Search

The muscarinic M 2 receptor (M 2 R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M 2 R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M 2 R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at -80 and 0 mV. The acetylcholine EC 50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M 2 R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M 2 R., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A new risk gene associated with amyotrophic lateral sclerosis (ALS) has recently been identified: the Tank-binding kinase 1 (TBK1) gene. Up to now, 90 TBK1 variants have been described in ALS patients with or without frontotemporal dementia (FTD), thus making TBK1 the third or fourth most frequent genetic cause of ALS and FTD. A point mutation analysis in a cohort of 69 Italian ALS patients was performed in order to analyze the frequency of TBK1 mutations and the correlation with clinical phenotypes. The analysis identified the novel variant p.Tyr424Asp in a patient with a rapid progression of the disease. Our data supports the implication of TBK1 in ALS pathogenesis in Italy.

Trueperella pyogenes (T. pyogenes) is an important opportunistic pathogen. Pyolysin (PLO) importantly contributes to the pathogenicity of T. pyogenes. However, the relationship between the structure and function and the virulence of PLO is not well documented. In the current study, recombinant PLO (rPLO) and three rPLO mutants were prepared. rPLO D238R, a mutant with the 238th aspartic acid replaced with an arginine, showed impairment in oligomerization activity on cholesterol-containing liposome and pore-forming activity on sheep red blood cell membrane. Further study employing the prepared mutants confirmed that the pore-forming activity of PLO is essential for inducing excessive inflammation responses in mice by upregulating the expression levels of IL-1β, TNF-α, and IL-6. By contrast, rPLO P499F, another mutant with impaired cell membrane binding capacity, elicited an inflammation response that was dependent on pathogen-associated molecular pattern (PAMP) activity, given that the mutant significantly upregulated the expression of IL-10 in macrophages and in mice, whereas rPLO did not. Results indicated that domain 1 of the PLO molecule plays an important role in maintaining pore-forming activity. Moreover, the PLO pore-forming activity and not PAMP activity is responsible for the inflammation-inducing effect of PLO. The results of this study provided new information for research field on the structure, function, and virulence of PLO., Abbreviations: T. pyogenes: Trueperella pyogenes; PLO: Pyolysin; rPLO: recombinant PLO; PAMP: pathogen-associated molecular pattern; CDCs: cholesterol-dependent cytolysins; PLY: pneumolysin; NLRP3: NLR family pyrin domain containing protein 3; PRRs: pattern recognition receptors; Asp: aspartic acid; TLR4: Toll-like receptor 4; Arg: arginine; Asn: asparagine; IPTG: Isopropyl-β-d-thiogalactoside; PBS: phosphate-buffered saline; sRBCs: sheep red blood cells; TEM: Transmission electron microscopy; RBCM: red blood cell membrane; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NC membrane: nitrocellulose membrane; SDS-AGE: dodecyl sulfate agarose gel electrophoresis; MDBK cells: Madin-Darby bovine kidney cells; RPMI-1640 medium: Roswell Park Memorial Institute-1640 medium; FBS: fetal bovine serum; BMDMs: bone marrow-derived macrophages; TNF-α: tumor necrosis factor α; IL-1β: interleukin-1β; IFN-γ: interferon-γ; TGF-β: transforming growth factor-β; ELISA: enzyme-linked immunosorbent assay.

Background: The amyloid AV-45 (florbetapir) positron emission tomography (PET) has been used in the study of the familial Alzheimer's disease (FAD) with the D678H amyloid precursor protein (APP) mutation. In addition, the progress of the disease remains unknown., Objective: We aim to investigate the progression rate of amyloid accumulation in FAD patients with this mutation by neuroimages analysis., Methods: The clinical course, changes in cognitive function, brain magnetic resonance imaging (MRI) and 18F-AV-45 PET scan were investigated in FAD patients and sporadic AD (sAD) patients. We compared the amyloid deposition pattern in serial brain 18F-AV-45 PET scan among the FAD, familial mild cognitive impairment (FMCI), and sMCI and sAD patients., Results: Seven familial patients received a follow-up survey. The follow up duration for brain AV-45 PET was from 1.54 to 3.61 years. In 4 FMCI patients, an increased regional SUVR was noted, and the annual change rates were increased from 1.03% to 18.82%. However, a decreased regional SUVR was noted in 3 FAD patients and the annual change rates were from -2.62% to -16.03%. As compared with the sAD and sMCI patients, the annual change rate is statistically significant in FAD and FMCI patients respectively., Conclusions: The data indicate a biphasic course with an initial increase and then a decrease of SUVR in brain amyloid PET scan in familial APP mutation patients. The data also reveal that the novel Taiwan APP (D678H) mutation has a more amyloid burden than the sAD patients, particularly in an MCI stage.

Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α -helical content to CaMWt, and only modest differences in α -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N -terminal CaM and CyaA-ACD., Competing Interests: The authors declare no conflict of interest.

Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11 D126N in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11 D122N in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: Mutations in LMNA gene, encoding Lamin A/C, cause a diverse array of phenotypes, collectively referred to as laminopathies. The most common manifestation is dilated cardiomyopathy (DCM), occurring in conjunction with variable skeletal muscle involvement but without involvement of the coronary arteries. Much less commonly, LMNA mutations cause progeroid syndromes, whereby an early-onset coronary artery disease (CAD) is the hallmark of the disease. We report a hitherto unreported compound cardiac phenotype, dubbed as "non-syndromic cardiac progeria", in a young patient who carried a rare pathogenic variant in the LMNA gene and developed progressive degeneration of various cardiac structures, as seen in the elderly. The phenotype resembled the progeroid syndromes, except that it was restricted to the heart and did not involve other organs., Case Presentation: The patient was a well-developed Caucasian female who presented at age 29 years with an acute myocardial infarction (MI) and was found to have extensive CAD. She had none of the conventional risk factors for atherosclerosis. She underwent coronary artery bypass surgery but continued to require multiple percutaneous coronary interventions for symptomatic obstructive coronary lesions. During the course of next 10 years, she developed mitral regurgitation, degenerative mitral and aortic valve diseases, atrial flutter, and progressive conduction defects. She died from progressive heart failure with predominant involvement of the right ventricle and severe tricuspid regurgitation. Cardiac phenotype in this young patient resembled degenerative cardiac diseases of the elderly and the progeroid syndromes. However, in contrast to the progeroid syndromes, the phenotype was restricted to the heart and did not involve other organs. Thus, the phenotype was dubbed as a non-syndromic cardiac progeria. Genetic screening of several cardiomyopathy genes, including LMNA, which is a causal gene for progeroid syndromes, led to identification of a very rare pathogenic p.Asp300Asn variant in the LMNA gene., Conclusions: We infer that the LMNA p.Asp300Asn mutation is pathogenic in non-syndromic cardiac progeria. Mutations involving codon 300 in the LMNA gene have been associated with progeroid syndromes involving multiple organs. Collectively, the data provide credence to the causal role of p.Asp300Asn mutation in the pathogenesis of non-syndromic cardiac progeria.

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [ 3 H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.

Objective: To search for differences in prevalence of a CACNA1E variant between migraine without aura, various phenotypes of migraine with aura, and healthy controls., Background: Familial hemiplegic migraine type 1 (FHM1) is associated with mutations in the CACNA1A gene coding for the alpha 1A (Ca v 2.1) pore-forming subunit of P/Q voltage-dependent Ca 2+ channels. These mutations are not found in the common forms of migraine with or without aura. The alpha 1E subunit (Ca v 2.3) is the counterpart of Ca v 2.1 in R-type Ca 2+ channels, has different functional properties, and is encoded by the CACNA1E gene., Methods: First, we performed a total exon sequencing of the CACNA1E gene in three probands selected because they had no abnormalities in the three FHM genes. In a patient suffering from basilar-type migraine, we identified a single nucleotide polymorphism (SNP) in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760; Minor Allele Frequency 0.2241) hitherto not studied in migraine. In a second step, we determined its occurrence in four groups by direct sequencing on blood genomic DNA: migraine patients without aura (N = 24), with typical aura (N = 55), complex neurological auras (N = 19; hemiplegic aura: N = 15; brain stem aura: N = 4), and healthy controls (N = 102)., Results: The Asp859Glu - rs35737760 SNP of the CACNA1E gene was present in 12.7% of control subjects and in 20.4% of the total migraine group. In the migraine group it was significantly over-represented in patients with complex neurological auras (42.1%), OR 4.98 (95% CI: 1.69-14.67, uncorrected P = .005, Bonferroni P = .030, 2-tailed Fisher's exact test). There was no significant difference between migraine with typical aura (10.9%) and controls., Conclusions: We identified a polymorphism in exon 20 of the CACNA1E gene (Asp859Glu - rs35737760) that is more prevalent in hemiplegic and brain stem aura migraine. This missense variant causes a change from aspartate to glutamate at position 859 of the Ca v 2.3 protein and might modulate the function of R-type Ca 2+ channels. It could thus be relevant for migraine with complex neurological aura, although this remains to be proven., (© 2017 American Headache Society.)

CelA is the most abundant enzyme secreted by Caldicellulosiruptor bescii and has been shown to outperform mixtures of commercially available exo- and endoglucanases in vitro. CelA contains both a glycoside hydrolase family 9 endoglucanase and a glycoside hydrolase family 48 exoglucanase known to be synergistic in their activity, connected by three cellulose-binding domains via linker peptides. Here, repeated aspartate residues were introduced into the N-terminal ends of CelA GH9 and GH48 domains to improve secretion efficiency and/or catalytic efficiency of CelA. Among several constructs, the highest activity on carboxymethylcellulose (CMC), 0.81 ± 0.03 mg/mL was observed for the C. bescii strain containing CelA with 5-aspartate tag at the N-terminal end of GH9 domain-an 82% increase over wild type CelA. In addition, expression of CelA with N-terminal repeated aspartate residues in C. bescii results in a dramatic increase in its ability to grow on Avicel. Biotechnol. Bioeng. 2017;114: 945-950. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The affinity of ligands for G-protein-coupled receptors (GPCRs) is allosterically regulated by Na + via a highly conserved aspartate residue (Asp 2.50 ) in the second transmembrane domain of GPCRs. In the present study, we examined the Na + -mediated regulation of the affinity of ligands for G q/11 -protein-coupled human histamine H 1 receptors in Chinese hamster ovary cells. The affinities of 3 agonists and 20 antihistamines were evaluated by their displacement curves against the binding of [ 3 H]-mepyramine to membrane preparations in the presence or absence of 100mM NaCl. The affinities of most drugs including histamine, an agonist, and d-chlorpheniramine, a first-generation antihistamine, were reduced by NaCl, with the extent of NaCl-mediated changes varying widely between drugs. In contrast, the affinities of some second-generation antihistamines such as fexofenadine were increased by NaCl. These changes were retained in intact cells. The mutation of Asp 2.50 (Asp73) to asparagine abrogated NaCl-induced reductions in affinities for histamine and d-chlorpheniramine, but not NaCl-induced increases in the affinity for fexofenadine. Quantitative structure-activity relationship (QSAR) analyses showed that these Na + -mediated changes were explained and predicted by a combination of the molecular energies and implicit solvation energies of the compounds. These results suggest that Na + diversely regulates the affinity of ligands for H 1 receptors from the extracellular sites of receptors via Asp73-dependent and -independent mechanisms in a manner that depends on the physicochemical properties of ligands. These results may contribute to a deeper understanding of the fundamental mechanisms by which the affinity of ligands for their receptors is allosterically regulated by Na + ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1-cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1-cyclin-B1., Competing Interests: The authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Canavan disease is a leukodystrophy caused by aspartoacylase (ASPA) deficiency. The lack of functional ASPA, an enzyme enriched in oligodendroglia that cleaves N-acetyl-l-aspartate (NAA) to acetate and l-aspartic acid, elevates brain NAA and causes "spongiform" vacuolation of superficial brain white matter and neighboring gray matter. In children with Canavan disease, neuroimaging shows early-onset dysmyelination and progressive brain atrophy. Neuron loss has been documented at autopsy in some cases. Prior studies have shown that mice homozygous for the Aspa nonsense mutation Nur7 also develop brain vacuolation. We now report that numbers of cerebral cortical and cerebellar neurons are decreased and that cerebral cortex progressively thins in Aspa Nur7/Nur7 mice. This neuronal pathology is prevented by constitutive disruption of Nat8l, which encodes the neuronal NAA-synthetic enzyme N-acetyltransferase-8-like., Significance Statement: This is the first demonstration of cortical and cerebellar neuron depletion and progressive cerebral cortical thinning in an animal model of Canavan disease. Genetic suppression of N-acetyl-l-aspartate (NAA) synthesis, previously shown to block brain vacuolation in aspartoacylase-deficient mice, also prevents neuron loss and cerebral cortical atrophy in these mice. These results suggest that lowering the concentration of NAA in the brains of children with Canavan disease would prevent or slow progression of neurological deficits., (Copyright © 2017 the authors 0270-6474/17/370413-09$15.00/0.)

The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O 2 interact, substrate H 2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H 2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N-metal distance of 4.4 Å): its replacement with lysine lowers the H 2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H 2 /D 2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H 2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H 2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O 2 tolerance by stabilizing the oxidized resting Ni III -OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.

Bovine pancreatic ribonuclease A (RNase A) is the monomeric prototype of the so-called secretory 'pancreatic-type' RNase super-family. Like the naturally domain-swapped dimeric bovine seminal variant, BS-RNase, and its glycosylated RNase B isoform, RNase A forms N- and C-terminal 3D domain-swapped oligomers after lyophilization from acid solutions, or if subjected to thermal denaturation at high protein concentration. All mentioned RNases can undergo deamidation at Asn67, forming Asp or isoAsp derivatives that modify the protein net charge and consequently its enzymatic activity. In addition, deamidation slightly affects RNase B self-association through the 3D domain swapping (3D-DS) mechanism. We report here the influence of extensive deamidation on RNase A tendency to oligomerize through 3D-DS. In particular, deamidation of Asn67 alone slightly decreases the propensity of the protein to oligomerize, with the Asp derivative being less affected than the isoAsp one. Contrarily, the additional Asp and/or isoAsp conversion of residues other than N67 almost nullifies RNase A oligomerization capability. In addition, Gln deamidation, although less kinetically favorable, may affect RNase A self-association. Using 2D and 3D NMR we identified the Asn/Gln residues most prone to undergo deamidation. Together with CD spectroscopy, NMR also indicates that poly-deamidated RNase A generally maintains its native tertiary structure. Again, we investigated in silico the effect of the residues undergoing deamidation on RNase A dimers structures. Finally, the effect of deamidation on RNase A oligomerization is discussed in comparison with studies on deamidation-prone proteins involved in amyloid formation., (Copyright © 2016. Published by Elsevier B.V.)

The results of the study reported here are part of an ongoing integrated research programme aimed at producing additional, robust, evidence on the genetic resistance to classical scrapie in goats, with particular reference to codon 146. The study targeted animals aged ⩾6 years, which were born and raised in infected herds and were being culled for management reasons. A total of 556 animals were tested, and all positive animals (n = 117) were of the susceptible NN genotype. A total of 246 goats heterozygous or homozygous for putatively resistant alleles (S146 and D146) were screened with no positive results. The outcome of this study supports the hypothesis that the D146 and S146 alleles could be used as the basis for a nationwide strategy for breeding for resistance in the Cypriot goat population.

The plasma membrane Pma1 H+-ATPase of the yeast Saccharomyces cerevisiae contains conserved residue Asp739 located at the interface of transmembrane segment M6 and the cytosol. Its replacement by Asn or Val (Petrov et al. (2000) J. Biol. Chem., 275, 15709-15716) or by Ala (Miranda et al. (2011) Biochim. Biophys. Acta, 1808, 1781-1789) caused complete blockage of biogenesis of the enzyme, which did not reach secretory vesicles. It was proposed that a strong ionic bond (salt bridge) could be formed between this residue and positively charged residue(s) in close proximity, and the replacement D739A disrupted this bond. Based on a 3D homology model of the enzyme, it was suggested that the conserved Arg811 located in close proximity to Asp739 could be such stabilizing residue. To test this suggestion, single mutants with substituted Asp739 (D739V, D739N, D739A, and D739R) and Arg811 (R811L, R811M, R811A, and R811D) as well as double mutants carrying charge-neutralizing (D739A/R811A) or charge-swapping (D739R/R811D) substitutions were used. Expression of ATPases with single substitutions R811A and R811D were 38-63%, and their activities were 29-30% of the wild type level; ATP hydrolysis and H+ transport in these enzymes were essentially uncoupled. For the other substitutions including the double mutations, the biogenesis of the enzyme was practically blocked. These data confirm the important role of Asp739 and Arg811 residues for the biogenesis and function of the enzyme, suggesting their importance for defining H+ transport determinants but ruling out, however, the existence of a strong ionic bond (salt bridge) between these two residues and/or importance of such bridge for structure-function relationships in Pma1 H+-ATPase.

Breakdown of neuro-glial N-acetyl-aspartate (NAA) metabolism results in the failure of developmental myelination, manifest in the congenital pediatric leukodystrophy Canavan disease caused by mutations to the sole NAA catabolizing enzyme aspartoacylase. Canavan disease is a major point of focus for efforts to define NAA function, with available evidence suggesting NAA serves as an acetyl donor for fatty acid synthesis during myelination. Elevated NAA is a diagnostic hallmark of Canavan disease, which contrasts with a broad spectrum of alternative neurodegenerative contexts in which levels of NAA are inversely proportional to pathological progression. Recently generated data in the nur7 mouse model of Canavan disease suggests loss of aspartoacylase function results in compromised energetic integrity prior to oligodendrocyte death, abnormalities in myelin content, spongiform degeneration, and motor deficit. The present study utilized a next-generation "oligotropic" adeno-associated virus vector (AAV-Olig001) to quantitatively assess the impact of aspartoacylase reconstitution on developmental myelination. AAV-Olig001-aspartoacylase promoted normalization of NAA, increased bioavailable acetyl-CoA, and restored energetic balance within a window of postnatal development preceding gross histopathology and deteriorating motor function. Long-term effects included increased oligodendrocyte numbers, a global increase in myelination, reversal of vacuolation, and rescue of motor function. Effects on brain energy observed following AAV-Olig001-aspartoacylase gene therapy are shown to be consistent with a metabolic profile observed in mild cases of Canavan disease, implicating NAA in the maintenance of energetic integrity during myelination via oligodendroglial aspartoacylase., Competing Interests: Potential Conflicts of Interest AAV Oligo patent (TM, SJG) applied for by UNC. RJS is the founder of AskBio and Bamboo Therapeutics. PL is scientific co-founder of Bamboo Therapeutics and scientific advisory Board member of Agilis Biotherapeutics., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The UDP glucuronosyltransferase (UGT) superfamily comprises glycoproteins that reside in the endoplasmic reticulum membranes and that undergo post-translational modifications (PTMs). UGT2B7 is of particular interest because of its action on a wide variety of drugs. Most studies currently survey common variants and examine only a small fraction of the genetic diversity; however, rare variants (frequency <1%) might have a significant effect because they are predicted to greatly outnumber common variants in the human genome. We discovered a rare single nucleotide UGT2B7 variant of potential pharmacogenetic relevance that encodes a nonconservative amino acid substitution at codon 121. This low-frequency variation, found in two individuals of a population of 305 healthy volunteers, leads to the translation of an asparagine instead of an aspartic acid (UGT2B7 p.D 121 N). This amino acid change was predicted to create a putative N-glycosylation motif NX(S/T) subsequently validated upon endoglycosidase H treatment of microsomal fractions and inhibition of N-glycosylation of endogenously produced UGT2B7 with tunicamycin in human embryonic kidney (HEK293) cells. The presence of an additional N-linked glycan on the UGT2B7 enzyme, likely affecting proper protein folding, resulted in a significant decrease of 49% and 40% in the formation of zidovudine and mycophenolic acid glucuronides, respectively. A systematic survey of the Short Genetic Variations database uncovered 32 rare, naturally occurring missense variations predicted to create or disrupt N-glycosylation sequence motifs in the other UGT2B enzymes. Collectively, these variants have the potential to increase the proportion of variance explained in the UGT pathway resulting from changes in PTMs, such as N-linked glycosylation with consequences on drug metabolism., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate., Competing Interests: The authors declare no conflict of interest.

Cell death and survival signaling pathways have opposed but fundamental functions for various cellular processes and maintain cell homeostasis through cross talk. Here we report a novel mechanism of interaction between these two pathways through the cleavage of RNF31 by caspases. RNF31, a component of the linear ubiquitin chain assembly complex (LUBAC), regulates cell survival by inducing linear ubiquitination of NF-κB signaling components. We found that RNF31 is cleaved under apoptosis conditions through various stimulations. The effector caspases caspase 3 and caspase 6 are responsible for this event, and aspartates 348, 387, and 390 were identified as target sites for this cleavage. Cleavage of RNF31 suppressed its ability to activate NF-κB signaling; thus, mutation of cleavage sites inhibited the induction of apoptosis by treatment with tumor necrosis factor alpha (TNF-α). Our findings elucidate a novel regulatory loop between cell death and the survival signal and may provide guidance for the development of therapeutic strategies for cancers through the sensitization of tumor cells to death-inducing drugs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

TRPA1 (transient-receptor-potential-related ion channel with ankyrin domains) is a direct receptor or indirect effector for a wide variety of nociceptive signals, and thus is a compelling target for development of analgesic pharmaceuticals such as channel blockers. Recently, the structure of TRPA1 was reported, providing insights into channel assembly and pore architecture. Here we report whole-cell and single-channel current recordings of wild-type human TRPA1 as well as TRPA1 bearing point mutations of key charged residues in the outer pore. These measurements demonstrate that the glutamate at position 920 plays an important role in collecting cations into the mouth of the pore, by changing the effective surface potential by ~16 mV, while acidic residues further out have little effect on permeation. Electrophysiology experiments also confirm that the aspartate residue at position 915 represents a constriction site of the TRPA1 pore and is critical in controlling ion permeation., Competing Interests: Authors declare no competing interests.

Misfolding of the prion protein (PrP) is the key step in the transmission of spongiform pathologies in humans and several animals. Although PrP is highly conserved in mammals, a few changes in the sequence of endogenous PrP are proposed to confer protection to dogs, which were highly exposed to prion during the mad-cow epidemics. D159 is a unique amino acid found in PrP from dogs and other canines that was shown to alter surface charge, but its functional relevance has never been tested in vivo. Here, we show in transgenic Drosophila that introducing the N159D substitution on mouse PrP decreases its turnover. Additionally, mouse PrP-N159D demonstrates no toxicity and accumulates no pathogenic conformations, suggesting that a single D159 substitution is sufficient to prevent PrP conformational change and pathogenesis. Understanding the mechanisms mediating the protective activity of D159 is likely to lessen the burden of prion diseases in humans and domestic animals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the study was to evaluate the association between the angiotensin-converting enzyme ACE I/D (rs4340) polymorphism and DNA damage in pati-ents with essential hypertension (EH). The I/D polymorphism of ACE was determined by polymerase chain reaction in 170 male hypertensive patients and 64 normotensive blood donors. We used flow cytometry to determine the levels of cell death, micronuclei and accumulation of peripheral blood leukocytes in G1/G0, S, G2/M phases of the cell cycle. Additionally, the whole blood samples were incubated in vitro at 4 ºC for 24 h to investigate the genotype effects on the susceptibility of cells to DNA damage. We found lower frequency of cells in DNA synthesis S phase and higher levels of micronuclei in the hypertensive compared to normotensive group (p<0.05); increased formation of micronuclei was seen due to elevated micronuclei fre-quencies in patients with the ACE II genotype (p < 0.05), but not in ID or DD genotype carriers. Incubation of whole blood samples of normotensive individuals lead to the most active cell death (p < 0.05) and micronuclei formation (p > 0.05) in the II genotype carriers too. However, hypertensive patients displayed different cellular response to incubation-induced DNA damages in the ACE I/D genotype groups; after incubation, the frequencies of micronuclei were significantly higher in the DD genotype carriers (p < 0.05). To conclude, the study suggests that the ACE I/D polymorphism may contribute to mechanisms and intensity of DNA damages in hypertensive and normotensive individuals.

Although the Oct4/Sox2 complex is crucial for maintaining the pluripotency of stem cells, the molecular basis underlying its regulation during lineage-specific differentiation remains unknown. Here, we revealed that the highly conserved Oct4/Lys-156 is important for maintaining the stability of the Oct4 protein and the intermolecular salt bridge between Oct4/Lys-151 and Sox2/Asp-107 that contributes to the Oct4/Sox2 interaction. Post-translational modifications at Lys-156 and K156N, a somatic mutation detected in bladder cancer patients, both impaired the Lys-151-Asp-107 salt bridge and the Oct4/Sox2 interaction. When produced as a recombinant protein or overexpressed in pluripotent stem cells, Oct4/K156N, with reduced binding to Sox2, significantly down-regulated the stemness genes that are cooperatively controlled by the Oct4/Sox2 complex and specifically up-regulated the mesendodermal genes and the SNAIL family genes that promote the epithelial-mesenchymal transition. Thus, we conclude that Oct4/Lys-156-modulated Oct4/Sox2 interaction coordinately controls the epithelial-mesenchymal transition and mesendoderm specification induced by specific differentiation signals., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5., Competing Interests: There are no conflicts of interest.

Development of potent and isoform selective nitric oxide synthase (NOS) inhibitors is challenging because of the structural similarity in the heme active sites. One amino acid difference between NOS isoforms, Asp597 in rat neuronal NOS (nNOS) versus Asn368 in bovine endothelial NOS (eNOS), has been identified as the structural basis for why some dipeptide amide inhibitors bind more tightly to nNOS than to eNOS. We now have found that the same amino acid variation is responsible for substantially different binding modes and affinity for a new class of aminopyridine-based inhibitors.

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including (13)C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

To favor their replication, viruses express proteins that target diverse mammalian cellular pathways. Due to the limited size of many viral genomes, such proteins are endowed with multiple functions, which require targeting to different subcellular compartments. One salient example is the X protein of Borna disease virus, which is expressed both at the mitochondria and in the nucleus. Moreover, we recently demonstrated that mitochondrial X protein is neuroprotective. In this study, we sought to examine the mechanisms whereby the X protein transits between subcellular compartments and to define its localization signals, to enhance its mitochondrial accumulation and thus, potentially, its neuroprotective activity. We transfected plasmids expressing fusion proteins bearing different domains of X fused to enhanced green fluorescent protein (eGFP) and compared their subcellular localization to that of eGFP. We observed that the 5-16 domain of X was responsible for both nuclear export and mitochondrial targeting and identified critical residues for mitochondrial localization. We next took advantage of these findings and constructed mutant X proteins that were targeted only to the mitochondria. Such mutants exhibited enhanced neuroprotective properties in compartmented cultures of neurons grown in microfluidic chambers, thereby confirming the parallel between mitochondrial accumulation of the X protein and its neuroprotective potential.-Ferré C. A., Davezac, N., Thouard, A., Peyrin, J. M., Belenguer, P., Miquel, M.-C., Gonzalez-Dunia, D., Szelechowski, M. Manipulation of the N-terminal sequence of the Borna disease virus X protein improves its mitochondrial targeting and neuroprotective potential., (© FASEB.)

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome., (© 2016 by The American Society of Hematology.)

Objective: AuMan5A is a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii. To improve its enzymatic properties, we have previously constructed a mutant with loop substitution, AuMan5A/Af, by substituting a loop of seven residues (316KSPDGGN322) in its substrate binding groove with the corresponding region (PSPNDHF) of A. fumigatus GH family 5 β-mannanase. To reveal the correlation between the superior enzymatic properties of AuMan5A/Af and its residue Asp320, site-directed mutagenesis was used to obtain a new mutant enzyme AuMan5A/Af(D320G)., Methods: Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/Af(D320G) by mutating an Asp320 -encoding codon GAC of Auman5A/Af into a Gly320 -encoding GGT. Then, Auman5A/Af(D320G) was extracellularly expressed in Pichia pastoris GS115, and the enzymatic properties of the expressed product were analyzed., Results: Analytical results indicated that the optimal and melting temperature of AuMan5A/Af(D320G) was 70.0 degrees C and 71.5 degrees C, repectively, higher than those of AuMan5A (T(opt) = 65.0 degrees C, T(m) = 64.5 degrees C) and lower than those of AuMan5A/Af (T(opt) = 75.0 degrees C, T(m) =76.6 degrees C); its half-life at 70.0 degrees C was 40 min, 10 min longer than that of AuMan5A but greatly shorter than 480 min of AuMan5A/Af. Besides, its specific activity was 2.7 fold and 0.3 fold that of AuMan5A and AuMan5A/Af, respectively, and its catalytic efficiency (k(cat)/K(m)) was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af., Conclusion: The mutation of ASP320 into Gly320 greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp320 plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.

KCNE β-subunits assemble with and modulate the properties of voltage-gated K(+) channels. In the colon, stomach, and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form K(+) channels important for K(+) and Cl(-) secretion that appear to be voltage-independent. How KCNE3 subunits turn voltage-gated KCNQ1 channels into apparent voltage-independent KCNQ1/KCNE3 channels is not completely understood. Different mechanisms have been proposed to explain the effect of KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE3 affects the voltage sensor S4 and the gate of KCNQ1. We find that S4 moves in KCNQ1/KCNE3 channels, and that inward S4 movement closes the channel gate. However, KCNE3 shifts the voltage dependence of S4 movement to extreme hyperpolarized potentials, such that in the physiological voltage range, the channel is constitutively conducting. By separating S4 movement and gate opening, either by a mutation or PIP2 depletion, we show that KCNE3 directly affects the S4 movement in KCNQ1. Two negatively charged residues of KCNE3 (D54 and D55) are found essential for the effect of KCNE3 on KCNQ1 channels, mainly exerting their effects by an electrostatic interaction with R228 in S4. Our results suggest that KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.

One of the major reasons for age-related cataract formation is an accumulation of insoluble lens proteins. In particular, higher-order α-crystallin aggregates, comprising αA and αB subunits, are insolubilized by the build up of various post-translational modifications over time. Although we previously found an exceptional amount of Asp96 isomerization in αB-crystallin from aged human lens, the biological effect remains unknown. To approximate the effect of Asp 96 isomerization in αB-crystallin, here residues 93-103 of αB-crystallin were chemically synthesized as peptides in which l-α-Asp was replaced with l-β-Asp, D-α-Asp, or D-β-Asp. The resulting peptides were then compared in a biological assay. The results showed that isomerization of Asp 96 altered both the local structure of peptide and its stability against enzymatic digestion. In addition, the synthesized peptides decreased the insoluble fraction of heated α-crystallin. The D-β-Asp-containing peptide further decreased heat-induced precipitation of α-crystallin, and a chaperone assay based on heated alcohol dehydrogenase implied differential interaction of the peptides with substrate depending on the Asp isomer present in each. Our results suggest that the formation of Asp isomers is likely to affect the higher-order oligomer structure of α-crystallin and thereby its chaperone functions in aged lens., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Gene expression of nonsegmented negative-strand RNA viruses (nsNSVs) such as parainfluenza viruses requires the RNA synthesis activity of their polymerase L protein; however, the detailed mechanism of this process is poorly understood. In this study, a parainfluenza minireplicon assay expressing secretory Gaussia luciferase (Gluc) was established to analyze large protein (L) activity. Measurement of Gluc expression in the culture medium of cells transfected with the minigenome and viral polymerase components enabled quick and concise calculation of L activity. By comparing the amino acid sequences in conserved region III (CRIII), a putative polymerase-active domain of the L protein, two strictly conserved aspartates were identified in all families of nsNSV. A series of L mutants from human parainfluenza virus type 2 and parainfluenza virus type 5 showed that these aspartates are necessary for reporter gene expression. It was also confirmed that these aspartates are important for the production of viral mRNA and antigenome cRNA, but not for a polymerase-complex formation. These findings suggest that these two aspartates are key players in the nucleotidyl transfer reaction using two metal ions., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

Asparaginase isozyme II from Escherichia coli is a popular enzyme that has been used as a therapeutic agent against acute lymphoblastic leukemia. Here, fusion tag systems consisting of the pelB signal sequence and various lengths of repeated aspartate tags were devised to highly express and to release active asparaginase isozyme II extracellularly in E. coli. Among several constructs, recombinant asparaginase isozyme II fused with the pelB signal sequence and five aspartate tag was secreted efficiently into culture medium at 34.6 U/mg cell of specific activity. By batch fermentation, recombinant E. coli produced 40.8 U/ml asparaginase isozyme II in the medium. In addition, deletion of the gspDE gene reduced extracellular production of asparaginase isozyme II, indicating that secretion of recombinant asparaginase isozyme II was partially ascribed to the recognition by the general secretion machinery. This tag system composed of the pelB signal peptide, and repeated aspartates can be applied to extracellular production of other recombinant proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Studies of amphioxus Go-opsin have demonstrated that Glu-181 functions as the counterion in this pigment. This finding has led to the proposal that Glu-181 may function as the counterion in other invertebrate visual pigments as well. Here we describe a series of mutagenesis experiments to test this hypothesis and to also test whether other conserved acidic amino acids in Drosophila Rhodopsin 1 (Rh1) may serve as the counterion of this visual pigment. Of the 5 Glu and Asp residues replaced by Gln or Asn in our experiments, none of the mutant pigments shift the absorption of Rh1 by more than 6 nm. In combination with prior studies, these results suggest that the counterion in Drosophila Rh1 may not be located at Glu-181 as in amphioxus, or at Glu-113 as in bovine rhodopsin. Conversely, the extremely low steady state levels of the E194Q mutant pigment (bovine opsin site Glu-181), and the rhabdomere degeneration observed in flies expressing this mutant demonstrate that a negatively charged residue at this position is essential for normal rhodopsin function in vivo. This work also raises the possibility that another residue or physiologic anion may compensate for the missing counterion in the E194Q mutant., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Polymorphisms in the endothelial nitric oxide synthase (eNOS) and in the plasminogen activator inhibitor -1 (PAI-1) genes have been implicated in stroke pathogenesis but results are still controversial. The aim of this study was to examine the possible contribution of Glu298Asp in the eNOS and 4G/5G in the PAI-1polymorphisms with ischemic stroke in a young Mexican population., Materials and Methods: In a case-control study, conducted between January 2006 and June 2010, 204 patients ≤45 years of age with ischemic stroke and 204 controls matched by age and gender, were recruited. The Glu298Asp and 4G/5G polymorphisms were determined in all participants by polymerase chain reaction-restriction fragment length polymorphism., Results: There was a significant difference in the Glu298Asp genotype distribution (P=0.001) and allele frequency between the two groups (P=0.001). The 4G/5G genotype distribution (P=0.40) and the allele frequency was similar between groups; (P=0.13). There were independent factors for ischemic stroke: Asp carriage (GluAsp+AspAsp) (P=0.02); smoking (P=0.01); hypertension (P=0.03), and familial history of atherothrombotic disease (P=0.04)., Conclusions: The Asp allele from the Gu298Asp gene represents an independent risk factor for ischemic stroke in a young Mexican population. In contrast, the 4G/5G was not associated with an increased risk for this disease in the same group of patients, as previously has been demonstrated in other populations.

Background: Apurinic/apyrimidinic endonuclease-1 (APE1) is a rate-limiting enzyme in DNA base excision repair and has been implicated in carcinogenesis. In this study, we summarize available data to examine the susceptibility of APE1 gene Asp148Glu variant to digestive cancer via a meta-analysis., Material and Methods: Study selection and data abstraction were conducted independently by 2 authors. Random-effects model was utilized to pool effect estimates. Heterogeneity and publication bias were addressed., Results: Sixteen articles involving 4916 digestive cancer patients and 7748 controls were qualified for this meta-analysis. Overall association showed an indicative association between Asp148Glu variant and digestive cancer under allelic (odds ratio or OR=1.11; 95% confidence interval or CI: 0.99-1.25; P=0.074) and dominant (OR=1.18; 95% CI: 1.00-1.40; P=0.056) models, with strong evidence of heterogeneity. Deviation from Hardy-Weinberg equilibrium was an obvious source of heterogeneity. In subgroup analyses by cancer sites, this variant was significantly associated with the increased risk for hepatocellular cancer under allelic (OR=1.50; 95% CI: 1.25-1.80; P<0.001) and homozygous genotypic (OR=1.55; 95% CI: 1.02-2.29; P=0.028) models. There were low probabilities of publication bias for the above comparisons., Conclusions: The results of this meta-analysis collectively suggest that APE1 gene Asp148Glu variant is not a risk-conferring factor for digestive cancer. Further large and well-designed studies are required.

Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids., (© 2015 American Society of Plant Biologists. All Rights Reserved.)