Feedback control of Wnt signaling based on ultrastable histidine cluster co-aggregation between Naked/NKD and Axin.

Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the ß-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses.

Histidine in Health and Disease: Metabolism, Physiological Importance, and Use as a Supplement.

L-histidine (HIS) is an essential amino acid with unique roles in proton buffering, metal ion chelation, scavenging of reactive oxygen and nitrogen species, erythropoiesis, and the histaminergic system. Several HIS-rich proteins (e.g., haemoproteins, HIS-rich glycoproteins, histatins, HIS-rich calcium-binding protein, and filaggrin), HIS-containing dipeptides (particularly carnosine), and methyl- and sulphur-containing derivatives of HIS (3-methylhistidine, 1-methylhistidine, and ergothioneine) have specific functions. The unique chemical properties and physiological functions are the basis of the theoretical rationale to suggest HIS supplementation in a wide range of conditions. Several decades of experience have confirmed the effectiveness of HIS as a component of solutions used for organ preservation and myocardial protection in cardiac surgery. Further studies are needed to elucidate the effects of HIS supplementation on neurological disorders, atopic dermatitis, metabolic syndrome, diabetes, uraemic anaemia, ulcers, inflammatory bowel diseases, malignancies, and muscle performance during strenuous exercise. Signs of toxicity, mutagenic activity, and allergic reactions or peptic ulcers have not been reported, although HIS is a histamine precursor. Of concern should be findings of hepatic enlargement and increases in ammonia and glutamine and of decrease in branched-chain amino acids (valine, leucine, and isoleucine) in blood plasma indicating that HIS supplementation is inappropriate in patients with liver disease.

Zinc-Mediated Binding of Nucleic Acids to Amyloid-ß Aggregates: Role of Histidine Residues.

Amyloid-ß peptide (Aß) plays a central role in Alzheimer's disease (AD) pathogenesis. Besides extracellular Aß, intraneuronal Aß (iAß) has been suggested to contribute to AD onset and development. Based on reported in vitro Aß-DNA interactions and nuclear localization of iAß, the interference of iAß with the normal DNA expression has recently been proposed as a plausible pathway by which Aß can exert neurotoxicity. Employing the sedimentation assay, thioflavin T fluorescence, and dynamic light scattering we have studied effects of zinc ions on binding of RNA and single- and double-stranded DNA molecules to Aß42 aggregates. It has been found that zinc ions significantly enhance the binding of RNA and DNA molecules to pre-formed ß-sheet rich Aß42 aggregates. Another type of Aß42 aggregates, the zinc-induced amorphous aggregates, was demonstrated to also bind all types of nucleic acids tested. To evaluate the role of the Aß metal-binding domain's histidine residues in Aß-nucleic acid interactions mediated by zinc, Aß16 mutants with substitutions H6R and H6A-H13A and rat Aß16 lacking histidine residue 13 were used. The zinc-induced interaction of Aß16 with DNA was shown to critically depend on histidine residues 6 and 13. However, the inclusion of H6R mutation in Aß42 peptide did not affect DNA binding to Aß42 aggregates. Since oxidative and/or nitrosative stresses implicated in AD pathogenesis are known to release zinc ions from metallothioneins in cytoplasm and cell nuclei, our findings suggest that intracellular zinc can be an important player in iAß-nucleic acid interactions.

The active transport of histidine and its role in ATP production in Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of T. cruzi, suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in T. cruzi, showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.

Contribution of two conserved histidines to the dual activity of archaeal RNA guide-dependent and -independent pseudouridine synthase Cbf5.

In all organisms, several distinct stand-alone pseudouridine synthase (PUS) family enzymes are expressed to isomerize uridine into pseudouridine (Ψ) by specific recognition of RNAs. In addition, Ψs are generated in Archaea and Eukaryotes by PUS enzymes which are organized as ribonucleoprotein particles (RNP)--the box H/ACA s/snoRNPs. For this modification system, a unique TruB-like catalytic PUS subunit is associated with various RNA guides which specifically target and secure substrate RNAs by base-pairing. The archaeal Cbf5 PUS displays the special feature of exhibiting both RNA guide-dependent and -independent activities. Structures of substrate-bound TruB and H/ACA sRNP revealed the importance of histidines in positioning the target uridine in the active site. To analyze the respective role of H60 and H77, we have generated variants carrying alanine substitutions at these positions. The impact of the mutations was analyzed for unguided modifications U(55) in tRNA and U2603 in 23S rRNA, and for activity of the box H/ACA Pab91 sRNP enzyme. H77 (H43 in TruB), but not H60, appeared to be crucial for the RNA guide-independent activity. In contrast to earlier suggestions, H60 was found to be noncritical for the activity of the H/ACA sRNP, but contributes together with H77 to the full activity of H/ACA sRNPs. The data suggest that a similar catalytic process was conserved in the two divergent pseudouridylation systems.

The dimethylarginine (ADMA)/nitric oxide pathway in the brain and periphery of rats with thioacetamide-induced acute liver failure: Modulation by histidine.

Hepatic encephalopathy (HE) is related to variations in the nitric oxide (NO) synthesis and oxidative/nitrosative stress (ONS), and asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases (NOSs). In the present study we compared the effects of acute liver failure (ALF) in the rat TAA model on ADMA concentration in plasma and cerebral cortex, and on the activity and expression of the ADMA degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), in brain and liver. ALF increased blood and brain ADMA, and the increase was correlated with decreased DDAH activity in both brain and liver. An i.p. administration of histidine (His), an amino acid reported to alleviate oxidative stress associated with HE (100 mg/kg b.w.), reversed the increase of brain ADMA, which was accompanied by the recovery of brain DDAH activity (determined ex vivo), and with an increase of the total NOS activity. His also activated DDAH ex vivo in brain homogenates derived from control and TAA rats. ALF in this model was also accompanied by increases of blood cyclooxygenase activity and blood and brain TNF-α content, markers of the inflammatory response in the periphery, but these changes were not affected by His, except for the reduction of TNF-α mRNA transcript in the brain. His increased the total antioxidant capacity of the brain cortex, but not of the blood, further documenting its direct neuroprotective power.

Extended secondary structures in proteins.

Super secondary structures of proteins have been systematically searched and classified, but not enough attention has been devoted to such large edifices beyond the basic identification of secondary structures. The objective of the present study is to show that the association of secondary structures that share some of their backbone residues is a commonplace in globular proteins, and that such deeper fusion of secondary structures, namely extended secondary structures (ESSs), helps stabilize the original secondary structures and the resulting tertiary structures. For statistical purposes, a set of 163 proteins from the protein databank was randomly selected and a few specific cases are structurally analyzed and characterized in more detail. The results point that about 30%of the residues from each protein, on average, participate in ESS. Alternatively, for the specific cases considered,our results were based on the secondary structures produced after extensive Molecular Dynamics simulation of a protein­aqueous solvent system. Based on the very small width of the time distribution of the root mean squared deviations, between the ESS taken along the simulation and the ESS from the mean structure of the protein, for each ESS, we conclude that the ESSs significantly increase the conformational stability by forming very stable aggregates.The ubiquity and specificity of the ESS suggest that the role they play in the structure of proteins, including the domains formation, deserves to be thoroughly investigated.

Characterization of singlet oxygen production and its involvement in photodamage of Photosystem II in the cyanobacterium Synechocystis PCC 6803 by histidine-mediated chemical trapping.

Singlet oxygen production in intact cells of the cynobacterium Synechocystis 6803 was studied using chemical trapping by histidine, which leads to O2 uptake during illumination. The rate of O2 uptake, measured by a standard Clark-type electrode, is enhanced in the presence of D2O, which increases the lifetime of (1)O2, and suppressed by the (1)O2 quencher NaN3. Due to the limited mobility of (1)O2 these data demonstrate that exogenous histidine reaches close vicinity of (1)O2 production sites inside the cells. Flash induced chlorophyll fluorescence measurements showed that histidine does not inhibit Photosystem II activity up to 5mM concentration. By applying the histidine-mediated O2 uptake method we showed that (1)O2 production linearly increases with light intensity even above the saturation of photosynthesis. We also studied (1)O2 production in site directed mutants in which the Gln residue at the 130th position of the D1 reaction center subunit was changed to either Glu or Leu, which affect the efficiency of nonradiative charge recombination from the primary radical pair (Rappaport et al. 2002, Biochemistry 41: 8518-8527; Cser and Vass 2007, BBA 1767:233-243). We found that the D1-Gln130Glu mutant showed decreased (1)O2 production concomitant with decreased rate of photodamage relative to the WT, whereas both (1)O2 production and photodamage were enhanced in the D1-Gln130Leu mutant. The data are discussed in the framework of the model of photoinhibition in which (3)P680 mediated (1)O2 production plays a key role in PSII photodamage, and nonradiative charge recombination of the primary charge separated state provides a photoprotective pathway.

Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.

The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow performance. The experiment was conducted for 12 wk with 48 Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: -317 g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3 kg/d, respectively). Milk yield was decreased by DMP (35.2 kg/d), but was similar to ADMP (38.8 kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3 ± 0.75 kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased DMI and milk yield in dairy cows. Supplementation of the MP-deficient diet with RPLys and RPMet diminished the difference in DMI and milk yield compared with ADMP and additional supplementation with RPHis eliminated it. As total-tract fiber digestibility was decreased with the DMP diets, but DMI tended to increase with RP AA supplementation, we propose that, similar to monogastric species, AA play a role in DMI regulation in dairy cows. Our data implicate His as a limiting AA in high-producing dairy cows fed corn silage- and alfalfa haylage-based diets, deficient in MP. The MP-deficient diets clearly increased milk N efficiency and decreased dramatically urinary N losses.

Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases.

STUDY OBJECTIVES: Neuronal histamine shows diurnal rhythms in rodents and plays a major role in the maintenance of vigilance. No data are available on its diurnal fluctuation in humans, either in health or in neurodegenerative disorders such as Parkinson disease (PD), Alzheimer disease (AD), or Huntington disease (HD), all of which are characterized by sleep-wake disturbances. DESIGN: Quantitative in situ hybridization was used to study the mRNA expression of histidine decarboxylase (HDC), the key enzyme of histamine production in the tuberomammillary nucleus (TMN) in postmortem human hypothalamic tissue, obtained from 33 controls and 31 patients with a neurodegenerative disease-PD (n = 15), AD (n = 9), and HD (n = 8)-and covering the full 24-h cycle with respect to clock time of death. RESULTS: HDC-mRNA levels in controls were found to be significantly higher during the daytime than at night (e.g., 08:01-20:00 versus 20:01-08:00, P = 0.004). This day-night fluctuation was markedly different in patients with neurodegenerative diseases. CONCLUSION: The diurnal fluctuation of HDC-mRNA expression in human TMN supports a role for neuronal histamine in regulating day-night rhythms. Future studies should investigate histamine rhythm abnormalities in neurodegenerative disorders. CITATION: Shan L; Hofman MA; van Wamelen DJ; Van Someren EJW; Bao AM; Swaab DF. Diurnal fluctuation in histidine decarboxylase expression, the rate limiting enzyme for histamine production, and its disorder in neurodegenerative diseases.

Role of individual histidines in the pH-dependent global stability of human chloride intracellular channel 1.

Chloride intracellular channel proteins exist in both a soluble cytosolic form and a membrane-bound form. The mechanism of conversion between the two forms is not properly understood, although one of the contributing factors is believed to be the variation in pH between the cytosol (~7.4) and the membrane (~5.5). We systematically mutated each of the three histidine residues in CLIC1 to an alanine at position 74 and a phenylalanine at positions 185 and 207. We examined the effect of the histidine-mediated pH dependence on the structure and global stability of CLIC1. None of the mutations were found to alter the global structure of the protein. However, the stability of H74A-CLIC1 and H185F-CLIC1, as calculated from the equilibrium unfolding data, is no longer dependent on pH because similar trends are observed at pH 7.0 and 5.5. The crystal structures show that the mutations result in changes in the local hydrogen bond coordination. Because the mutant total free energy change upon unfolding is not different from that of the wild type at pH 7.0, despite the presence of intermediates that are not seen in the wild type, we propose that it may be the stability of the intermediate state rather than the native state that is dependent on pH. On the basis of the lower stability of the intermediate in the H74A and H185F mutants compared to that of the wild type, we conclude that both His74 and His185 are involved in triggering the pH changes to the conformational stability of wild-type CLIC1 via their protonation, which stabilizes the intermediate state.

Role of histidine 225 in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

Pyridoxal 5'-phosphate (PLP), in the active site of ornithine 4,5-aminomutase (OAM), forms a Schiff base with N(δ) of the d-ornithine side chain and facilitates interconversion of the amino acid to (2R, 4S) 2,4-diaminopentanoic acid via a radical-based mechanism. The crystal structure of OAM reveals that His225 is within hydrogen bond distance to the PLP phenolic oxygen, and may influence the pK(a) of the Schiff base during radical rearrangement. To evaluate the role of His225 in radical stabilization and catalysis, the residue was substituted with a glutamine and alanine. The H225Q and H225A variants have a 3- and 10-fold reduction in catalytic turnover, respectively, and a decrease in catalytic efficiency (7-fold for both mutants). Diminished catalytic performance is not linked to an increase in radical-based side reactions leading to enzyme inactivation. pH-dependence studies show that k(cat) increases with the ionization of a functional group, but it is not attributed to His225. Binding of 2,4-diaminobutyric acid to native OAM leads to formation of an overstabilized 2,4-diaminobutyryl-PLP derived radical. In the H225A and the H225Q mutants, the radical forms and then decays, as evidenced by accumulation of cob(III)alamin. From these data, we propose that His225 enhances radical stability by acting as a hydrogen bond acceptor to the phenolic oxygen, which favors the deprotonated state of the imino nitrogen and leads to greater resonance stabilization of the 2,4-diaminobutyryl-PLP radical intermediate. The potential role of His225 in lowering the activation energy barrier to mediate PLP-dependent radical rearrangement is discussed.

Assignment of function to histidines 260 and 298 by engineering the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase complex; substitutions that lead to acceptance of substrates lacking the 5-carboxyl group.

The first component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the "gatekeeper" for specificity for these two multienzyme complexes in bacteria, E1p for pyruvate but E2o for 2-oxoglutarate.

Residues R282 and D341 act as electrostatic gates in the proton-dependent oligopeptide transporter PepT1.

The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.

Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR.

The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip-assisted imidazole deprotonation is the rate-limiting step.

Solution structure of physiological Cu(His)2: novel considerations into imidazole coordination.

A disagreement on the mode of histidine binding to copper and the structure of [Cu(2+)(His)(2)] in solution still exists. Spectroscopic data in solution support a six-coordinate species with N4O2 donor atoms, while X-ray crystallography reveals five-coordinate N(3)O(2) donor atoms. We modified [Cu(2+)(His)(2)] in solution using diethyl pyrocarbonate (DEPC) and monitored the products spectrophotometrically and by mass spectrometry. Our spectrophotometric study indicates the presence of a free imidazole in the [Cu(2+)(His)(2)] complex in solution. Mass spectral characterization of a DEPC-modified [Cu(2+)(His)(2)] complex yielded a peak at 587.8 amu corresponding to three DEPC adducts. Taken together, our data indicate that the [Cu(2+)(His)(2)] complex in solution exists as a neutral five-coordinate structure with N3O2 donor atoms.

Extracellular acidification exerts opposite actions on TREK1 and TREK2 potassium channels via a single conserved histidine residue.

Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

Molecular basis of multidrug transport by ABC transporters.

Multidrug ABC transporters such as the human multidrug resistance P-glycoprotein (ABCB1) play an important role in the extrusion of drugs from the cell and their overexpression can be a cause of failure of anticancer and antimicrobial chemotherapy. These transport systems contain two nucleotide-binding domains (NBDs) where ATP is bound and hydrolyzed and two membrane domains (MDs) which mediate vectorial transport of substrates across the cell membrane. Recent crystal structures of the bacterial ABCB1 homologues Sav1866 from Staphylococcus aureus and MsbA from Salmonella typhimurium and other organisms shed light on the possible conformational states adopted by multidrug ABC transporters during transport. These structures help to interpret cellular and biochemical data gathered on these transport proteins over the past three decades. However, there are contradictory views on how the catalytic cycle of ATP binding and hydrolysis by the NBDs is linked to the change in drug binding affinity at the MDs, which underlies the capture (high affinity) of the transported drug on one side of the membrane and its release (low affinity) on the other. This review provides an overview of the current evidence for the different transport models and establishes the most recent structure-function relationships in multidrug ABC transporters.