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Recently, substantial levels of acidic D-amino acids, such as D-aspartate and D-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic D-amino acids have multiple physiological significances. Although acidic D-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the D-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic D-amino acids are primarily degraded by the flavoenzyme D-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic D-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic D-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed. Key Points ・Occurrence and distribution ofd-aspartate oxidase. ・Fundamental properties of d -aspartate oxidase of various eukaryotic organisms. ・Biotechnological applications and potential engineering ofd-aspartate oxidase.

Mammalian Na

We investigated if the therapeutic switching of sofalcone (SFC), a gastroprotective agent, to an anticolitic agent is feasible using colon-targeted drug delivery. SFC can activate the anti-inflammatory nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-hemeoxygenase-1 (HO-1) pathway in human colon epithelial cells and murine macrophages. For the efficient treatment of colitis, SFC was coupled with acidic amino acids to yield SFC-aspartic acid (SFC-AA) and SFC-glutamic acid, and their colon targetability and therapeutic effects were assessed as an anticolitic agent in a 2,4-dinitrobenezenesulfonic acid-induced rat colitis model. The SFC derivatives were decoupled up to 72% in the cecal contents but remained stable in the small intestinal contents. Oral gavage of SFC-AA (oral SFC-AA, equivalent to 1.67 mg/kg of SFC) delivered SFC (maximal cecal concentration: 57.36 μM) to the cecum, while no SFC was detected with oral gavage of SFC (oral SFC, 1.67 mg/kg). Moreover, oral SFC-AA (equivalent to 10 mg/kg of SFC) did not afford detectable concentration of SFC in the blood but detected up to 4.64 μM with oral SFC (10 mg/kg), indicating efficient colonic delivery and limited systemic absorption of SFC upon oral SFC-AA. Oral SFC-AA ameliorated colonic damage and inflammation in rat colitis with elevating colonic levels of HO-1 and nuclear Nrf2 protein, and the anticolitic effects of SFC-AA were significantly undermined by an HO-1 inhibitor. At an equivalent dose of SFC, oral SFC-AA but not oral SFC increased colonic HO-1 and nuclear Nrf2 levels, and oral SFC-AA was more effective than oral SFC in treating rat colitis. Moreover, oral SFC-AA was as effective against colitis as oral sulfasalazine being used for the treatment of inflammatory bowel disease. In conclusion, colon-targeted delivery of SFC facilitated the therapeutic switching of the drug to an anticolitic drug via Nrf2 activation.

Adequate protein intake is crucial for the survival and well-being of animals. How animals assess prospective protein sources and ensure dietary amino acid intake plays a critical role in protein homeostasis. By using a quantitative feeding assay, we show that three amino acids, L-glutamate (L-Glu), L-alanine (L-Ala) and L-aspartate (L-Asp), but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promote food consumption in the fruit fly Drosophila melanogaster. This feeding-promoting effect of dietary amino acids is independent of mating experience and internal nutritional status. In vivo and ex vivo calcium imagings show that six brain neurons expressing diuretic hormone 44 (DH44) can be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44+ neurons abolishes the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons is sufficient to promote feeding, suggesting that DH44+ neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining reveal that a putative amino acid transporter, CG13248, is enriched in DH44+ neurons. Knocking down CG13248 expression in DH44+ neurons blocks the increase in food consumption and eliminates calcium responses induced by dietary amino acids. Therefore, these data identify DH44+ neuron as a key sensor to detect amino acids and to enhance food intake via a putative transporter CG13248. These results shed critical light on the regulation of protein homeostasis at organismal levels by the nervous system.

CRSBP-1 (mammalian LYVE-1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF-BB and VEGF-A) containing CRS motifs (clusters of basic amino-acid residues). The ligand binding activities are mediated by Link module and acidic-amino-acid-rich (AAAR) domains, respectively. These CRSBP-1/LYVE-1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild-type mice. We hypothesize that CRSBP-1/LYVE-1 ligands, particularly CRS-containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE-1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE-1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE-1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP-1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF-BB and VEGF-A, respectively, specifically bind to CRSBP-1 but do not interact with either PDGFβR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus (PRV) vaccine in pigs. These results support the notion that LYVE-1 is involved in adaptive immunity in mammals.

ADP-ribosylation, a posttranslational modification catalyzed by a family of enzymes known as poly(ADP- ribose) polymerases (PARPs, 17 in humans), regulates diverse cellular processes. To aid in understanding the functions of ADP-ribosylation in cells, we developed a clickable aminooxy probe, AO-alkyne, which detects ADP-ribosylation of acidic amino acids. AO-alkyne can be used to detect auto-ADP-ribosylation of PARP10 in cells following Cu-catalyzed click conjugation to an azide reporter. This method can be extended to other PARP family members that catalyze ADP-ribosylation on acidic amino acids, providing a convenient and direct readout of PARP activity in cells.

Conserved acidic and aromatic residues in the four sodium channel voltage-sensor domains make domain-specific functional contributions., Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation–pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.

Many ion channels have been shown to be regulated by the membrane signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here, we demonstrate that the binding of PIP(2) to SpIH, a sea urchin hyperpolarization-activated cyclic nucleotide-gated ion channel (HCN), has a dual effect: potentiation and inhibition. The potentiation is observed as a shift in the voltage dependence of activation to more depolarized voltages. The inhibition is observed as a reduction in the currents elicited by the partial agonist cGMP. These two effects were separable and arose from PIP(2) binding to two different regions. Deletion of the C-terminal region of SpIH removed PIP(2)-induced inhibition but not the PIP(2)-induced shift in voltage dependence. Mutating key positively charged amino acids in the C-terminal region adjacent to the membrane selectively disrupted PIP(2)-induced inhibition, suggesting a direct interaction between PIP(2) in the membrane and amino acids in the C-terminal region that stabilizes the closed state relative to the open state in HCN channels.

The P2Y1 receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y1 receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.

In order to investigate the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as a carboxylic acid bioisoster at ionotropic glutamate receptors (iGluRs), a series of acidic alpha-aminocarboxylic acids in which the distal carboxy group was replaced by the 4-hydroxy-1,2,5-oxadiazol-3-yl group was synthesized. Ionization constants were determined. All target compounds, except the Asp analogue 12, were resolved using chiral HPLC. Whereas 12 showed good affinity exclusively at NMDA receptors, the Glu analogue, (+)-10, was an unselective, though potent AMPA receptor preferring agonist (EC(50) = 10 microM at iGluR2) showing only low stereoselectivity. The two higher Glu homologues, (+)-15 and (+)-18, turned out to be weak agonists at iGluR2 as well as weak antagonists at NR1/NR2A, whereas the corresponding (-)-isomers were selective NR1/NR2A antagonists with somewhat higher potency. The results proved the 4-hydroxy-1,2,5-oxadiazol-3-yl moiety to be a useful bioisoster at all three classes of iGluRs, capable of being integrated into agonists as well as antagonists.

Effects of neutralizing individual negatively charged (acidic [E,D]) and innermost positively charged (basic [K,R]) residues in transmembrane segments S2 (D230Q, E240Q), S3 (D263Q) and S4 (K299A/Q, R302A/Q) of the K(V)4.3 putative voltage sensing domain (VSD) were determined. S2 D230Q generated large macroscopic currents, depolarized steady-state activation ("a(4)") and isochronal (1 sec) inactivation ("i") relationships, and significantly accelerated kinetics of deactivation and recovery (from both macroscopic and closed state inactivation [CSI]). D230Q thus stabilized non-inactivated closed states. These effects were attributable to structural perturbations, and indicated D230 is not primarily involved in voltage sensing. Both S2 E240Q and S3 D263Q failed to generate measurable ionic currents, suggesting deletion of negative charges at these putatively more intracellular acidic positions were functionally "lethal" to macroscopic K(V)4.3 function. S4 innermost positive charge deletion mutants K299A/Q and R032A/Q generated functional currents with reduced peak amplitudes. While reduced K299A/Q and R302A/Q currents prevented accurate determination of "a(4)" and estimates of potential electrostatic perturbations, both sets of mutants: (i) depolarized potentials at which currents could be macroscopically detected, consistent with stabilization of closed states (structural perturbations); and (ii) accelerated macroscopic recovery. These results provide further evidence that: (i) basic residues in S4 are involved not only in regulating K(V)4.3 activation and deactivation, but also CSI and recovery; and (ii) suggest putative electrostatic interactions between acidic S2/S3 and basic S4 residues may be different in K(V)4.3 from those proposed to exist in Shaker. Functional implications are discussed.

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A–expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.

Aimed at optional ESR label 2-(4'-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into the guanido of L-Arg-OH, the omega-amino group of L-Lys-OH with methylcarboxyl as a linker, and into the beta-carboxyl of L-Asp-OH and the gamma-carboxyl of L-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR optionally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides confirmed that this strategy was practical.

HERG1 K(+) channels are critical for modulating the duration of the cardiac action potential. The role of hERG1 channels in maintaining electrical stability in the heart derives from their unusual gating properties: slow activation and fast inactivation. HERG1 channel inactivation is intrinsically voltage sensitive and is not coupled to activation in the same way as in the Shaker family of K(+) channels. We recently proposed that the S4 transmembrane domain functions as the primary voltage sensor for hERG1 activation and inactivation and that distinct regions of S4 contribute to each gating process. In this study, we tested the hypothesis that S4 rearrangements underlying activation and inactivation gating may be associated with distinct cooperative interactions between a key residue in the S4 domain (R531) and acidic residues in neighboring regions (S1 - S3 domains) of the voltage sensing module. Using double-mutant cycle analysis, we found that R531 was energetically coupled to all acidic residues in S1-S3 during activation, but was coupled only to acidic residues near the extracellular portion of S2 and S3 (D456, D460 and D509) during inactivation. We propose that hERG1 activation involves a cooperative conformational change involving the entire voltage sensing module, while inactivation may involve a more limited interaction between R531 and D456, D460 and D509.

alpha-Lactalbumin (alpha-La) undergoes considerable structural changes upon loss of bound Ca2+ at acidic pH, leaving alpha-La in a molten globule structure. Using fluorescence the present work provides more insight into the structural transition of alpha-La at acidic pH leading to protein aggregation, most likely caused by a combination of hydrophobic and electrostatic interactions. The rate of aggregation is determined by the protein concentration and temperature applied. Availability of Ca2+ stabilises the protein, and thus prevent aggregation at pH values as low as pH 2.9. In contrast, presence of Cu2+ induces a destabilisation of the protein, which can be explained by a binding to the Zn2+ binding site in alpha-La, possibly resulting in structural alterations of the protein. In general, presence of anions destabilize alpha-La at pH values below pI, with SO4(2-) exhibiting the strongest effect on the protein stability, thus correlating well with the Hofmeister series. At more acidic pH values far from pI, alpha-La becomes more stable towards ion induced aggregation, since higher ion activity is required to efficiently screen the charges on the protein surface. The results presented in this paper provide detailed knowledge on the external parameters leading to aggregation of alpha-La at acidic pH, thus permitting rational design of the aggregation process.

TASK2 is a member of the two-pore domain K + channel family that plays a role in acid–base homeostasis; TASK2 knockout animals have plasma electrolyte patterns typical of the human clinical condition of renal tubular acidosis. It is expressed preferentially in epithelia, including the proximal tubules of the kidney. In common with the other TASK channels, TASK2 is sensitive to changes in extracellular pH, although the molecular mechanism of such pH sensing is not understood. We have examined the role of charged residues in the extracellular domains in pH sensing using a mutational approach. Mutant channels were expressed in CHO cells and studied by whole-cell and single-channel patch clamp. Neutralization of no single amino acid in isolation gave complete loss of pH sensitivity. However, the combined removal of five charged amino acids in the large extracellular loop linking the first transmembrane and pore domains, the M1–P1 loop, resulted in an essentially pH-insensitive channel, stabilized in the open state. Wild-type channels contain two such loops, but a concatemeric construct, comprised of one wild-type subunit and one containing the five mutations, was fully pH-sensitive, indicating that only one M1–P1 loop is required to yield a fully pH-sensitive channel, demonstrating a regulatory role of this distinctive structure in two-pore domain K + channels. Thus, pH sensing in TASK2 channels is conferred by the combined action of several charged residues in the large extracellular M1–P1 loop.

The amino terminus and third loop regions of class B G protein-coupled receptors play critical roles in ligand docking and action. For the prototypic secretin receptor, the hormone amino terminus is spatially approximated with receptor region high in transmembrane segment 6 (TM6), whereas residues ranging from position 6 through 26 label the amino terminus. Here, we focus on the role of charge of the secretin amino terminus, using a series of full-agonist, acetylated probes. Sites of covalent labeling were examined using sequential purification, chemical and enzymatic cleavage, and Edman degradation. High-affinity amino-terminally-blocked probes labeled the distal amino-terminal tail, rather than TM6, while adding a basic residue, again labeled TM6. These data suggest that the secretin amino terminus docks between the amino terminus and TM6 of the receptor, with this region of secretin likely interacting with an acidic residue within the receptor TM6 and the third extracellular loop. To explore this, candidate acidic residues were mutated to Ala (E341A, D342A, E345A, E351A). The E351A mutant markedly interfered with binding, biological activity, and internalization, whereas all others bound secretin and signaled and internalized normally. This supports the possibility that there is a charge-charge interaction between this residue and the amino terminus of secretin that is critical to its normal docking.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the α7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating., This work was supported by Grants BMC2002-00972 and SAF200200209 from the Ministry of Science and Technology of Spain and Grants CTIDIB/2002/138 and GRUPOS03/038 from the Generalitat Valenciana.

Crystals that form in tubular fluid must be retained in the kidney to become stones. Nucleolin-related protein (NRP) is found on the surface of inner medullary collecting duct (IMCD) cells in culture (cIMCD) and selectively adsorbs to calcium oxalate (CaOx). We proposed that NRP mediates attachment to the renal tubular epithelium of Ca stone crystals through an electrostatic interaction with a highly acidic region (acidic fragment [AF]) similar to those of other proteins that have been reported to affect urinary crystal formation. The current studies demonstrate that nucleolin is expressed on both apical and basolateral cell surfaces of cIMCD, reaching a peak in the late stages of mitosis and gradually declining to undetectable levels with maturation of the polarized epithelium. Scraping areas of mature monolayers stimulated the cells surrounding the defects to migrate and proliferate so as to repair them, and these areas demonstrate surface NRP expression and enhanced attachment of CaOx monohydrate crystals. Surface expression of the NRP AF was produced by cloning the NRP AF into a display vector. Transfected cIMCD demonstrating copious surface expression of AF enhanced CaOx attachment 6.7-fold compared with control cIMCD, whereas cells transfected with a vector without the AF did not differ from control. AF was also cloned into a replication-deficient adenovirus and expressed in 293 cells, resulting in AF secretion into the nutrient medium. This medium inhibited CaOx attachment to cIMCD, compared with conditioned medium from cells infected with wild-type virus. These results demonstrate that surface-bound AF can mediate CaOx attachment and that secreted AF can inhibit attachment. These results support the notion that surface-associated NRP could mediate attachment of CaOx to the renal tubule epithelium, thereby causing retention of crystals that might eventually become kidney stones.

The blood-brain barrier (BBB) segregates the circulating blood from interstitial fluid in the brain, and restricts drug permeability into the brain. Our latest studies have revealed that the BBB transporters play important physiological roles in maintaining the brain milieu. The BBB supplies creatine to the brain for an energy-storing system, and creatine transporter localized at the brain capillary endothelial cells (BCECs) is involved in BBB creatine transport. The BBB is involved in the brain-to-blood efflux transport of the suppressive neurotransmitter, gamma-aminobutyric acid, and GAT2/BGT-1 mediates this transport process. BCECs also express serotonin and norepinephrine transporters. Organic anion transporter 3 (OAT3) and ASCT2 are localized at the abluminal membrane of the BCECs. OAT3 is involved in the brain-to-blood efflux of a dopamine metabolite, a uremic toxin and thiopurine nucleobase analogs. ASCT2 plays a role in L-isomer-selective aspartic acid efflux transport at the BBB. Dehydroepiandrosterone sulfate and small neutral amino acids undergo brain-to-blood efflux transport mediated by organic anion transporting polypeptide 2 and ATA2, respectively. The BBB transporters are regulated by various factors, ATA2 by osmolarity, taurine transporter by TNF-alpha, and L-cystine/L-glutamic acid exchange transporter by oxidative stress. Clarifying the physiological roles of BBB transport systems should give us important information allowing the development of better CNS drugs and improving our understanding of the relationship between CNS disorders and BBB function.

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor bound protein 2 (Grb2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferative agents due to its potential to shut down the Ras activation pathway. Generally, the pTyr-X-Asn minimal binding motif is required for a high-affinity ligand binding to the Grb2-SH2 domain. Using phage-display techniques, we discovered a non-pTyr-containing cyclic peptide G1 with moderate binding affinity from 107 different sequences. To understand the structural basis for the high-affinity binding of these novel non-phosphorus-containing inhibitors to the Grb2-SH2 domain, we extensively studied herein the unique functional requirements of the acidic side chain in Tyr-2 position due to the absence of the phosphate group in these non-phosphorylated peptides. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low nanomolar range binding affinity, such as cyclic peptide 20 with an IC 50 =0.026 μ M, which is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. Whole cell assays indicate that peptide 20 can penetrate the cell membranes and inhibit the association of Grb2 with p185erbB2 in erbB2-overexpressing MDA-MA-453 cancer cells at low micromolar concentrations.

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein–protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

There is evidence that ligand binding to and ligand-mediated signaling by the luteinizing hormone receptor (LHR) are influenced by buffer conditions, including ionic type and strength, an issue that becomes important in comparing functional parameters obtained on receptor mutants under different conditions. In order to study this phenomenon, we performed binding (kinetic and saturation) and signaling studies of human chorionic gonadotropin (hCG) with wild type (wt) LHR and several mutants expressed in COS-7 cells using two common buffer systems. One buffer was of low ionic strength and contained a low concentration of Na+, while the other had a near-physiological concentration of Na+. Emphasis was placed on mutations of two amino acid residues in the hinge region of the ectodomain (E332 and D333). It was found that the buffer of higher ionic strength, primarily from Na+, led to an increase of about 4-fold in the Kd of hCG binding to wt and mutant LHRs. The reduced binding affinities were attributable to a comparable reduction in the rate constants of association, with no significant differences in the calculated rate constants of dissociation in the two buffers. Analysis of the signaling properties of these mutants showed that, when corrected for the amount of hCG bound under the conditions of the signaling assay, the maximal ligand-mediated cAMP produced in cells maintained in the buffer of low ionic strength was comparable for wt LHR and the mutants, only the D333A mutant being somewhat elevated. In the buffer of higher ionic strength, however, the response by wt LHR was significantly greater than that of the mutants. These results show that E332 and D333 are important in hormone-mediated signaling, but only in the buffer of higher Na+ concentration. In addition to mutants of these two residues, the buffer of higher ionic strength also led to reduced binding to a number of mutants throughout the receptor. Since these mutants included additional replacements in the ectodomain and transmembrane helices 6 and 7, the general nature of the buffer effect on wt and mutant LHRs suggests that electrostatic effects are contributing to ligand binding and/or that the LHR ectodomain may exist in two conformations, one being more accessible to ligand at reduced ionic strength.

Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pK(a) that varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo.

SummaryRecent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (KD ~ nM range) and recognised sequences V357-F360 on the acidic a1 region and E724-L731 on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC50: 77 nM and 40 nM respectively), and (b) VWF (IC50: 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab’)2 fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.

A simple mix-and-detect photoluminescence method was developed for the turn-on detection of acidic amino acids. To achieve this, graphene quantum dots (GQDs), which emit both down-conversion and up-conversion photoluminescence were prepared by solvothermal synthesis. The carboxylic acid-rich surface not only increases the water solubility of the prepared GQDs, but also makes Eu(3+)-triggered GQDs aggregation possible, thus causing the photoluminescence quenching of GQDs. The quenched photoluminescence can be recovered by the competition between acidic amino acids and GQDs for Eu(3+). Under optimized conditions, sensitive and specific acidic amino acids quantitation can be achieved by utilizing the changes in either down-conversion or up-conversion photoluminescence. Up-conversion mode gives a little lower detection limit than the down-conversion one. Nearly overlapped calibration curves were obtained for the two acidic amino acids, glutamic acid (Glu) and aspartic acid (Asp), thus suggesting that the proposed method can be used not only for the quantitation of individual acidic amino acids, but also for the detection of total amount of them.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

TLRs are divided into two groups based on their subcellular localization patterns. TLR1, 2, 4, 5, and 6 are expressed on the cell surface, whereas the nucleotide-sensing TLRs, such as TLR3, 7, 8, and 9 stay mainly inside cells. The polytopic membrane protein UNC93B1 physically interacts with the nucleotide-sensing TLRs and delivers them from the endoplasmic reticulum to endolysosomes, where the TLRs recognize their ligands and initiate signaling. In cells with nonfunctional UNC93B1, the nucleic acid–sensing TLRs fail to exit the endoplasmic reticulum and consequently do not signal. However, the detailed molecular mechanisms that underlie the UNC93B1-mediated TLR trafficking remain to be clarified. All nucleotide-sensing TLRs contain acidic amino acid residues in the juxtamembrane region between the leucine-rich repeat domain and the transmembrane segment. We show that the D812 and E813 residues of TLR9 and the D699 and E704 residues of TLR3 help to determine the interaction of these TLRs with UNC93B1. Mutation of the acidic residues in TLR3 and TLR9 prevents UNC93B1 binding, as well as impairs TLR trafficking and renders the mutant receptors incapable of transmitting signals. Therefore, the acidic residues in the juxtamembrane region of the nucleotide-sensing TLRs have important functional roles.

Purpose Recent results showed that plasminogen kringle 5 (K5) has improved inhibitory effect on human umbilical vein endothelial cells (HUVECs) viability when 5 acidic amino acids in NH2 terminal outside kringle domain were replaced by 5 serine residues (mutant K5, mK5). This study was designed to identify the enhanced antiangiogenic activity of mK5 in corneal neovascularization (CNV). Methods Alkali burn-induced CNV was induced and treated with K5 and mK5 for 11 days. CNV and inflammation were evaluated by the CNV area and the inflammatory index, respectively. At the end of treatment, the corneas were removed for terminal deoxynucleotidyl transferase dUTP nick end labeling detection and immunohistochemistry. The effects of mK5 and K5 on HUVECs apoptosis were tested by MTT, BrdU, and flow cytometry. The expression levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were detected by Western blot. Results In a rat model of CNV induced by alkali, topical treatment with mK5 significantly decreased the neovascular area and inflammation compared with the wild-type K5-treated group. Meanwhile, mK5 and K5 specifically inhibited the HUVECs proliferation and induced vascular endothelial cell apoptosis in vitro and in vivo, and mK5 exerted higher apoptosis induction. Toward the mechanism of action, both mK5 and K5 significantly upregulated the expression of PEDF and mildly downregulated the expression of VEGF. The elevation of PEDF/VEGF ratio induced by mK5 was higher than that by K5. Conclusions These findings suggest that mK5 has more effective therapeutic potential in CNV than wild-type K5.

Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine–borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus . The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.

This study determined changes in plasma amino acid concentration in late-gestating (beginning 58 ± 1.02 days prior to calving), primiparous, winter-grazing range heifers receiving wheat middling-based supplement without (CON) or with rumen-protected methionine (MET) to provide 15 g DL-MET each day. Plasma was collected on days -2 and 0 (start of MET supplementation just prior to individually receiving supplement at 0700 hours). Plasma was sampled again on days 40, 42 and 44 prior to supplementation at 0700 and 1100 hours (4 h after receiving daily supplement). Data were analyzed with cow as the experimental unit. Continuous variables were analyzed by the main effects of treatment, date, or time and their interaction when appropriate. Comparable BW (P = 0.32) and BCS (P = 0.83) over the 44-day metabolism trial were found between both CON- and MET-fed heifers. MET-supplemented heifers had greater (P < 0.01) plasma concentrations of methionine indicating that the rumen-protection technology successfully delivered methionine to the small intestine. Supplementation with rumen-protected DL-MET caused a significant supplement × date interaction for glutamine (P = 0.03), glycine (P = 0.02), methionine (P < 0.01), and serine (P = 0.05). In addition, trends for supplement × date interactions were detected for leucine (P = 0.07), threonine (P = 0.09), valine (P = 0.08), total amino acids (TAA; P = 0.08), non essential amino acids (NEAA; P = 0.08), branched chain amino acids (BCAA; P = 0.08), and glucogenic amino acids (GLUCO; P = 0.08). These results suggest that the BCAA (leucine and valine) were utilized more efficiently with MET supplemented heifers compared to CON supplemented heifers. Plasma AA concentrations for glutamic acid (P < 0.01), histidine (P = 0.01), tyrosine (P < 0.01), and EAA (P < 0.01), all decreased throughout the study. These results further confirm methionine is a limiting amino acid in forage fed late-gestating heifers and further suggests the limitation when grazing dormant range forages as shown by improved utilization of other plasma amino acids when supplemental methionine was provided.

The P-type ATPases are responsible for the transport of cations across cell membranes. The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) transports two Ca²⁺ ions from the cytoplasm to the lumen of the sarco(endo)plasmic reticulum and countertransports two or three protons per catalytic cycle. Two binding sites for Ca²⁺ ions have been located via protein crystallography, including four acidic amino acid residues that are essential to the ion coordination. In this study, we present molecular dynamics (MD) simulations examining the protonation states of these amino acid residues in a Ca²⁺-free conformation of SERCA. Such knowledge will be important for an improved understanding of atomistic details of the transport mechanism of protons and Ca²⁺ ions. Eight combinations of the protonation of four central acidic residues, Glu309, Glu771, Asp800, and Glu908, are tested from 10 ns MD simulations with respect to protein stability and ability to maintain a structure similar to the crystal structure. The trajectories for the most prospective combinations of protonation states were elongated to 50 ns and subjected to more detailed analysis, including prediction of pK(a) values of the four acidic residues over the trajectories. From the simulations we find that the combination leaving only Asp800 as charged is most likely. The results are compared to available experimental data and explain the observed destabilization upon full deprotonation, resulting in the entry of cytoplasmic K⁺ ions into the Ca²⁺ binding sites during the simulation in which Ca²⁺ ions are absent. Furthermore, a hypothesis for the exchange of protons from the central binding cavity is proposed.

Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143--Gln and Lys192--Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.

The Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding family of monomeric clathrin adaptors (GGAs) is known to bind cargo molecules through short C-terminal peptide motifs conforming to the sequence DXXLL (X = any amino acid), while the heterotetrameric adaptors AP-1 and AP-2 utilize a similar but discrete sorting motif of the sequence [D,E]XXXL[L,I]. While it has been established that a single cargo molecule may contain either or both types of these acidic cluster-dileucine (AC-LL) sorting signals, there are no examples of cargo with overlapping GGA and AP-1/AP-2-binding motifs. In this study, we report that the cytosolic tail of low-density lipoprotein receptor-related protein (LRP)9 contains a bifunctional GGA and AP-1/AP-2-binding motif at its carboxy-terminus (EDEPLL). We further demonstrate that the internal EDEVLL sequence of LRP9 also binds to GGAs in addition to AP-2. Either AC-LL motif of LRP9 is functional in endocytosis. These findings represent the first study characterizing the trafficking of LRP9 and also have implications for the identification of additional GGA cargo molecules.

We have tested an acidic oligopeptide-based targeting system for delivery of enzymes to tissues, especially bone and brain, in a murine mucopolysaccharidosis type VII (MPS VII) model. This strategy is based upon tagging a short peptide consisting of acidic amino acids (AAA) to N terminus of human β-glucuronidase (GUS). The pharmacokinetics, biodistribution, and the pathological effect on MPS VII mouse after 12 weekly infusions were determined for recombinant human untagged and tagged GUS. The tagged GUS was taken up by MPS VII fibroblasts in a mannose 6-phosphate receptor-dependent manner. Intravenously injected AAA-tagged enzyme had five times more prolonged blood clearance compared with the untagged enzyme. The tagged enzyme was delivered effectively to bone, bone marrow, and brain in MPS VII mice and was effective in reversing the storage pathology. The storage in osteoblasts was cleared similarly with both enzyme types. However, cartilage showed a little response to any of the enzymes. The tagged enzyme reduced storage in cortical neurons, hippocampus, and glia cells. A highly sensitive method of tandem mass spectrometry on serum indicated that the concentration of serum dermatan sulfate and heparan sulfate in mice treated with the tagged enzyme decreased more than the untagged enzyme. These preclinical studies suggest that this AAA-based targeting system may enhance enzyme-replacement therapy.

Variants and homologs of bovine pancreatic ribonuclease (RNase A) can exhibit cytotoxic activity. This toxicity relies on cellular internalization of the enzyme. Residues Glu49 and Asp53 form an anionic patch on the surface of RNase A. We find that replacing these two residues with arginine does not affect catalytic activity or affinity for the cytosolic ribonuclease inhibitor (RI) protein. This 'arginine graft' does, however, increase toxicity towards human cancer cells. Appending a nonaarginine domain to this cationic variant results in an additional increase in cytotoxicity, providing one of the most cytotoxic known variants of RNase A. These findings correlate the potency of a ribonuclease with its deliverance of ribonucleolytic activity to the cytosol, and indicate a rational means to enhance the efficacy of ribonucleases and other cytotoxic proteins.

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP(C) and the pathological isoform PrP(Sc). The conformational change of the alpha-helical PrP(C) into beta-sheet-rich PrP(Sc) is the fundamental event of prion disease. The transition of recombinant PrP from a PrP(C)-like into a PrP(Sc)-like conformation can be induced in vitro by submicellar concentrations of SDS. An alpha-helical dimer was identified that might represent either the native state of PrP(C) or the first step from the monomeric PrP(C) to highly aggregated PrP(Sc). In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.

The endocytic trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) involves multiple sorting steps. A cluster of acidic amino acids followed by a dileucine motif in the cytoplasmic tail has been proposed to mediate receptor sorting from the trans Golgi network (TGN) to late endosomes. Mutations in this motif impair lysosomal enzyme sorting by preventing association of CI-MPR with coat proteins. The role of the acidic cluster/dileucine motif in the post-endocytic transport of the receptor was examined using the CI-MPR mutants, AC01 and D160E (Chen HJ, Yuan J, Lobel P. J Biol Chem 1997;272:7003–7012). Following internalization, wild type (WT) CI-MPR is transported through sorting endosomes into the endocytic recycling compartment (ERC), after which it traffics to the TGN and other organelles. However, the mutants localize mostly to the ERC and only a small portion reaches the TGN, suggesting that the sorting of the CI-MPR mutants from the ERC into the TGN is severely impaired. We observed no defect in receptor internalization or in the rate of tail mutant recycling to the cell surface compared to the WT. These results demonstrate that the acidic cluster/dileucine motif of CI-MPR is critical for receptor sorting at early stages of intracellular transport following endocytosis.

Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ( 5 S and 53 S) and at least three minor phosphorylation sites ( 4 S, 7 S, and 59 T) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of 5 S and 53 S by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19–30 and containing 26 S and 27 S), it phosphorylated 26 S efficiently when this peptide contained a chemically synthesized phospho- 27 S modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclin-dependent and Cdc7 kinases is essential for the initiation of DNA replication.

A recent crystal structure, at atomic resolution, of the NO38-core chaperone has revealed a decamer comprised of a dimer of pentamers, with each pentamer consisting of closely coupled eight-stranded beta-barrel monomers. This N-terminal core domain of the chaperone shares the Nucleoplasmin family fold and is presumed to assist the binding of the core histones in their assembly into nucleosomes during DNA replication and repair. The present work provides a measure of the hydrophobic residue burial about the different interfaces and centers of the NO38-core multimeric structure. While the hydrophobic "pentameric ring," comprised of the hydrophobic cores of the monomers and prevalence of non-polar residues at their interfaces is observed, a hydrophobic bias with respect to the center of the pentamer is also found, and consequently also expected to contribute to the thermostability of the multimer. Structural and chromatographic analysis had shown the NO38-core chaperone to bind (H3-H4)2 histone tetramers as well as H2A-H2B dimers. The acidic dipole, which reflects the spatial disposition of the acidic residues of the core monomer points to the lateral region of the monomers comprising the oligomers, and thereby, shows it to be the region of charge that would optimally complement the basic charge of the histones in their electrostatic binding to the chaperone. It is also pointed out that the prevalence of basic residues on the short helices of the histone cores also provides regions of charge that would complement histone binding to the chaperone.

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate approximately 25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated approximately 9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated approximately 2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a K(m) value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than K(i) values (approximately 9-18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341-345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

The translation elongation factor 2 in eukaryotes (eEF-2) contains a unique posttranslationally modified histidine residue, termed diphthamide, which serves as the only target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Diphthamide biosynthesis is carried out by five highly conserved proteins, Dph1 to Dph5, and an as-yet-unidentified amidating enzyme. The evolutionary conservation of the complex diphthamide biosynthesis pathway throughout eukaryotes implies a key role for diphthamide in normal cellular physiology. Of the proteins required for diphthamide synthesis, Dph3 is the smallest, containing only 82 residues. In addition to having a role in diphthamide biosynthesis, Dph3 is also involved in modulating the functions of the Elongator complex in yeast. To explore the physiological roles of Dph3 and to begin to investigate the function of diphthamide, we generated dph3 knockout mice and showed that dph3+/- mice are phenotypically normal, whereas dph3-/- mice, which lack the diphthamide modification on eEF-2, are embryonic lethal. Loss of both dph3 alleles causes a general delay in embryonic development accompanied by lack of allantois fusion to the chorion and increased degeneration and necrosis in neural tubes and is not compatible with life beyond embryonic day 11.5. The dph3-/- placentas also developed abnormally, showing a thinner labyrinth lacking embryonic erythrocytes and blood vessels. These results attest to the physiological importance of Dph3 in development. The biological roles of Dph3 are also discussed.

International audience; Beta-scorpion toxins specifically modulate the voltage dependence of sodium channel activation by acting through a voltage-sensor trapping model. We used mutagenesis, functional analysis and the action of beta-toxin as tools to investigate the existence and role in channel activation of molecular interactions between the charged residues of the S2, S3 and S4 segments in domain II of sodium channels. Mutating to arginine the acidic residues of the S2 and S3 transmembrane segments in domain II, or making charge-reversal mutation of the two outermost gating charges of the IIS4 voltage sensor, shifts the voltage dependence of channel activation to more positive potentials and enhances the effect of beta-scorpion toxin. Thus, mutations of acidic residues in IIS2 and IIS3 segments are able to promote voltage-sensor trapping in a way that is similar to the mutations of the arginines in the IIS4 segment. In order to disclose the network of interactions among acidic and basic residues we performed functional analysis of charge-inversion double mutants: our data suggest that the first arginine of the voltage sensor S4 in domain II (R850) interacts specifically with E805, D814 and E821 in the S2 and S3 segments, whereas the second arginine (R853) only interacts with D827 in the S3 segment. Our results suggest that the S2, S3 and S4 segments in domain II form a voltage-sensing structure, and that molecular interactions between the charged residues of this structure modulate the availability of the IIS4 voltage sensor for trapping by beta-toxins. They also provide unique insights into the molecular events that occur during channel activation, as well as into the structure of the channel.

Ca2+ exerts both a stimulatory and inhibitory effect on type-I IP3R channel activity. However, the structural determinants of Ca2+ sensing in IP3Rs are not fully understood. Previous studies by others have identified eight domains of the type-I IP3R that bind 45Ca2+ when expressed as GST-fusion proteins. We have mutated six highly conserved acidic residues within the second of these domains (aa378-450) in the full-length IP3R and measured the Ca2+ regulation of IP3-mediated Ca2+ release in COS-7 cells. 45Ca2+ flux assays measured with a maximal [IP3] (1 microM) indicate that one of the mutants retained a Ca2+ sensitivity that was not significantly different from control (E411Q), three of the mutants show an enhanced Ca2+ inhibition (D426N, E428Q and E439Q) and two of the mutants were relatively insensitive to Ca2+ inhibition (D442N and D444N). IP3 dose-response relationships indicated that the sensitivity to Ca2+ inhibition and affinity for IP3 were correlated for three of the constructs. Other mutants with enhanced IP3 sensitivity (e.g. R441Q and a type-II/I IP3R chimera) were also less sensitive to Ca2+ inhibition. We conclude that the acidic residues within the aa378-450 segment are unlikely to represent a single functional Ca2+ binding domain and do not contribute to Ca2+ activation of the receptor. The different effects of the mutations may be related to their location within two clusters of acidic residues identified in the crystal structure of the ligand-binding domain [I. Bosanac, J.R. Alattia, T.K. Mal, et al., Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Nature 420 (2002) 696-700]. The data support the view that all IP3R isoforms may display a range of Ca2+ sensitivities that are determined by multiple sites within the protein and markedly influenced by the affinity of the receptor for IP3.

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Coactosin is a small (MW approximately 15 kDa) evolutionarily conserved actin filament binding protein. It displays remote sequence homology to ADF/cofilin proteins and to the ADF-H domains of twinfilin and Abp1/drebrin. However, biochemical analyses have demonstrated that coactosin has a very different role in actin dynamics from the ones of ADF/cofilin, twinfilin or Abp1/drebrin. To elucidate the molecular mechanism of coactosin/actin interaction, we determined the three-dimensional structure of mouse coactosin by multidimensional NMR spectroscopy. We find that the coactosin structure is homologous to ADF/cofilin and to the ADF-H domains of twinfilin. Furthermore, the regions that have been shown to be important for actin filament interactions in ADF/cofilins are structurally conserved in coactosin suggesting that these two proteins interact with F-actin through a conserved interface. Our analysis also identifies key structural differences between these proteins that may account for the differences in biochemical activities and cellular roles of these proteins.