The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the �� Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N^G-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).² Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO^•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO^• and peroxynitrite (ONOO⁻) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO^• (11–13). However, the UV-induced NOS activation and NO^• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α.     

Catalytic Role of the Metal Ion in the Metallo-��-lactamase GOB

Metallo-β-lactamases (MβLs) stand as one of the main mechanisms of bacterial resistance toward carbapenems. The rational design of an inhibitor for MβLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. Here we show that the MβL GOB from Elizabethkingia meningoseptica is active as a monometallic enzyme by using different divalent transition metal ions as surrogates of the native Zn(II) ion. Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give rise to the most active enzymes and are shown to occupy the same binding site as the native ion. However, Zn(II) is the only metal ion capable of stabilizing an anionic intermediate that accumulates during nitrocefin hydrolysis, in which the C–N bond has already been cleaved. This finding demonstrates that the catalytic role of the metal ion in GOB is to stabilize the formation of this intermediate prior to nitrogen protonation. This role may be general to all MβLs, whereas nucleophile activation by a Zn(II) ion is not a conserved mechanistic feature.     

Specific Role of Neuronal Nitric-oxide Synthase when Tethered to the Plasma Membrane Calcium Pump in Regulating the ��-Adrenergic Signal in the Myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates β-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser¹⁶ phospholamban (PLB) phosphorylation as well as Ser²² and Ser²³ cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the β-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.Neuronal nitric-oxide synthase (nNOS)⁵ is involved in a number of key processes in cardiomyocytes including calcium cycling (1), the β-adrenergic contractile response (2, 3), post-infarct left ventricular remodeling (4), and the regulation of redox equilibrium (5). Moreover, a polymorphism in an nNOS-interacting protein, CAPON, has been found to form a quantitative trait for the determination of the QT interval in humans (6), whereas a mutation in α1-syntrophin (SNTA1), another interacting partner of nNOS, has been associated with long QT syndrome (7). The signaling events downstream of the nNOS-CAPON (8) and nNOS-SNTA1 (7) complexes, which are responsible for mediating cardiac repolarization and sodium current respectively, have been elucidated. The nNOS-containing protein complex is therefore of immediate relevance to human pathology.In recent years, we have shown that the sarcolemmal calcium pump, which ejects calcium to the extracellular compartment (reviewed in Refs. 9 and 10), is an important molecule involved in signal regulation and transmission in the heart (11). We have demonstrated that isoform 4b of the sarcolemmal calcium pump (also known as PMCA4b for plasma membrane calcium/calmodulin-dependent ATPase 4b) modulates signaling through a tight molecular interaction with nNOS, leading to the modulation of β-adrenergic responsiveness in the heart (12). However, the events following signaling through the PMCA4b-nNOS complex remain unknown.In myocardial cells, nNOS has been localized to the sarcolemma (13), sarcoplasmic reticulum (2), and mitochondria (14), and translocation between compartments has been demonstrated (15). It has been speculated that these various localizations provide specificity to NO signaling, but the exact mechanisms have yet to be elucidated. In this study, we show a mechanism by which one fraction of nNOS serves highly specific functions through binding to PMCA4b. As PMCA4b is confined to the sarcolemma and is a calcium pump, it is the first identified protein to fulfill these aggregate functions. 1) It acts as an anchoring protein; 2) it regulates nNOS activity; and 3) it modulates a process at the plasma membrane, i.e. β-adrenergic signaling.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Molecular Basis of the Recognition of Nephronectin by Integrin ��8��1

Integrin α8β1 interacts with a variety of Arg-Gly-Asp (RGD)-containing ligands in the extracellular matrix. Here, we examined the binding activities of α8β1 integrin toward a panel of RGD-containing ligands. Integrin α8β1 bound specifically to nephronectin with an apparent dissociation constant of 0.28 ± 0.01 nm, but showed only marginal affinities for fibronectin and other RGD-containing ligands. The high-affinity binding to α8β1 integrin was fully reproduced with a recombinant nephronectin fragment derived from the RGD-containing central “linker” segment. A series of deletion mutants of the recombinant fragment identified the LFEIFEIER sequence on the C-terminal side of the RGD motif as an auxiliary site required for high-affinity binding to α8β1 integrin. Alanine scanning mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a critical motif ensuring the high-affinity integrin-ligand interaction. Although a synthetic LFEIFEIER peptide failed to inhibit the binding of α8β1 integrin to nephronectin, a longer peptide containing both the RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was ∼2,000-fold more potent than a peptide containing only the RGD motif. Furthermore, trans-complementation assays using recombinant fragments containing either the RGD motif or LFEIFEIER sequence revealed a clear synergism in the binding to α8β1 integrin. Taken together, these results indicate that the specific high-affinity binding of nephronectin to α8β1 integrin is achieved by bipartite interaction of the integrin with the RGD motif and LFEIFEIER sequence, with the latter serving as a synergy site that greatly potentiates the RGD-driven integrin-ligand interaction but has only marginal activity to secure the interaction by itself.Integrins are a family of adhesion receptors that interact with a variety of extracellular ligands, typically cell-adhesive proteins in the extracellular matrix (ECM).² They play mandatory roles in embryonic development and the maintenance of tissue architectures by providing essential links between cells and the ECM (1). Integrins are composed of two non-covalently associated subunits, termed α and β. In mammals, 18 α and 8 β subunits have been identified, and combinations of these subunits give rise to at least 24 distinct integrin heterodimers. Based on their ligand-binding specificities, ECM-binding integrins are classified into three groups, namely laminin-, collagen- and RGD-binding integrins (2, 3), of which the RGD-binding integrins have been most extensively investigated. The RGD-binding integrins include α5β1, α8β1, αIIbβ3, and αV-containing integrins, and have been shown to interact with a variety of ECM ligands, such as fibronectin and vitronectin, with distinct binding specificities.The α8 integrin subunit was originally identified in chick nerves (4). Integrin α8β1 is expressed in the metanephric mesenchyme and plays a crucial role in epithelial-mesenchymal interactions during the early stages of kidney morphogenesis. Disruption of the α8 gene in mice was found to be associated with severe defects in kidney morphogenesis (5) and stereocilia development (6). To date, α8β1 integrin has been shown to bind to fibronectin, vitronectin, osteopontin, latency-associated peptide of transforming growth factor-β1, tenascin-W, and nephronectin (also named POEM) (7–13), among which nephronectin is believed to be an α8β1 integrin ligand involved in kidney development (10).Nephronectin is one of the basement membrane proteins whose expression and localization patterns are restricted in a tissue-specific and developmentally regulated manner (10, 11). Nephronectin consists of five epidermal growth factor-like repeats, a linker segment containing the RGD cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor protein-tyrosine phosphatase μ (MAM) domain (see Fig. 3A). Although the physiological functions of nephronectin remain only poorly understood, it is thought to play a role in epithelial-mesenchymal interactions through binding to α8β1 integrin, thereby transmitting signals from the epithelium to the mesenchyme across the basement membrane (10). Recently, mice deficient in nephronectin expression were produced by homologous recombination (14). These nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype reminiscent of α8 integrin knock-out mice (14), despite the fact that other RGD-containing ligands, including fibronectin and osteopontin, were expressed in the embryonic kidneys (9, 15). The failure of the other RGD-containing ligands to compensate for the deficiency of nephronectin in the developing kidneys suggests that nephronectin is an indispensable α8β1 ligand that plays a mandatory role in epithelial-mesenchymal interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its fragments. A, schematic diagrams of full-length nephronectin (NN) and its fragments. RGD-linker and RGD-linker (GST), the central RGD-containing linker segments expressed in mammalian and bacterial expression systems, respectively; PRGDV, a short RGD-containing peptide modeled after nephronectin and expressed as a GST fusion protein (see Fig. 4A for the peptide sequence). The arrowheads indicate the positions of the RGD motif. B, purified recombinant proteins were analyzed by SDS-PAGE in 7–15% gradient (left and center panels) and 12% (right panels) gels, followed by Coomassie Brilliant Blue (CBB) staining, immunoblotting with an anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for blotting with anti-FLAG and PNA) in the left and center panels;1 μg in the right panel. C, recombinant proteins (10 nm) were coated on microtiter plates and assessed for their binding activities toward α8β1 integrin (10 nm) in the presence of 1 mm Mn²⁺. The backgrounds were subtracted as described in the legend to Fig. 2. The results represent the mean ± S.D. of triplicate determinations. D, titration curves of α8β1 integrin bound to full-length nephronectin (NN, closed squares), the RGD-linker segments expressed in 293F cells (RGD-linker, closed triangles) and E. coli (RGD-linker (GST), open triangles), the MAM domain (MAM, closed diamonds), and the PRGDV peptide expressed as a GST fusion protein in E. coli (PRGDV (GST), open circles). The assays were performed as described in the legend to Fig. 2B. The results represent the means of duplicate determinations.Although ligand recognition by RGD-binding integrins is primarily determined by the RGD motif in the ligands, it is the residues outside the RGD motif that define the binding specificities and affinities toward individual integrins (16, 17). For example, α5β1 integrin specifically binds to fibronectin among the many RGD-containing ligands, and requires not only the RGD motif in the 10th type III repeat but also the so-called “synergy site” within the preceding 9th type III repeat for fibronectin recognition (18). Recently, DiCara et al. (19) demonstrated that the high-affinity binding of αVβ6 integrin to its natural ligands, e.g. foot-and-mouth disease virus, requires the RGD motif immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to align the two conserved hydrophobic residues along the length of the helix. Given the presence of many naturally occurring RGD-containing ligands, it is conceivable that the specificities of the RGD-binding integrins are dictated by the sequences flanking the RGD motif or those in neighboring domains that come into close proximity with the RGD motif in the intact ligand proteins. However, the preferences of α8β1 integrin for RGD-containing ligands and how it secures its high-affinity binding toward its preferred ligands remain unknown.In the present study, we investigated the binding specificities of α8β1 integrin toward a panel of RGD-containing cell-adhesive proteins. Our data reveal that nephronectin is a preferred ligand for α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side of its RGD motif serves as a synergy site to ensure the specific high-affinity binding of nephronectin to α8β1 integrin.     

Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to ��-Difluoromethylornithine

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes for ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are enzymes needed for the synthesis of putrescine, spermidine, and spermine, are lethal at early stages of embryonic development (1, 2). There is convincing evidence that the formation of hypusine in eIF5A, which requires spermidine as a precursor, is essential for eukaryotes (3). However, the function(s) of spermine is not so well established. Yeast mutants with inactivated spermine synthase grow at a normal rate (4). Mammalian cells in culture also grow normally in the presence of inhibitors of spermine synthase (5) or after inactivation of the spermine synthase gene (SpmS) (6–8). Inactivation of both of the genes that were originally described as encoding spermine synthases in plants leads to profound developmental defects (9–11), but recently it was discovered that one of these genes actually encodes a thermospermine synthase, and it appears that the lack of thermospermine may be responsible for these defects (12).In contrast, spermine is clearly required for normal development in mammals. The rare human Snyder-Robinson syndrome is caused by mutations in SpmS located in the X chromosome that drastically reduces the amount of spermine synthase (13, 14). This leads to mental retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry, thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X chromosomal deletion that includes SpmS and have no detectable spermine synthase activity, do survive but are only viable on the B6C3H background (15–17). This mouse strain having an X-linked dominant mutation was isolated from a female offspring of an irradiated mouse and was termed gyro (Gy)² based on a circling behavior pattern in affected males (18). Subsequent studies have shown that the Gy mice have a deletion of part of the X chromosome that inactivates both Phex, a gene that regulates phosphate metabolism, and SpmS (16, 19). The lack of SpmS causes a total absence of spermine (6, 7, 15, 16). Such Gy mice suffer from hypophosphatemia, have a greatly reduced size, sterility, and neurological abnormalities, and have a short life span (6, 16, 18). All of these changes except the hypophosphatemia are reversed when spermine synthase activity is restored (20).The original characterization of Gy mice also reported preliminary indications that these mice had hearing defects lacking the Preyer reflex (21, 22). This is of particular interest in the context of polyamine metabolism because a drug, α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine decarboxylase has been shown to cause occasional hearing loss in some patients (23–26). Although DFMO was ineffective for cancer treatment, it is an extremely promising agent for cancer chemoprevention (27, 28). When combined with sulindac, DFMO treatment produced a substantial reduction in the recurrence of colorectal adenomas in a large clinical trial (27). DFMO is a major drug for the treatment of African sleeping sickness caused by Trypanosoma brucei (29, 30). It is also used as a topically applied cream for treatment of unwanted facial hair in women (31, 32). DFMO is generally well tolerated even at high doses, but reversible hearing loss has been reported in multiple clinical trials (25, 33), and a rarer irreversible defect has also been reported (34). These side effects are not observed at lower doses of DFMO (26, 27).Ototoxicity has been demonstrated to occur in experimental animals treated with DFMO including rats (35), guinea pigs (36), gerbils (37), and mice (38). Using immunohistochemistry, a high level of ornithine decarboxylase was observed in the inner ear of the rat, with the highest in the organ of Corti and lateral wall followed by the cochlear nerve (39). Measurements of polyamines in the relevant structures are very difficult due to the small amount of tissue available, but as expected, DFMO treatment reduced polyamine levels and ornithine decarboxylase activity in the inner ear of the guinea pig (36). A plausible explanation for the importance of polyamines in auditory physiology is based on their well documented role as regulators of potassium channels (38). The inward rectification of Kir channels is caused by blockage of the outward current by polyamines (40–42). Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel showed that inward rectification was reduced and that there was a marked reduction in endocochlear potential (EP). It was proposed that DFMO treatment increases the outward Kir4.1 current, resulting in a drop in EP (38).In the experiments reported here, we have studied in more detail the role of polyamines in auditory physiology using Gy mice and crosses of these mice with transgenic CAG/SpmS mice (43). These mice express spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter, which was designed to provide ubiquitous expression (44–46). Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that there was a high level of expression of the transgene in many different organs and that this level was maintained for at least 1 year (43). Our studies confirm that Gy mice are totally deaf and that this condition is reversed by the expression of the SpmS gene. These changes are due to a virtually complete loss of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy mice. Unexpectedly, it was found that these mice show a rapid and profound toxicity to this drug, leading to death within a few days. Within 5 days of exposure to DFMO in the drinking water, the DFMO-treated mice suffered a catastrophic loss of balance due to inner ear effects. This toxicity was also prevented by the transgenic expression of spermine synthase in the Gy background.     

The N-terminal Amphipathic ��-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α₁-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.Type I interferons are the first line of defense against viral infections. The significance of the interferon pathway is illustrated by the susceptibility of interferon signaling mutants to infection and by viral mechanisms that counteract this pathway (1, 2). Although many genes are induced upon interferon stimulation, very few of these genes have been functionally characterized. Viperin is highly induced by both Type I and II interferons and has a broad range of antiviral activity, inhibiting DNA viruses, notably human cytomegalovirus (3); RNA viruses such as influenza, hepatitis C virus (HCV),² and alphaviruses (4-6); and retroviruses such as human immunodeficiency virus (7). Upon expression, viperin localizes to the endoplasmic reticulum (ER), where it interacts with farnesyl-diphosphate synthase, an enzyme involved in lipid biosynthesis. This interaction appears to result in the disruption of lipid raft microdomains and prevention of influenza virus from budding from the plasma membrane (4).Although recent studies have explored the antiviral functions of viperin, the general biochemical properties of this protein remain largely undefined. Viperin is highly conserved across both mammals and lower vertebrates and shares homology with the MoaA family of “radical S-adenosylmethionine” enzymes that bind Fe-S clusters (3, 8). In addition to a putative Fe-S cluster-binding domain, viperin has a 42-amino acid residue N-terminal amphipathic α-helix, and similar domains in other proteins have been shown to bind membranes and induce membrane curvature (9, 10).In this study, we examined the role of the viperin N-terminal α-helical domain in both cellular localization and ER membrane morphology and analyzed the biochemical properties of viperin. We discovered that viperin forms dimers and induces a tightly ordered, visually striking array of ER membranes, known as crystalloid ER(11-13), upon overexpression. In addition, viperin expression impedes the secretion of a variety of soluble proteins. Although the N-terminal amphipathic α-helix is not sufficient to induce crystalloid ER formation, it is both necessary and sufficient to mediate ER localization and to inhibit protein secretion.     

The Novel S527F Mutation in the Integrin ��3 Chain Induces a High Affinity ��IIb��3 Receptor by Hindering Adoption of the Bent Conformation

Three heterozygous mutations were identified in the genes encoding platelet integrin receptor αIIbβ3 in a patient with an ill defined platelet disorder: one in the β3 gene (S527F) and two in the αIIb gene (R512W and L841M). Five stable Chinese hamster ovary cell lines were constructed expressing recombinant αIIbβ3 receptors bearing the individual R512W, L841M, or S527F mutation; both the R512W and L841M mutations; or all three mutations. All receptors were expressed on the cell surface, and mutations R512W and L841M had no effect on integrin function. Interestingly, the β3 S527F mutation produced a constitutively active receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound to non-activated αIIbβ3 receptors carrying the S527F mutation, indicating that the conformation of this receptor was altered and corresponded to the high affinity ligand binding state. In addition, the conformational change induced by S527F was evident from basal anti-ligand-induced binding site antibody binding to the receptor. A molecular model bearing this mutation was constructed based on the crystal structure of αIIbβ3 and revealed that the S527F mutation, situated in the third integrin epidermal growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor from adopting a wild type-like bent conformation. Movement of I-EGF3 into a cleft in the bent conformation may be hampered both by steric hindrance between Phe⁵²⁷ in β3 and the calf-1 domain in αIIb and by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin receptors that consist of noncovalently linked α/β-heterodimers. They are cell-surface receptors that play a role in cell-cell and cell-matrix interactions. Under resting conditions, integrin receptors adopt the low affinity conformation and do not interact with their ligands. Inside-out signaling turns the receptor into a high affinity conformation capable of ligand binding. Ligand binding itself induces additional conformational changes resulting in exposure of neoantigenic sites called ligand-induced binding sites (LIBS)³ and generates in turn outside-in signaling, which triggers a range of downstream signals (1, 2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In flowing blood under resting conditions, αIIbβ3 does not interact with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere at sites of vascular injury and become activated. As a consequence, αIIbβ3 adopts the high affinity conformation and binds fibrinogen. This results in platelet aggregation and thrombus formation, which eventually will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal ligand-binding head domain (the β-propeller domain in the αIIb chain and the βI domain in the β3 chain) standing on two long legs or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial growth factor-like (I-EGF), and β-tail domains in the β3 chain), followed by transmembrane and cytoplasmic domains (1, 2). X-ray crystal structures of the extracellular domain of non-activated αVβ3 revealed that the legs are severely bent, putting the head domain next to the membrane-proximal portions of the legs (4, 5). The bending occurs between I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1 domains in the α-subunit. This bent conformation represents the low affinity state of the receptor. The high affinity state of the receptor is induced by activation and is associated with a large-scale conformational rearrangement in which the integrin extends with a switchblade-like motion (2). Recently, the crystal structure of the entire extracellular domain of αIIbβ3 in its low affinity conformation was resolved and revealed that this integrin also adopts the bent conformation under resting conditions (6). Structural rearrangements in αIIbβ3 between the bent and extended conformations are similar to what has been reported for other integrins (7).We report here that the S527F mutation in the I-EGF3 region of the β3 polypeptide chain of the αIIbβ3 receptor induces a constitutively active receptor adopting an extended high affinity conformation. This was evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding. These data were further corroborated by modeling the replacement of Ser⁵²⁷ with Phe in the crystal structure of the extracellular domain of αIIbβ3. In this model, the S527F mutation decreases the flexibility of I-EGF3 and appears to prevent movement of the lower β-leg into the cleft between the upper β-leg and the lower α-leg. As a consequence, formation of the bent conformation of the non-activated receptor is hampered.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

Novel Role of RanBP9 in BACE1 Processing of Amyloid Precursor Protein and Amyloid �� Peptide Generation

Accumulation of the amyloid β (Aβ) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Aβ generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Aβ generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced β-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Aβ generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.The major defining pathological hallmark of Alzheimer disease (AD)² is the accumulation of amyloid β protein (Aβ), a neurotoxic peptide derived from β- and γ-secretase cleavages of the amyloid precursor protein (APP). The vast majority of APP is constitutively cleaved in the middle of the Aβ sequence by α-secretase (ADAM10/TACE/ADAM17) in the non-amyloidogenic pathway, thereby abrogating the generation of an intact Aβ peptide. Alternatively, a small proportion of APP is cleaved in the amyloidogenic pathway, leading to the secretion of Aβ peptides (37–42 amino acids) via two proteolytic enzymes, β- and γ-secretase, known as BACE1 and presenilin, respectively (1).The proteolytic processing of APP to generate Aβ requires the trafficking of APP such that APP and BACE1 are brought together in close proximity for β-secretase cleavage to occur. We and others have shown that the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytosis receptor (2), binds to APP and alters its trafficking to promote Aβ generation. The loss of LRP substantially reduces Aβ release, a phenotype that is reversed when full-length (LRP-FL) or truncated LRP is transfected in LRP-deficient cells (3, 4). Specifically, LRP-CT lacking the extracellular ligand binding regions but containing the transmembrane domain and the cytoplasmic tail is capable of rescuing amyloidogenic processing of APP and Aβ release in LRP deficient cells (3). Moreover, the LRP soluble tail (LRP-ST) lacking the transmembrane domain and only containing the cytoplasmic tail of LRP is sufficient to enhance Aβ secretion (5). This activity of LRP-ST is achieved by promoting APP/BACE1 interaction (6), although the precise mechanism is unknown. Although we had hypothesized that one or more NPXY domains in LRP-ST might underlie the pro-amyloidogenic processing of APP, we recently found that the 37 C-terminal residues of LRP (LRP-C37) lacking the NPXY motif was sufficient to robustly promote Aβ production independent of FE65 (7). Because LRP-C37 likely acts by recruiting other proteins, we used the LRP-C37 region as bait in a yeast two-hybrid screen, resulting in the identification of 4 new LRP-binding proteins (7). Among these, we focused on Ran-binding protein 9 (RanBP9) in this study, which we found to play a critical role in the trafficking and processing of APP. RanBP9, also known as RanBPM, acts as a multi-modular scaffolding protein, bridging interactions between the cytoplasmic domains of a variety of membrane receptors and intracellular signaling targets. These include Axl and Sky (8), MET receptor protein-tyrosine kinase (9), and β2-integrin LFA-1 (10). Similarly, RanBP9 interacts with Plexin-A receptors to strongly inhibit axonal outgrowth (11) and functions to regulate cell morphology and adhesion (12, 13). Here we show that RanBP9 robustly promotes BACE1 processing of APP and Aβ generation.     

Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

The Block of DNA Polymerase �� Strand Displacement Activity by an Abasic Site Can Be Rescued by the Concerted Action of DNA Polymerase �� and Flap Endonuclease 1

Abasic (AP) sites are very frequent and dangerous DNA lesions. Their ability to block the advancement of a replication fork has been always viewed as a consequence of their inhibitory effect on the DNA synthetic activity of replicative DNA polymerases (DNA pols). Here we show that AP sites can also affect the strand displacement activity of the lagging strand DNA pol δ, thus preventing proper Okazaki fragment maturation. This block can be overcome through a polymerase switch, involving the combined physical and functional interaction of DNA pol β and Flap endonuclease 1. Our data identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism and suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling.Loss of purine and pyrimidine bases is a significant source of DNA damage in prokaryotic and eukaryotic organisms. Abasic (apurinic and apyrimidinic) lesions occur spontaneously in DNA; in eukaryotes it has been estimated that about 10⁴ depurination and 10² depyrimidation events occur per genome per day. An equally important source of abasic DNA lesions results from the action of DNA glycosylases, such as uracil glycosylase, which excises uracil arising primarily from spontaneous deamination of cytosines (1). Although most AP sites are removed by the base excision repair (BER)⁵ pathway, a small fraction of lesions persists, and DNA with AP lesions presents a strong block to DNA synthesis by replicative DNA polymerases (DNA pols) (2, 3). Several studies have been performed to address the effects of AP sites on the template DNA strand on the synthetic activity of a variety of DNA pols. The major replicative enzyme of eukaryotic cells, DNA pol δ, was shown to be able to bypass an AP lesion, but only in the presence of the auxiliary factor proliferating cell nuclear antigen (PCNA) and at a very reduced catalytic efficiency if compared with an undamaged DNA template (4). On the other hand, the family X DNA pols β and λ were shown to bypass an AP site but in a very mutagenic way (5). Recent genetic evidence in Saccharomyces cerevisiae cells showed that DNA pol δ is the enzyme replicating the lagging strand (6). According to the current model for Okazaki fragment synthesis (7–9), the action of DNA pol δ is not only critical for the extension of the newly synthesized Okazaki fragment but also for the displacement of an RNA/DNA segment of about 30 nucleotides on the pre-existing downstream Okazaki fragment to create an intermediate Flap structure that is the target for the subsequent action of the Dna2 endonuclease and the Flap endonuclease 1 (Fen-1). This process has the advantage of removing the entire RNA/DNA hybrid fragment synthesized by the DNA pol α/primase, potentially containing nucleotide misincorporations caused by the lack of a proofreading exonuclease activity of DNA pol α/primase. This results in a more accurate copy synthesized by DNA pol δ. The intrinsic strand displacement activity of DNA pol δ, in conjunction with Fen-1, PCNA, and replication protein A (RP-A), has been also proposed to be essential for the S phase-specific long patch BER pathway (10, 11). Although it is clear that an AP site on the template strand is a strong block for DNA pol δ-dependent synthesis on single-stranded DNA, the functional consequences of such a lesion on the ability of DNA pol δ to carry on strand displacement synthesis have never been investigated so far. Given the high frequency of spontaneous hydrolysis and/or cytidine deamination events, any detrimental effect of an AP site on the strand displacement activity of DNA pol δ might have important consequences both for lagging strand DNA synthesis and for long patch BER. In this work, we addressed this issue by constructing a series of synthetic gapped DNA templates with a single AP site at different positions with respect to the downstream primer to be displaced by DNA pol δ (see Fig. 1A). We show that an AP site immediately upstream of a single- to double-strand DNA junction constitutes a strong block to the strand displacement activity of DNA pol δ, even in the presence of RP-A and PCNA. Such a block could be resolved only through a “polymerase switch” involving the concerted physical and functional interaction of DNA pol β and Fen-1. The closely related DNA pol λ could only partially substitute for DNA pol β. Based on our data, we propose that stalling of a replication fork by an AP site not only is a consequence of its ability to inhibit nucleotide incorporation by the replicative DNA pols but can also stem from its effects on strand displacement during Okazaki fragment maturation. In summary, our data suggest the existence of a novel repair pathway that might be important in preventing replication fork stalling and identify a previously unnoticed deleterious effect of the AP site lesion on normal cell metabolism.Open in a separate windowFIGURE 1.An abasic site immediately upstream of a double-stranded DNA region inhibits the strand displacement activity of DNA polymerase δ. The reactions were performed as described under “Experimental Procedures.” A, schematic representation of the various DNA templates used. The size of the resulting gaps is indicated in nt. The position of the AP site on the 100-mer template strand is indicated relative to the 3′ end. Base pairs in the vicinity of the lesion are indicated by dashes. The size of the gaps (35–38 nt) is consistent with the size of ssDNA covered by a single RP-A molecule, which has to be released during Okazaki fragment synthesis when the DNA pol is approaching the 5′-end of the downstream fragment. When the AP site is covered by the downstream terminator oligonucleotide (Gap-3 and Gap-1 templates) the nucleotide placed on the opposite strand is C to mimic the situation generated by spontaneous loss of a guanine or excision of an oxidized guanine, whereas when the AP site is covered by the primer (nicked AP template), the nucleotide placed on the opposite strand is A to mimic the most frequent incorporation event occurring opposite an AP site. B, human PCNA was titrated in the presence of 15 nm (lanes 2–4 and 10–12) or 30 nm (lanes 6–8 and 14–16) recombinant human four subunit DNA pol δ, on a linear control (lanes 1–8) or a 38-nt gap control (lanes 9–16) template. Lanes 1, 5, 9, and 13, control reactions in the absence of PCNA. C, human PCNA was titrated in the presence of 60 nm DNA pol δ, on a linear AP (lanes 2–4) or 38-nt gap AP (lanes 6–9) template. Lanes 1 and 5, control reactions in the absence of PCNA.     

Engineering of Ion Sensing by the Cystathionine ��-Synthase Module of the ABC Transporter OpuA

We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I_in). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q₁₀ was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state.In their natural habitats microorganisms are often exposed to changes in the concentration of solutes in the environment (1). A sudden increase in the medium osmolality results in loss of water from the cell, loss of turgor, a decrease in cell volume, and an increase in intracellular osmolyte concentration. Osmoregulatory transporters such as OpuA in Lactococcus lactis, ProP in Escherichia coli, and BetP in Corynebacterium glutamicum diminish the consequences of the osmotic stress by mediating the uptake of compatible solutes upon an increase in extracellular osmolality (2–4). For the ATP-binding cassette (ABC)⁵ transporter OpuA, it has been shown that the system, reconstituted in proteoliposomes, is activated by increased concentrations of lumenal ions (increased internal ionic strength) (2, 5, 6). This activation is instantaneous both in vivo and in vitro and only requires threshold levels of ionic osmolytes. Moreover, the ionic threshold for activation is highly dependent of the ionic lipid content (charge density) of the membrane and requires the presence of so-called cystathionine β-synthase (CBS) domains, suggesting that the ionic signal is transduced to the transporter via critical interactions of the protein with membrane lipids.The ABC transporter OpuA consists of two identical nucleotide-binding domains (NBD) fused to CBS domains and two identical substrate-binding domains fused to transmembrane domains. The NBD-CBS and substrate-binding domain-transmembrane domain subunits are named OpuAA and OpuABC, respectively. Two tandem CBS domains are linked to the C-terminal end of the NBD; each domain (CBS1 and CBS2) has a β-α-β-β-α secondary structure (5) (Fig. 1A). The CBS domains are widely distributed in most if not all species of life but their function is largely unknown. Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7–9, 32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg²⁺ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg²⁺ binding or release (10, 11). In general, ABC transporters are functional as dimers, which implies that two tandem CBS domains are present in the OpuA complex. Preliminary experiments with disulfides engineered at the interface of two tandem CBS domains in OpuA suggest that large structural rearrangements (association-dissociation of the interfaces) play a determining role in the ionic strength-regulated transport. Finally, a subset of CBS-containing proteins has a C-terminal extension, which in OpuA is highly anionic (sequence: ADIPDEDEVEEIEKEEENK) and modulates the ion sensing activity (6).Open in a separate windowFIGURE 1.Domain structure of CBS module of OpuA. A, sequence of tandem CBS domains. The predicted secondary structure is indicated above the sequence. The residues modified in this study are underlined. The amino acid sequence end-points of OpuAΔ61 and OpuAΔ119 are indicated by vertical arrows. B, homology model of tandem CBS domain of OpuA. The CBS domains were individually modeled on the crystal structure of the tandem CBS protein Ta0289 from T. acidophilum (PDB entry 1PVM), using Phyre. Ta0289 was used for the initial modeling, because its primary sequence was more similar to the CBS domains of OpuA than those of the other crystallized CBS proteins. The individual domain models were then assembled with reference to the atomic coordinates of the tandem CBS domains of IMPDH from Streptococcus pyogenes (PDB entry 1ZFJ) to form the tandem CBS pair, using PyMOL (DeLano). The positions of the (substituted) cationic residues are indicated.In this study, we have engineered the surface-exposed cationic residues of the CBS module and the C-terminal anionic tail of OpuA (Fig. 1B). The ionic strength and lipid dependence of the OpuA mutants were determined in vivo and in vitro. We show that substitution of five cationic residues for neutral amino acids is sufficient to inactivate the ionic strength sensor and convert OpuA into a constitutively active transporter. Moreover, by substituting six anionic plus four neutral residues of the C-terminal anionic tail for histidines, the transport reaction becomes strongly pH-dependent.