Loop 2 Structure in Glycine and GABAA Receptors Plays a Key Role in Determining Ethanol Sensitivity

The present study tests the hypothesis that the structure of extracellular domain Loop 2 can markedly affect ethanol sensitivity in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABA_ARs). To test this, we mutated Loop 2 in the α1 subunit of GlyRs and in the γ subunit of α1β2γ2GABA_ARs and measured the sensitivity of wild type and mutant receptors expressed in Xenopus oocytes to agonist, ethanol, and other agents using two-electrode voltage clamp. Replacing Loop 2 of α1GlyR subunits with Loop 2 from the δGABA_AR (δL2), but not the γGABA_AR subunit, reduced ethanol threshold and increased the degree of ethanol potentiation without altering general receptor function. Similarly, replacing Loop 2 of the γ subunit of GABA_ARs with δL2 shifted the ethanol threshold from 50 mm in WT to 1 mm in the GABA_A γ-δL2 mutant. These findings indicate that the structure of Loop 2 can profoundly affect ethanol sensitivity in GlyRs and GABA_ARs. The δL2 mutations did not affect GlyR or GABA_AR sensitivity, respectively, to Zn²⁺ or diazepam, which suggests that these δL2-induced changes in ethanol sensitivity do not extend to all allosteric modulators and may be specific for ethanol or ethanol-like agents. To explore molecular mechanisms underlying these results, we threaded the WT and δL2 GlyR sequences onto the x-ray structure of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC). In addition to being the first GlyR model threaded on GLIC, the juxtaposition of the two structures led to a possible mechanistic explanation for the effects of ethanol on GlyR-based on changes in Loop 2 structure.Alcohol abuse and dependence are significant problems in our society, with ∼14 million people in the United States being affected (1, 2). Alcohol causes over 100,000 deaths in the United States, and alcohol-related issues are estimated to cost nearly 200 billion dollars annually (2). To address this, considerable attention has focused on the development of medications to prevent and treat alcohol-related problems (3–5). The development of such medications would be aided by a clear understanding of the molecular structures on which ethanol acts and how these structures influence receptor sensitivity to ethanol.Ligand-gated ion channels (LGICs)² have received substantial attention as putative sites of ethanol action that cause its behavioral effects (6–12). Research in this area has focused on investigating the effects of ethanol on two large superfamilies of LGICs: 1) the Cys-loop superfamily of LGICs (13, 14), whose members include nicotinic acetylcholine, 5-hydroxytryptamine₃, γ-aminobutyric acid type A (GABA_A), γ-aminobutyric acid type C, and glycine receptors (GlyRs) (10, 11, 15–20) and 2) the glutamate superfamily, including N-methyl d-aspartate, α-amino-3-hydroxyisoxazolepropionic acid, and kainate receptors (21, 22). Recent studies have also begun investigating ethanol action in the ATP-gated P2X superfamily of LGICs (23–25).A series of studies that employed chimeric and mutagenic strategies combined with sulfhydryl-specific labeling identified key regions within Cys-loop receptors that appear to be initial targets for ethanol action that also can determine the sensitivity of the receptors to ethanol (7–12, 18, 19, 26–30). This work provides several lines of evidence that position 267 and possibly other sites in the transmembrane (TM) domain of GlyRs and homologous sites in GABA_ARs are targets for ethanol action and that mutations at these sites can influence ethanol sensitivity (8, 9, 26, 31).Growing evidence from GlyRs indicates that ethanol also acts on the extracellular domain. The initial findings came from studies demonstrating that α1GlyRs are more sensitive to ethanol than are α2GlyRs despite the high (∼78%) sequence homology between α1GlyRs and α2GlyRs (32). Further work found that an alanine to serine exchange at position 52 (A52S) in Loop 2 can eliminate the difference in ethanol sensitivity between α1GlyRs and α2GlyRs (18, 20, 33). These studies also demonstrated that mutations at position 52 in α1GlyRS and the homologous position 59 in α2GlyRs controlled the sensitivity of these receptors to a novel mechanistic ethanol antagonist (20). Collectively, these studies suggest that there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).Subsequent studies revealed that the polarity of the residue at position 52 plays a key role in determining the sensitivity of GlyRs to ethanol (20). The findings with polarity in the extracellular domain contrast with the findings at position 267 in the TM domain, where molecular volume, but not polarity, significantly affected ethanol sensitivity (9). Taken together, these findings indicate that the physical-chemical parameters of residues at positions in the extracellular and TM domains that modulate ethanol effects and/or initiate ethanol action in GlyRs are not uniform. Thus, knowledge regarding the physical-chemical properties that control agonist and ethanol sensitivity is key for understanding the relationship between the structure and the actions of ethanol in LGICs (19, 31, 34–40).GlyRs and GABA_ARs, which differ significantly in their sensitivities to ethanol, offer a potential method for identifying the structures that control ethanol sensitivity. For example, α1GlyRs do not reliably respond to ethanol concentrations less than 10 mm (32, 33, 41). Similarly, γ subunit-containing GABA_ARs (e.g. α1β2γ2), the most predominantly expressed GABA_ARs in the central nervous system, are insensitive to ethanol concentrations less than 50 mm (42, 43). In contrast, δ subunit-containing GABA_ARs (e.g. α4β3δ) have been shown to be sensitive to ethanol concentrations as low as 1–3 mm (44–51). Sequence alignment of α1GlyR, γGABA_AR, and δGABA_AR revealed differences between the Loop 2 regions of these receptor subunits. Since prior studies found that mutations of Loop 2 residues can affect ethanol sensitivity (19, 20, 39), the non-conserved residues in Loop 2 of GlyR and GABA_AR subunits could provide the physical-chemical and structural bases underlying the differences in ethanol sensitivity between these receptors.The present study tested the hypothesis that the structure of Loop 2 can markedly affect the ethanol sensitivity of GlyRs and GABA_ARs. To accomplish this, we performed multiple mutations that replaced the Loop 2 region of the α1 subunit in α1GlyRs and the Loop 2 region of the γ subunit of α1β2γ2 GABA_ARs with corresponding non-conserved residues from the δ subunit of GABA_AR and tested the sensitivity of these receptors to ethanol. As predicted, replacing Loop 2 of WT α1GlyRs with the homologous residues from the δGABA_AR subunit (δL2), but not the γGABA_AR subunit (γL2), markedly increased the sensitivity of the receptor to ethanol. Similarly, replacing the non-conserved residues of the γ subunit of α1β2γ2 GABA_ARs with δL2 also markedly increased ethanol sensitivity of GABA_ARs. These findings support the hypothesis and suggest that Loop 2 may play a role in controlling ethanol sensitivity across the Cys-loop superfamily of receptors. The findings also provide the basis for suggesting structure-function relationships in a new molecular model of the GlyR based on the bacterial Gloeobacter violaceus pentameric LGIC homologue (GLIC).     

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag⁺ and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag⁺ sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag⁺ sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd²⁺, a membrane-impermeant soft metal ion with properties similar to Ag⁺. Cd²⁺ inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.F₁F₀ ATP synthase utilizes the energy stored in an H⁺ or Na⁺ electrochemical gradient to synthesize ATP in bacteria, mitochondria, and chloroplasts (1–4). The ATP synthase complex is composed of two sectors, i.e. a water-soluble F₁ sector that is bound to a membrane-embedded F₀ sector. In bacteria, F₁ is composed of five subunits in an α₃β₃γδϵ ratio and contains three catalytic sites for ATP synthesis and/or hydrolysis centered at the α-β subunit interfaces. F₀ is composed of three subunits in an a₁b₂c_10–15 ratio and functions as the ion-conducting pathway (5–9). Ion translocation through F₀ drives rotation of a cylindrical ring of c-subunits that is coupled to rotation of the γ subunit within the (αβ)₃ hexamer of F₁ to force conformational changes in the three active sites and in turn drive synthesis of ATP by the binding change mechanism (1–4, 10–13).Subunit c of F₀ folds in the membrane as a hairpin of two extended α-helices. In Escherichia coli, 10 copies of subunit c pack together to form a decameric ring with TMH1² on the inside and TMH2 on the periphery (6, 14). An atomic resolution structure of the Na⁺-translocating c₁₁-ring from Ilyobacter tartaricus was recently published by Meier et al. (8). In the c₁₁ structure, the Na⁺ binding site is formed by two interacting c subunits. The essential Na⁺-binding Glu residue, which corresponds to Asp-61 in E. coli, is located in TMH2 at the middle of the lipid bilayer. Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (15–18). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (18–21). During ion translocation through F₀, the essential Arg-210 on TMH4 of subunit a is postulated to facilitate the protonation/deprotonation cycle at Asp-61 of subunit c and cause the rotation of the c-ring past the stationary subunit a (3, 4, 19).Chemical modification of cysteine-substituted transmembrane proteins has been widely used as a means of probing the aqueous accessible regions (22–24). The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F₀ has been probed using Ag⁺ and NEM (19, 25–27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2–5 extending from the periplasm to the center of the membrane. Protons entering through this periplasmic access channel are postulated to bind to the essential Asp-61 residues of the c-ring and exit to the cytoplasm by a still uncertain pathway at the peripheral face of aTMH4 with protonation/deprotonation of Asp-61 driving c-ring rotation.During H⁺-driven ATP synthesis, two models for the pathway by which H⁺ or Na⁺ exit to the cytoplasm have been proposed. The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25–27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19, 25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na⁺ can access Glu-65 in the absence of other F₀ subunits, suggesting an intrinsic channel in subunit c (28, 29). However, no such channel was apparent in the crystal structure of the c₁₁-ring (8). In a previous study (30), we probed the thiolate reactivity of Cys substitutions in the cytoplasmic half of TMH2 in subunit c. These experiments revealed extensive reactivity to sulfhydryl-directed reagents on the peripheral face of cTMH2, supporting the presence of the cytoplasmic exit channel at the a-c interface. In this study, we extended the survey of aqueous accessibility in transmembrane regions by probing thiolate reactivity of Cys substitutions in TMH1 and in the periplasmic half of TMH2. The reactivity of Cys substituted into these regions proved to be more limited. Only a small region of TMH1, lying directly behind Asp-61, was reactive with Ag⁺. In addition to Ag⁺, we used Cd²⁺ as a complementary, membrane-impermeant probe for aqueous accessibility. The survey of Cd²⁺ sensitivity confirmed that aqueous accessibility from the cytoplasm is much greater for residues packing at the periphery of the c-ring. The experiments reported here distinguish the aqueous accessible and inaccessible regions of the c-ring and strengthen evidence that the cytoplasmic H⁺ exit channel is situated at the a-c interface.     

The regulatory C-terminal domain of subunit ε of F₀F₁ ATP synthase is dispensable for growth and survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F_oF₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.F_oF₁ ATP synthases are ubiquitous enzymes that synthesize ATP using a transmembrane electrochemical potential of protons or proton motive force (PMF) generated by the respiratory chain across the cytoplasmic membrane of bacteria, the thylakoid membrane of chloroplasts, or the mitochondrial inner membrane (4, 5, 37). The enzyme consists of two parts: membrane-embedded F_o subcomplex (a complex of subunits a, b, and c in bacteria) and hydrophilic F₁ subcomplex (composed of subunits α, β, γ, δ, and ε). The enzyme is also known as a molecular motor, which is composed of the stator subcomplex (α, β, δ, a, and b) and the rotor subcomplex (γ, ε, and c), and its rotation is coupled to ATP synthesis and proton flow across the membrane (20, 31, 52). The reaction of the enzyme is reversible; ATP is hydrolyzed into ADP and inorganic phosphate, the rotor subcomplex rotates in reverse, and protons are extruded to the periplasmic side, resulting in the generation of PMF. Although some bacteria utilize the reverse reaction under particular conditions, the primary function of F_oF₁ ATP synthase is generation of ATP from the PMF. Therefore, the direction of the activity of F_oF₁ ATP synthase is regulated to avoid wasteful ATP hydrolysis.Subunit ε in bacterial F_oF₁ has been known to be an intrinsic inhibitor of F₁ and F_oF₁ complex (18, 21, 23) and is proposed to have a regulatory function (10, 11, 42). Although the inhibitory effects of subunit ε vary among species, in general, ε inhibits ATP hydrolysis activity while repressing ATP synthesis activity to a lesser degree (14, 27). This regulatory function of the ε subunit is mediated almost exclusively by the C-terminal region of ε, which is comprised of two antiparallel α-helices (18, 49, 50). Biochemical and crystallographic studies have revealed that the C-terminal helices can adopt two different conformations (34, 46, 47, 48). In the retracted conformation, the α-helices form a hairpin-like structure and sit on the N-terminal β-sandwich domain of the ε subunit. When the ε subunit exhibits an inhibitory effect, it adopts a more extended conformation in which the C-terminal α-helices extend along the γ subunit, which composes the central stalk. It has also been shown that basic, positively charged residues on the second α-helix of the ε subunit interact with negatively charged residues in the DELSEED segment of subunit β to exert the inhibitory effect (12).Escherichia coli mutants deleted in the entire ε subunit exhibit a reduced growth rate and growth yield, and this effect is proposed to be a result of a deficiency in assembly of the F_o and F₁ complexes (21). The N-terminal β-sandwich domain of the ε subunit is responsible for the assembly of F_o and F₁ and is therefore important for efficient coupling between proton translocation through F_o and ATP synthesis/hydrolysis in F₁ (15, 39). Deletion of the ε subunit leads to dissociation of the F_oF₁ complex and wasteful ATP hydrolysis by free (cytoplasmic) F₁ and dissipation of PMF through free F_o (21, 22, 51).While the importance of the entire ε subunit in the whole-cell physiology of E. coli is fairly well established, the role of the regulatory C-terminal region of ε has received little attention and warrants investigation to determine if the regulatory functions (e.g., inhibition of ATP hydrolysis) observed in vitro are manifested in the physiology of E. coli under various growth conditions. To address this question, we constructed isogenic E. coli mutants that were deleted in the C-terminal region of ε subunit (ε^DC) and used these strains to compare physiological properties of wild-type versus ε^DC cells under a wide range of environmental conditions and stressors.     

PR55��, a Regulatory Subunit of PP2A, Specifically Regulates PP2A-mediated ��-Catenin Dephosphorylation

A central question in Wnt signaling is the regulation of β-catenin phosphorylation and degradation. Multiple kinases, including CKIα and GSK3, are involved in β-catenin phosphorylation. Protein phosphatases such as PP2A and PP1 have been implicated in the regulation of β-catenin. However, which phosphatase dephosphorylates β-catenin in vivo and how the specificity of β-catenin dephosphorylation is regulated are not clear. In this study, we show that PP2A regulates β-catenin phosphorylation and degradation in vivo. We demonstrate that PP2A is required for Wnt/β-catenin signaling in Drosophila. Moreover, we have identified PR55α as the regulatory subunit of PP2A that controls β-catenin phosphorylation and degradation. PR55α, but not the catalytic subunit, PP2Ac, directly interacts with β-catenin. RNA interference knockdown of PR55α elevates β-catenin phosphorylation and decreases Wnt signaling, whereas overexpressing PR55α enhances Wnt signaling. Taken together, our results suggest that PR55α specifically regulates PP2A-mediated β-catenin dephosphorylation and plays an essential role in Wnt signaling.Wnt/β-catenin signaling plays essential roles in development and tumorigenesis (1–3). Our previous work found that β-catenin is sequentially phosphorylated by CKIα⁴ and GSK3 (4), which creates a binding site for β-Trcp (5), leading to degradation via the ubiquitination/proteasome machinery (3). Mutations in β-catenin or APC genes that prevent β-catenin phosphorylation or ubiquitination/degradation lead ultimately to cancer (1, 2).In addition to the involvement of kinases, protein phosphatases, such as PP1, PP2A, and PP2C, are also implicated in Wnt/β-catenin regulation. PP2C and PP1 may regulate dephosphorylation of Axin and play positive roles in Wnt signaling (6, 7). PP2A is a multisubunit enzyme (8–10); it has been reported to play either positive or negative roles in Wnt signaling likely by targeting different components (11–21). Toward the goal of understanding the mechanism of β-catenin phosphorylation, we carried out siRNA screening targeting several major phosphatases, in which we found that PP2A dephosphorylates β-catenin. This is consistent with a recent study where PP2A is shown to dephosphorylate β-catenin in a cell-free system (18).PP2A consists of a catalytic subunit (PP2Ac), a structure subunit (PR65/A), and variable regulatory B subunits (PR/B, PR/B′, PR/B″, or PR/B‴). The substrate specificity of PP2A is thought to be determined by its B subunit (9). By siRNA screening, we further identified that PR55α, a regulatory subunit of PP2A, specifically regulates β-catenin phosphorylation and degradation. Mechanistically, we found that PR55α directly interacts with β-catenin and regulates PP2A-mediated β-catenin dephosphorylation in Wnt signaling.     

Mass Spectrometry Reveals Differences in Stability and Subunit Interactions between Activated and Nonactivated Conformers of the (���¦æ�)4 Phosphorylase Kinase Complex

Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, β, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the β subunit, the predominant subunit responsible for PhK''s activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple β subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αβγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.In the cascade activation of glycogenolysis in skeletal muscle, phosphorylase kinase (PhK),¹ upon becoming activated through phosphorylation, subsequently phosphorylates glycogen phosphorylase in a Ca²⁺-dependent reaction. This phosphorylation of glycogen phosphorylase activates its phosphorolysis of glycogen, leading to energy production (1). The 1.3 MDa (αβγδ)₄ PhK complex was the first protein kinase to be characterized and is among the largest and most complex enzymes known (2). As such, the intact complex has proved to be refractory to high resolution x-ray crystallographic or NMR techniques; however, low resolution structures of the nonactivated and Ca²⁺-saturated conformers of PhK have been deduced through modeling (3) and solved by means of three-dimensional electron microscopic (EM) reconstruction (4–7), and they show that the complex is a bilobal structure with interconnecting bridges. Approximate locations of small regions of each subunit in the complex are known (8–10) and show that the subunits pack head-to-head as apparent αβγδ protomers that form two octameric (αβγδ)₂ lobes associating in D₂ symmetry (11), although direct evidence that the αβγδ protomers are discrete, functional subcomplexes has been lacking until now.Approximately 90% of the mass of the PhK complex is involved in its regulation. Its kinase activity is carried out by the catalytic core of the γ subunit (44.7 kDa), with the k_cat being enhanced up to 100-fold by multiple metabolic, hormonal, and neural stimuli that are integrated through allosteric sites on PhK''s three regulatory subunits, α, β, and δ (12). The small δ subunit (16.7 kDa), which is tightly bound integral calmodulin (13), binds to at least the C-terminal regulatory domain of the γ subunit (γCRD) (14, 15), thereby mediating activation of the catalytic subunit by the obligate activator Ca²⁺ (16). The α and β subunits, as deduced from DNA sequencing, are polypeptides of 1237 and 1092 amino acids, respectively, with calculated masses prior to post-translational modifications of 138.4 and 125.2 kDa (17, 18). Both subunits can be phosphorylated by numerous protein kinases, including cAMP-dependent protein kinase and PhK itself (2). The α and β subunits are also homologous (38% identity and 61% similarity); however, each subunit has unique phosphorylatable regions that contain nearly all the phosphorylation sites found in these subunits (17, 18).The regulation of PhK activity by both Ca²⁺ (19–23) and phosphorylation has been studied extensively (reviewed in Ref. 24); however, only the structural effects induced by Ca²⁺ are well characterized (25), primarily through comparison of the non-activated and Ca²⁺-activated conformers using three-dimensional EM reconstructions (4), small angle x-ray scattering modeling (3), and biophysical (26–28) and chemical crosslinking methods (29–32). In contrast to the Ca²⁺-activated versus non-activated conformers, there are no reported structures of phosphorylated PhK to compare against the non-activated form. A very small amount of structural information for phospho-activated PhK derived from chemical crosslinking raises the possibility of phosphorylation-dependent communication between the β and γ subunits: Arg-18 in the N-terminal phosphorylatable region of β was found to be relatively near the γCRD (33). Several lines of evidence suggest that transduction of the activating phosphorylation signal in PhK occurs concomitantly with conformational changes in β (33) that are detected via various methods (10, 34), including chemical crosslinking (35). For example, crosslinking of only the phosphorylated conformer by the short-span crosslinker 1,5-difluoro-2,4-dinitrobenzene results in the formation of β homodimers (35). Correspondingly, more recent two-hybrid screens of the full length β subunit against itself yielded positive binding interactions only for point mutants in which the N-terminal phosphorylatable serine residues were mutated to phosphomimetic glutamates (33). It should be noted, however, that both chemical crosslinking and two-hybrid screening have potential drawbacks in the study of subunit interactions within a multisubunit complex. In the case of the latter, it is difficult when observing homodimeric two-hybrid interactions to determine whether they correspond to naturally occurring interactions between two like subunits within a complex or between two interacting regions within a single subunit of that complex. Studying subunit interactions in a complex through chemical crosslinking comes with its own inherent limitations. For example, an initial mono-derivatization can potentially cause a conformational change in one subunit that might affect the subsequent crosslinking reaction. This is particularly the case if the crosslinker contains a functionality, such as an aromatic group, that can unexpectedly direct it to a specific locus on the protein complex (36, 37). In addition, the spacer arms on many crosslinkers are sufficiently long to confound interpretation as to whether two subunits within a complex are actually in contact. Similarly, it should be proved that any observed crosslinked conjugate is formed from subunits within a complex, as opposed to between complexes (38, 39), a control that is often not run. Thus, it is prudent to analyze subunit interactions within a complex using a variety of approaches.To corroborate, complement, and expand the previous two-hybrid screening and chemical crosslinking studies of PhK''s subunit interactions and to investigate changes in the pattern of subunit interactions induced by phosphorylation, we carried out comparative MS analyses of both intact and partially denatured forms of nonactivated and phospho-activated PhK using mass spectrometers modified specifically to enhance the transmission of large noncovalently bound protein complexes (40–42). The array of subunit interactions detected for the nonactivated PhK complex largely replicated those reported in the crosslinking literature for this conformer, both corroborating those earlier studies and validating the use of these MS approaches to study subunit interactions within the PhK complex. Additionally, several novel subcomplexes of PhK were revealed, most notably an αβγδ protomer, which corroborates the observed packing of this subcomplex in the D₂ symmetrical (αβγδ)₄ native complex (9, 11). Moreover, we show herein that the array of subunit interactions detected for phospho-activated PhK differs significantly from that observed for the nonactivated conformer, with only the former showing extensive self-interactions between and among the regulatory β subunits. As is discussed, this suggests that activation through phosphorylation is associated with increased interprotomeric interactions in the bridged core of the PhK complex (33, 35).     

The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membrane-bound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V₀ subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V₁ and V₀(−d) that failed to form V₁V₀, and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V₁V₀ assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V₁V₀ was delivered to the vacuole and active. It retained 63–71% of the wild-type activity and was responsive to glucose. Tether-less V₁V₀ disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V₁ subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V₀ assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.V-ATPase⁴ proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1–6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1–3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V₁ (peripheral) and V₀ (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V₁V₀ assembly (8). Deletion of a peripheral V₁ subunit leads to disruption of the entire V₁ domain in yeast. Loss of a V₀ subunit does not affect V₁ assembly but disrupts the entire V₀ domain, which also prevents V₁ from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requires disruption of both genes (9).Eight different subunits (A-H) compose the V₁ domain where ATP hydrolysis takes place at a catalytic hexamer A₃B₃ (1). Six subunits (a, c, c′, c′′, d, e) form V₀, the membrane intrinsic domain that holds V₁ and forms the path for proton transport via a hydrophobic ring structure (c-ring). V₁ and V₀ subunits contribute to the formation of one central and three peripheral stalks that connect the c-ring and the catalytic hexamer A₃B₃ (1). ATP hydrolysis drives rotation of the central stalk (connected to the c-ring) (10). Protons are transferred from the cytosol to each subunit of the c-ring and from the c-ring to the other side of the membrane passing through subunit a (11). As many protons, as subunits forming the c-ring, get transferred against a concentration gradient when hydrolysis of three ATP molecules powers 360° rotation.V-ATPases are related to F-ATP (F₁F₀ ATP) synthases. Both proteins work as molecular motors (10, 12–14). It is postulated that the asymmetry imposed by having a 3-fold symmetry in F₁ (and V₁) and an apparent 10-fold symmetry in the c-ring of F₀ (and V₀) requires energy to be transiently stored. The energy of coupling is thought to be stored in the peripheral (stator) and central (rotor) stalk structures of F₁F₀ (15–17). Subunit a is the only peripheral stalk component of the V-ATPase complex that is secured in the membrane (18). It is key for maintaining structural stability when relative rotation of subunits occurs during catalysis. Thus, the tether of subunit a in V₀ could be functionally comparable with the tether of subunit b in F₀ (Escherichia coli), although subunits a (V₀) and b (F₀) do not share sequence homology.Subunit a is a 95-kDa protein that consists of two domains that are structurally and functionally distinguishable. The hydrophilic N-terminal domain (∼45 kDa) is oriented toward the cytosolic side of the membrane and contains the information necessary to deliver V-ATPases to different compartments (19). The N terminus interacts with multiple V₁ subunits, including the catalytic subunit A (20) and peripheral stalk-forming subunits H, C, E, and G of V₁ (18, 21). It is through these interactions that the N-terminal domain serves as a stator, which prevents rotation of the A₃B₃ hexamer during catalysis. The other half of subunit a, the C-terminal domain (∼50 kDa), is buried in the membrane by multiple transmembrane-spanning regions (9). The C-terminal domain interacts with the periphery of the c-ring (22) and contributes to the path for proton transport (11, 19) by providing access to cytosolic protons and directing their exit to the luminal side of the membrane.In contrast to its role as stator during catalysis, the N-terminal domain of subunit a is a movable element that switches positions when V₁V₀ is regulated by disassembly and reassembly in vivo (1, 2, 23, 24). Inactivation of V-ATPases by disassembly is a rapid response to glucose starvation in yeast (23, 25). In the absence of glucose the V-ATPase complex dissociates into three parts: V₁ subunit C, V₁ (without subunit C), and V₀ (23). Disassembly is reversible, and the three components reassociate immediately after glucose addition, restoring ATP hydrolysis and proton transport. As V₁V₀ disassembles and reassembles, the N-terminal domain of subunit a alternates between V₁V₀ and V₀ (26, 27). In V₁V₀ it contributes to stabilizing the stator-forming V₁ subunits (1, 18). In V₀, its role has yet to be determined.As its functional and regulatory roles emerge, it becomes clear that the cytosolic N terminus of V₀ subunit a is key for V₁V₀ activity, assembly, and regulation. In this study deletions were made at amino acids that connect the N-terminal and C-terminal domains of subunit a Vph1. Shrinking of the tether that anchors subunit a to the membrane harmed assembly of subunit d into V₀, making yeast cells sensitive to pH (vma growth phenotype). Growth defects were rescued by exogenous VMA6, the gene encoding subunit d. Remarkably, subunit d restored assembly and significant function of V-ATPase proton pumps that had up to 46 residues of the tether removed. Because V₁V₀ containing tether-less vph1 assembled with peripheral V₁ subunits and with the glycolytic enzyme phosphofructokinase, we concluded that no major structural changes were generated at the N- and C-terminal domains. Early steps of V₀ assembly, and trafficking were likely impaired by shorter tethers and rescued by VMA6. The potential mechanisms by which overexpression of subunit d rescued subunit a deletions are discussed.     

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of ��-�� Subunit Interactions in the Catalytic Dwell

The temperature-dependent rotation of F₁-ATPase γ subunit was observed in V_max conditions at low viscous drag using a 60-nm gold bead (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126–4131). The Arrhenius slopes of the speed of the individual 120° steps and reciprocal of the pause length between rotation steps were very similar, indicating a flat energy pathway followed by the rotationally coupled catalytic cycle. In contrast, the Arrhenius slope of the reciprocal pause length of the γM23K mutant F₁ was significantly increased, whereas that of the rotation rate was similar to wild type. The effects of the rotor γM23K substitution and the counteracting effects of βE381D mutation in the interacting stator subunits demonstrate that the rotor-stator interactions play critical roles in the utilization of stored elastic energy. The γM23K enzyme must overcome an abrupt activation energy barrier, forcing it onto a less favored pathway that results in uncoupling catalysis from rotation.F-ATPase (F_oF₁), consisting of the catalytic sector F₁ (α₃β₃γδϵ) and the transmembrane proton transport sector F_o (ab₂c₁₀), synthesizes or hydrolyzes ATP coupled with proton transport (for reviews, see Ref. 1–6). As Abrahams et al. (7) discovered in the first high resolution x-ray structure, a critical feature of the F₁-ATPase is the inherent asymmetry of the three β subunits in different conformations, β_TP, β_DP, and β_E, referring to the nucleotide bound in each catalytic site, ATP, ADP, or empty, respectively. A rotational mechanism has been firmly established mostly based on direct observation in single molecule experiments of the behavior of the rotor complex ϵγc₁₀, relative to the stator complex α₃β₃δab₂ (reviewed in Ref. 1). ATP hydrolysis-dependent rotation of the γ and ϵ subunits in purified bacterial F₁ (8, 9), the ϵγc₁₀ complex in detergent solubilized F_oF₁ (10–13), and the ϵγc₁₀ complexin F_oF₁ in lipid bilayers (14) were shown experimentally by single molecule observations using fluorescent actin filament as a probe. Relative rotation of the single copy F_o a subunit was also shown in F₀F₁, which was immobilized through the ring of ∼10 c subunits, suggesting that the rotor and stator are interchangeable mechanical units (14). ATP synthesis by F-ATPase is believed to follow the reverse mechanism of ATP hydrolysis because mechanically induced rotation of the γ subunit in immobilized F₁ in the presence of ADP and P_i results in net ATP synthesis (15, 16). There remain many questions about the mechanism of coupling between catalysis and transport via mechanical rotation. In particular, the mechanism of coupling H⁺ transport to rotation of the subunit c₁₀ ring is still not well understood (4).In contrast, there is considerably more information on the mechanism of coupling catalysis to γ and ϵ subunit rotation. Observations of γ subunit rotation in the catalytic F₁ sector are consistent with Boyer''s binding change model (17); thus coupling between the chemistry and rotation can be assessed by studies of the soluble F₁, and these findings relate to the mechanism of the entire ATP synthase complex. The γ subunit rotates relative to the α₃β₃ hexamer in distinct 120° steps. A 120° rotation step consisting of pause and rotation substeps appears to correspond to the hydrolysis of one ATP, assuming that three ATP molecules are hydrolyzed per 360° revolution (18). Additional pauses observed at low ATP concentrations are attributed to the “ATP waiting” dwell (19). Yasuda et al. (19) and Shimabukuro et al. (20) further resolved that each 120° step occurred in two substeps: an 80° substep whose onset was dependent upon the Mg·ATP concentration, and a 40° substep, which was not affected by substrate concentration (19). The pause before the 80° substep, the ATP waiting dwell became shorter with increasing [Mg·ATP]. In contrast, the pause duration before the 40° rotation step was modulated by the slow hydrolysis rate of ATPγS² or by the catalytic site mutant βE190D (in the Bacillus PS3 F₁), which was found to significantly increase the length of the catalytic dwell (20). These data together indicate that the dwell before the 40° step is the “catalytic dwell” (20) and defines the order of the substeps during the 120° rotation step observed in high Mg·ATP concentrations (21).In this paper, we address the question of when the rate-limiting step of steady state catalysis occurs, with respect to the rotational behavior. Pre-steady state analysis of the burst kinetics of ATP hydrolysis at nearly V_max conditions demonstrated that the rate-limiting transition state occurs after the reversible hydrolysis/synthesis step and before release of phosphate (P_i) (22, 23). The rate-limiting step is likely associated with a rotation step because a γ-β cross-linked enzyme is still able to undergo the initial ATP hydrolysis, but the rotation-impeded enzyme is unable to release P_i (23). Significantly, the kinetics of steady state hydrolysis can only be assessed when the Mg·ATP concentration is high enough to fill all three catalytic sites. The only model consistent with these data is one that involves all three catalytic sites. During each 120° catalytic cycle, one site binds ATP, a different site carries out reversible hydrolysis/synthesis, and the third site releases product P_i and ADP (22, 23).Steady state analyses, which take advantage of a particular γ subunit mutation γM23K (24), are consistent with this model. Replacement of the conserved γMet-23 with lysine causes an uncoupling between catalysis and γ subunit rotation caused by altered interactions between γ and β subunits (25). Importantly, Al-Shawi and Nakamoto (26) and Al-Shawi et al. (25, 27) found that the γM23K mutation strongly affected the rate-limiting transition state of steady state ATP hydrolysis and ATP synthesis. The slope of the Arrhenius plots and thus the energy of activation were significantly increased in the mutant enzyme. Several second site suppressor mutations, mostly in the γ subunit (28, 29) but also in the β subunits (30, 31), were genetically identified because they restored coupled ATP synthesis. Significantly, all were in the γ-β interface. Thermodynamic analyses found that the second site suppressors generally compensated for the primary γM23K mutations by reducing the increased activation energy (25, 27, 31). Although most of the second site mutations were found distant from the γM23K site, the x-ray crystal structures (7) suggested that γM23K may directly interact with conserved βGlu-381. As expected, replacement of βGlu-381 with aspartate also suppressed the uncoupling effects of γM23K (31).To identify the rate-limiting transition state step in the rotational behavior, we analyzed the temperature dependence of the γM23K mutant in V_max conditions observed in single molecule experiments. Interestingly, direct observation of this mutant using the micron-length actin filaments did not detect differences in the rotation behavior at room temperature (9). In contrast, we find in the data presented here that there is dramatic effect of the mutation on the temperature dependence of the length of the catalytic dwell or pause between the 120° rotation steps. This is likely because of two factors: first, we used a bead small enough not to invoke a drag on the rotation (32), and second, the temperature dependence of the rate of the rotation steps is critical for the analyses of the mechanism.     

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

The structure of the membrane integral rotor ring of the proton translocating F₁F₀ ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c₁₁ rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu⁶¹ in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu⁶¹ is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu⁶¹ by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.ATP synthases found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts catalyze ATP synthesis and ATP hydrolysis coupled with transmembrane proton or sodium ion transport. The enzymes are multi-subunit complexes composed of an extra-membranous catalytic F₁ domain and an interconnected integral membrane F₀ domain. The hydrophilic F₁ domain consists of five different polypeptides with a stoichiometry of α₃β₃γδϵ. Detailed structural information obtained with the mitochondrial enzyme (1–3) in combination with biochemical (4), biophysical (5), and single molecule studies (6–9) revealed that synthesis or hydrolysis of ATP in the F₁ domain is accomplished via a rotary catalytic mechanism. In addition to information on the catalytic mechanism, structure analysis and single molecule studies of the mitochondrial or the chloroplast F₁ complex have also unraveled the molecular mechanism of several F₁-specific inhibitors (10–14). Less detailed information is available on the integral membrane F₀ domain, which consists of three different polypeptides (a, b, and c) and mediates the transfer of protons or sodium ions across the membrane. Subunits a and b were shown to reside at the periphery of a cylindrical complex formed by multiple copies of the c subunit (15–18). The number of c subunits in the cylindrical subcomplex shows substantial variation in different organisms. Ten protomers are found in ATP synthases from yeast, Escherichia coli and Bacillus PS3 (19–21), 11 in Ilyobacter tartaricus, Propionigenium modestum, and Clostridium paradoxum (22–24), 13 in the thermoalkalophilic Bacillus TA2.TA1 (25), 14 in spinach chloroplasts (26), and 15 in the cyanobacterium Spirulina platensis (27). The structure of isolated subunits a, b, and c from E. coli has been studied by mutagenesis analysis and by NMR spectroscopy in a mixed solvent that was suggested to mimic the membrane environment (28–32). These studies showed that subunit a folds with five membrane-spanning helices. The fourth of these helices directly interacts with subunit c and contains a conserved arginine (Arg²¹⁰), which is thought to be involved in proton transfer (33). Subunit b, which is present in two copies in the intact F₀, contains a single transmembrane helix. Cross-linking data support a direct interaction of the two copies of the b subunit (29). Subunit c was studied at two different pH values to obtain the protonated and deprotonated form of a conserved carboxylate (Asp⁶¹ in E. coli) that was shown to be essential for proton transport (34). NMR spectroscopy revealed that the isolated c subunit consists of two long hydrophobic membrane spanning segments connected by a short hydrophilic loop (30, 35). This loop is located close to the γ and ϵ subunit on the F₁ side of the membrane (36, 37). Low resolution x-ray crystallography, cryo-electron microscopy, and atomic force microscopy showed that the membrane-spanning helices of the multiple copies of subunit c in the intact F₀ complex are tightly packed in two concentric rings (19, 22, 26). Atomic resolution of the c ring was recently provided for the Na⁺-translocating F-type ATPase from I. tartaricus (38) and the related Na⁺-translocating V-type ATPase from Enterococcus hirae (39). Rotation of the c ring was demonstrated by cross-linking (18), fluorescence studies (40), and single molecule visualization (41, 42). Based on the structural and biochemical information on F₁ and F₀, different mechanical models have been proposed describing how the rotation of the c ring is coupled to the rotation of the F₁ rotor subunits. This rotation in turn drives sequential conformational shifts at the three catalytic β subunits that result in ATP synthesis (43–45). Vice versa hydrolysis of ATP in the F₁ domain is thought to drive rotation of the γϵc_10–15 subcomplex and transports protons or sodium ions across the membrane.Here we describe the crystal structure of the chloroplast c₁₄ rotor, which is the first structure of an isolated c ring rotor from a proton driven ATPase. The structure was solved by molecular replacement using a tetradecameric search model that was generated from a monomer taken from the I. tartaricus c₁₁ structure. The imposition of noncrystallographic symmetry restraints during refinement substantially improved electron density and structure determination.     

Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution

Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.Myofilament proteins comprise the fundamental contractile apparatus of the heart, the cardiac sarcomere. They are subdivided into thin filament proteins, including actin, tropomyosin, the troponin complex (troponin C, troponin I, and troponin T), and thick filament proteins, including myosin heavy chains, myosin light chains, and myosin-binding protein C. Although calcium is the principal regulator of cardiac contraction through the excitation-contraction coupling process that culminates in calcium binding to troponin C, myofilament function is also significantly modulated by phosphorylation of constituent proteins, such as cardiac troponin I (cTnI),1 cardiac myosin-binding protein C (cMyBP-C), and myosin regulatory light chain (MLC-2). “Skinned” myocyte preparations from rodent hearts, in which the sarcolemmal envelope is disrupted through the use of detergents, have been invaluable in providing mechanistic information on the functional consequences of myofilament protein phosphorylation following exposure to neurohormonal stimuli that activate pertinent kinases prior to skinning or direct exposure to such kinases in active form after skinning (for recent examples, see studies on the phosphorylation of cTnI (1–3), cMyBP-C (4–6), and MLC-2 (7–9)). Nevertheless, to date, only a few myofilament proteins have been studied using proteomics (10–19), and a detailed proteomic characterization of the myofilament subproteome and its associated proteins from skinned myocytes has not been performed. In the present analysis, we used an LTQ Orbitrap XL equipped with ETD (20) to analyze the subproteome of skinned cardiomyocytes with or without prior stimulation. Endothelin-1 and isoproterenol were used to activate the endothelin receptor/protein kinase C and β-adrenoreceptor/protein kinase A pathway, respectively (21, 22). Importantly, the mass accuracy of the Orbitrap mass analyzer helped to distinguish true phosphorylation sites from false assignments, and the sensitivity of the ion trap provided novel insights into the translocation of phosphatase regulatory and targeting subunits following β-adrenergic stimulation.     

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a ��-Carotene 15,15��-Dioxygenase

Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved β-carotene at its central double bond (15,15′) to yield two molecules of all-trans-retinal. The molecular mass of the native purified enzyme was ∼64 kDa as a dimer of 32-kDa subunits. The K_m, k_cat, and k_cat/K_m values for β-carotene as substrate were 37 μm, 3.6 min⁻¹, and 97 mm⁻¹ min⁻¹, respectively. The enzyme exhibited the highest activity for β-carotene, followed by β-cryptoxanthin, β-apo-4′-carotenal, α-carotene, and γ-carotene in decreasing order, but not for β-apo-8′-carotenal, β-apo-12′-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₅ seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a β-carotene 15,15′-dioxygenase. Although the Blh protein and β-carotene 15,15′-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian β-carotene 15,15′-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial β-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of β-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian β-carotene 15,15′-monooxygenases (EC 1.14.99.36). β-Carotene is cleaved by the enzyme symmetrically into two molecules of all-trans-retinal, and retinal is then converted to vitamin A in vivo (3–5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15′-double bond of β-carotene to produce β-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These β-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).β-Carotene 15,15′-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of β-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from β-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of β-carotene 15,15′-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert β-carotene to retinal include bacterioopsin-related protein (Brp) and bacteriorhodopsin-related protein-like homolog protein (Blh) (16). Brp protein is expressed from the bop gene cluster, which encodes the structural protein bacterioopsin, consisting of at least three genes as follows: bop (bacterioopsin), brp (bacteriorhodopsin-related protein), and bat (bacterioopsin activator) (17). brp genes were reported in Haloarcula marismortui (18), Halobacterium sp. NRC-1 (19), Halobacterium halobium (17), Haloquadratum walsbyi, and Salinibacter ruber (20). Blh protein is expressed from the proteorhodopsin gene cluster, which contains proteorhodopsin, crtE (geranylgeranyl-diphosphate synthase), crtI (phytoene dehydrogenase), crtB (phytoene synthase), crtY (lycopene cyclase), idi (isopentenyl diphosphate isomerase), and blh gene (21). Sources of blh genes were previously reported in Halobacterium sp. NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). β-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of β-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23–28). Blh protein has been proposed to catalyze or regulate the conversion of β-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.In this study, we used codon optimization to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli, and we performed a detailed biochemical and enzymological characterization of the expressed Blh protein. In addition, the properties of the enzyme were compared with those of mammalian β-carotene 15,15′-monooxygenases.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.