production by eNOS appears to be determined by the availability of BH4 versus the abundance of 7,8-dihydrobiopterin (BH2, that is inactive for NOS cofactor function and may compete with BH4 for NOS binding (1). Intracellular biopterin levels are regulated principally by the activity of the de novo biosynthetic pathway (Fig. 1). Guanosine triphosphate cyclohydrolase I (GTPCH; EC 3.5.4.16) catalyzes the formation of dihydroneopterin triphosphate from GTP, and BH4 is generated by two further steps through 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. GTPCH appears to be the rate-limiting enzyme in BH4 biosynthesis, and overexpression of GTPCH is sufficient to augment BH4 levels in cultured endothelial cells (2). Electron paramagnetic resonance spectroscopy studies have shown that BH4 both stabilizes and donates electrons to the ferrous-dioxygen complex in the oxygenase domain, as the initiating step of l-arginine oxidation (3–5). In this reaction BH4 forms the protonated trihydrobiopterin cation radical, which is subsequently reduced by electron transfer from NOS flavins. When BH4 availability is limiting, electron transfer from NOS flavins becomes uncoupled from l-arginine oxidation, eNOS generates superoxide rather than NO, BH4 becomes oxidized to catalytically incompetent BH2, and a futile feed-forward cascade of BH4 destruction proceeds (1). Recent studies reveal that BH4 and BH2 bind eNOS with equal affinity and that BH2 can efficiently replace eNOS-bound BH4, resulting in eNOS uncoupling (6). Indeed, we have previously shown that the relative abundance of eNOS versus BH4 together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling (7), a hypothesis supported by a recent publication where BH2 levels are elevated after exposure of bovine aortic endothelial cells to DHFR-specific siRNA (8). Thus, mechanisms that regulate the BH4:BH2 ratio independently of overall biopterin levels may play an equally important role in regulating eNOS coupling as the well established role of GTCPH, which regulates de novo BH4 biosynthesis. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR; EC 1.5.1.3) can reduce BH2, thus regenerating BH4 (9, 10). It is, therefore, likely that net BH4 bioavailability within the endothelium reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. In human liver extracts DHFR has been shown to reduce BH2 back to BH4 as part of the salvage pathway for biopterin synthesis (11). However, the role of this pathway and the extent to which it regulates intracellular BH4 levels in vivo remains unknown. Recent work by Chalupsky and Cai (2) investigated the functionality of endothelial DHFR in cultured bovine aortic endothelial cells. Exposure to angiotensin II down-regulated DHFR expression, decreased BH4 levels, and increased eNOS uncoupling, which was restored by overexpression of DHFR (2). A recent study also suggests that perturbation of BH4 metabolism differentially affects eNOS phosphorylation sites. Knockdown of DHFR by siRNA inhibits vascular endothelial growth factor-induced dephosphorylation of eNOS at Ser-116, an effect that is completely recovered by the addition of exogenous BH4 (8). However, the requirement for DHFR in regulating intracellular BH4 homeostasis and the quantitative relationships that relate BH4 de novo synthesis versus BH4 recycling to eNOS coupling remain uncertain. Accordingly, we sought to address these questions using both pharmacologic and genetic manipulation of DHFR activity and related these interventions to effects on eNOS coupling. We manipulated DHFR in both endothelial cells and in novel cell lines that stably express an eNOS-GFP fusion protein and where expression of human GTPCH can be regulated by doxycycline in order to test the effects of variations in intracellular BH4 biosynthesis (7). We report that although GTPCH is the key regulator of the total amount of intracellular biopterins, DHFR is critical to eNOS function by determining BH4:BH2 ratio and, thus, in maintaining eNOS coupling. In particular, DHFR is important in preventing “self-propagated” eNOS uncoupling in conditions of low total biopterin levels, when eNOS-dependent oxidation of BH4 that would further exacerbate eNOS uncoupling can be rescued by DHFR.Open in a separate windowFIGURE 1.Schematic representation of the BH4 recycling pathway and eNOS coupling. BH4 is synthesized from GTP via a series of reactions involving GTPCH, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase (SR) and DHFR. DHFR activity can be inhibited by MTX. GFRP, GTP cyclohydrolase feedback regulatory protein. PTPS, 6-pyruvoyl-tetrahydropterin synthase.     

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]     

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to P_fr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-R_C and P_fr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R_C substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.Phytochromes are red-light photoreceptors occurring in plants, bacteria, and fungi where they control important developmental processes (1–6). The discovery of microbial phytochromes from genome sequencing (7–9) provided new prospects for biochemical, spectroscopic and structural analyses of this light sensor family. Agp1 (AtBphP1)³ from the soil bacterium Agrobacterium tumefaciens is a typical member of the widespread family of proteobacterial phytochromes (10, 11) and is the subject of the present study.The domain arrangement of canonical phytochromes consists of an N-terminal photosensory domain, including PAS, GAF, and PHY domains and a C-terminal regulatory kinase domain (see, e.g. Ref. 3). Bacterial phytochromes lack the N-terminal extension, and the PAS module insertion of plant phytochromes (3). In most of the bacterial phytochromes, the C-terminal regulatory domain is a histidine kinase (4). These kinases form homodimers as functional units (12) where the subunits transphosphorylate each other (13). The cofactors are linear tetrapyrroles that are covalently attached via a thioether linkage (14) to the side chains of specific conserved cysteine residues. The native chromophore of plant phytochromes is phytochromobilin (PΦB) (14), some cyanobacterial phytochromes incorporate phycocyanobilin (PCB) (15, 16), and all other bacterial phytochromes bind biliverdin (BV) (10, 11). Whereas the chromophore binding site of the more reduced bilins PΦB and PCB is located in the GAF domain, the binding site of BV is close to the N terminus upstream of the PAS domain (4, 11). The two distinct binding sites apparently require a specific substituent at the C3 carbon of pyrrole ring A, either an ethylidene (PΦB and PCB) or a vinyl (BV) group, for covalent attachment of the bilin chromophore (4). The holophytochrome assembly that includes covalent attachment of the chromophore is an autocatalytic process implying an intrinsic bilin C-S lyase activity of the apophytochrome (17). Kinetic studies of the autoassembly in vitro showed that ligation of the chromophore is the ultimate step following incorporation in the binding pocket and internal protonation (18).Phytochromes display photochromicity involving two either thermally stable or long-lived states, P_r and P_fr (red and far-red absorbing forms), that can be reversibly converted by light of appropriate wavelengths. The P_r to P_fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19–22) leading within picoseconds to the formation of the Lumi-R intermediate (23, 24). The following thermal relaxations via Meta-R_A and Meta-R_C intermediates to P_fr proceed on the time scale of microseconds and milliseconds (25–28).Assembly of Agp1 with locked BV derivatives showed that the geometry of the C-D methine bridge is 15Z_anti in P_r and 15E_anti in P_fr (29) suggesting that this methine bridge remains in the anti conformation during photoconversion. The crystal structures of the chromophore binding domains of the bacteriophytochromes from Deinococcus radiodurans and Rhodopseudomonas palustris revealed that the BV chromophore adopts a 5Z_syn,10Z_syn,15Z_anti configuration/conformation in the P_r state (30–32). The 5Z_syn geometry of the A-B methine bridge in the P_r state was confirmed by assembly of Agp1 with the corresponding locked BV chromophore (33). Recently, heteronuclear NMR investigations and crystallographic studies on the complete photosensory domain of the cyanobacterial phytochrome Cph1 from Synechocystis showed that the PCB chromophore is also in the 5Z_syn,10Z_syn,15Z_anti geometry in P_r (34, 35).Because the locked 5Z_syn adduct of Agp1 did not show a P_fr-like photo-product, conformational changes of the A-B methine bridge in the thermal relaxation cascade have been predicted (33). Flash photolysis experiments with this adduct suggested that these changes occur in the Meta-R_A to Meta-R_C transition (36). The stereochemistry of the A-B methine bridge in the P_fr state and in the preceding intermediates could not be determined unambiguously yet. Recent studies with doubly locked chromophores suggest that the C5–C6 single bond undergoes a thermal rotation from syn to anti in the photoconversion of Agp1, whereas an additional Z/E isomerization around the C4C5 double bond (hula-twist mechanism) was postulated for Agp2 (37). However, the crystal structure of the photosensory domain of the bacteriophytochrome PaBphP in its P_fr-enriched dark-adapted state favors the 5Z_syn conformation of the BV chromophore (38). Structural changes of the A-B methine bridge were excluded for the PCB chromophore of Cph1 on the basis of heteronuclear NMR (34), whereas low temperature Fourier transform IR studies on plant phytochrome suggested an environmental change of the ring A carbonyl group and/or a twist of the A-B methine bridge (39).The mechanism by which the signal is transmitted from the bilin chromophore to the protein is still obscure. The recent three-dimensional structures of the complete photosensory domains of Cph1 (35) and PaBphP (38) reveal key interactions between GAF and PHY domains in the corresponding dark states reflecting P_r and P_fr, respectively. In view of the intrinsic differences between the two phytochromes, it is not trivial to differentiate which of the numerous structural differences arise from light-induced conformational changes and are thus potentially important for signal transmission. We note that many approaches to provide a clue on the mechanism of signal transmission from the bilin chromophore to its proximate environment imply that this process is exclusively coupled to the photo-isomerization localized at ring D and its environment and that the chromophore then remains a passive element in the thermal relaxation cascade. This point of view is supported by recent results from femtosecond stimulated Raman spectroscopy suggesting that the chromophore structures in Lumi-R and P_fr are very similar (24). On the other hand, size exclusion chromatography experiments demonstrated that the global conformational changes observed for the P_fr state of Agp1 WT are absent in constructs (locked 5Zs adduct and mutants D197A and H250A), where the formation of P_fr is inhibited but the primary photoreaction proceeds (33, 40). These results are difficult to explain in terms of an ultra-fast signal transmission from the chromophore to the surrounding residues in its pocket.Light-induced conformational changes at the surface of plant phytochrome were observed by using covalently attached labels that are sensitive to the polarity of the microenvironment (41, 42). Due to the accessibility of several binding sites (i.e. the sulfhydryl groups of cysteines) in these experiments, the labeling was unspecific preventing further assignment of the observed changes to particular regions of the protein. Time-resolved absorption measurements with a covalently attached fluorescein derivative showed that the changes occur in the Meta-R_C to P_fr transition (41). In the present work with Agp1 phytochrome, we take advantage of the highly reactive sulfhydryl group of Cys-20, the genuine binding site of the BV chromophore, to specifically attach a fluorescein derivative. We observed that this construct assembles with PCB and BV forming noncovalent photochromic adducts, spectrally and kinetically undisturbed by the fluorescein label. Upon photo-conversion, the absorption band of the label displays a bathochromic shift and increase in ellipticity suggesting that the label moves in a more hydrophobic and asymmetric environment in the P_fr state. The label thus serves as a polarity probe at the entrance of the binding pocket. We postulate that these polarity changes reflect conformational changes of the A-B methine of the bilin chromophore and/or the microenvironment of ring A at the entrance of the binding pocket. Time-resolved measurements reveal that the changes occur in the Meta-R_A to Meta-R_C and Meta-R_C to P_fr transitions. Analogous results were obtained with the V249C mutant of Agp1 in which both the fluorescein probe and the PCB chromophore are covalently attached.