Vesicular Inhibitory Amino Acid Transporter Is a Cl?/γ-Aminobutyrate Co-transporter

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of γ-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Δψ) (positive inside) but not ΔpH. VIAAT-mediated GABA uptake had an absolute requirement for Cl⁻ and actually accompanied Cl⁻ movement. Kinetic analysis indicated that one GABA molecule and two Cl⁻ equivalents were transported during one transport cycle. VIAAT in which Glu²¹³ was specifically mutated to alanine completely lost the ability to take up both GABA and Cl⁻. Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl⁻ transporter that co-transports Cl⁻ with GABA or glycine in a Δψ dependent manner. It is concluded that Cl⁻ plays an essential role in vesicular storage of GABA and glycine.     

Amino acid requirements for growth of the rabbit blastocyst in vitro

Ten amino acids, namely, arginine, histidine, lysine, tryptophane, methionine, phenylalanine, leucine, valine, threonine and serine were indispensable for growth of rabbit blastocysts in vitro; others were nonessential. Of all the essential amino acids, arginine and lysine were required in relatively high concentrations, 10^?2 M and 10^?3 M, respectively, for optimum growth. Complete omission of the non-essential amino acids from the medium markedly reduced blastocyst growth. Interaction between serine and glycine demonstrated a partial sparing action on serine by glycine, similar to that observed between methionine and cysteine. The amino acid composition of a culture medium capable of providing continuous and consistent growth of rabbit blastocysts in vitro is described.     

Glyphosate Resistance by Engineering the Flavoenzyme Glycine Oxidase

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and d-isomer of amino acids to yield the corresponding α-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the k_cat/K_m ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the α2-α3 loop (comprising residues 50–60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg⁵⁴ could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide.     

4-Carboxy-4-hydroxy-2-aminoadipic acid and other acidic amino acids in Caylusea abyssinica

Two diastereoisomers of 4-carboxy-4-hydroxy-2-aminoadipic acid have been isolated from leaves and inflorescences of Caylusea abyssinica. Green parts of the plant also contain appreciable amounts of the two diastereoisomers of 4-hydroxy-4-methylglutamic acid, 3-(3-carboxyphenyl)alanine, (3-carboxyphenyl)glycine, 3-(3-carboxy-4-hydroxyphenyl)alanine, (3-carboxy-4-hydroxyphenyl)glycine and in low concentration 2-aminoadipic acid, saccharopine [(2S, 2′S)-N⁶-(2-glutaryl)lysine] and some γ-glutamyl peptides. The acidic amino acids were separated from other amino acids on an Ecteola ion exchange column with M pyridine as eluant.     

Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation

Background

FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH^−/− mice.

Methodology/Principal Findings

FAAH^−/− mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH^−/− mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2–3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH^−/− mice. Dysregulated hepatic FAAH^−/− lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH^−/− acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH^−/− mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH^−/− mice was consistent with a compensating contribution from decreased acetylation of fed FAAH^−/− aldolase B. Fed FAAH^−/− alcohol dehydrogenase (ADH) acetylation was also decreased.

Conclusions/Significance

Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH^−/− mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver''s role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models.     

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C₂₇ and C₂₄ intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C₂₆ sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.     

Is the simple auger coring method reliable for below-ground standing biomass estimation in Eucalyptus forest plantations?

Background and Aims

Despite their importance for plant production, estimations of below-ground biomass and its distribution in the soil are still difficult and time consuming, and no single reliable methodology is available for different root types. To identify the best method for root biomass estimations, four different methods, with labour requirements, were tested at the same location.

Methods

The four methods, applied in a 6-year-old Eucalyptus plantation in Congo, were based on different soil sampling volumes: auger (8 cm in diameter), monolith (25 × 25 cm quadrate), half Voronoi trench (1·5 m³) and a full Voronoi trench (3 m³), chosen as the reference method.

Key Results

With the reference method (0–1m deep), fine-root biomass (FRB, diameter <2 mm) was estimated at 1·8 t ha⁻¹, medium-root biomass (MRB diameter 2–10 mm) at 2·0 t ha⁻¹, coarse-root biomass (CRB, diameter >10 mm) at 5·6 t ha⁻¹ and stump biomass at 6·8 t ha⁻¹. Total below-ground biomass was estimated at 16·2 t ha⁻¹ (root : shoot ratio equal to 0·23) for this 800 tree ha⁻¹ eucalypt plantation density. The density of FRB was very high (0·56 t ha⁻¹) in the top soil horizon (0–3 cm layer) and decreased greatly (0·3 t ha⁻¹) with depth (50–100 cm). Without labour requirement considerations, no significant differences were found between the four methods for FRB and MRB; however, CRB was better estimated by the half and full Voronoi trenches. When labour requirements were considered, the most effective method was auger coring for FRB, whereas the half and full Voronoi trenches were the most appropriate methods for MRB and CRB, respectively.

Conclusions

As CRB combined with stumps amounted to 78 % of total below-ground biomass, a full Voronoi trench is strongly recommended when estimating total standing root biomass. Conversely, for FRB estimation, auger coring is recommended with a design pattern accounting for the spatial variability of fine-root distribution.     

Uptake of aluminium into Arabidopsis root cells measured by fluorescent lifetime imaging

Background and Aims

Measuring the Al³⁺ uptake rate across the plasma membrane of intact root cells is crucial for understanding the mechanisms and time-course of Al toxicity in plants. However, a reliable method with the sufficient spatial and temporal resolution to estimate Al³⁺ uptake in intact root cells does not exist.

Methods

In the current study, fluorescent lifetime imaging (FLIM) analysis was used to quantify Al³⁺ uptake in the root-cell cytoplasm in vivo. This was performed via the estimation of the fluorescence lifetime of Al–lumogallion {5-chloro-3[(2,4-dihydroxyphenyl)azo]-2-hydroxybenzenesulfonic acid} complexes and measurements of intracellular pH while exposing arabidopsis seedlings to acidic and Al³⁺ stresses.

Key Results

The lifetime of Al–lumogallion complexes fluorescence is pH-dependent. The primary sites for Al³⁺ entry are the meristem and distal elongation zones, while Al³⁺ uptake via the cortex and epidermis of the mature root zone is limited. The maximum rates of Al uptake into the cytoplasm (2–3 µmol m⁻³ min⁻¹ for the meristematic root zone and 3–7 µmol m⁻³ min⁻¹ for the mature zone) were observed after a 30-min exposure to 100 µm AlCl₃ (pH 4·2). Intracellular Al concentration increased to 0·4 µm Al within the first 3 h of exposure to 100 µm AlCl₃.

Conclusions

FLIM analysis of the fluorescence of Al–lumogallion complexes can be used to reliably quantify Al uptake in the cytoplasm of intact root cells at the initial stages of Al³⁺ stress.Key words: Acid stress, Al³⁺, aluminium toxicity, Arabidopsis thaliana, low pH, fluorescent lifetime imaging (FLIM), lumogallion     

Beneficial Effect of Acetic Acid on the Xylose Utilization and Bacterial Cellulose Production by Gluconacetobacter xylinus

In this work, acetic acid was found as one promising substrate to improve xylose utilization by Gluconacetobacter xylinus CH001. Also, with the help of adding acetic acid into medium, the bacterial cellulose (BC) production by G. xylinus was increased significantly. In the medium containing 3 g l⁻¹ acetic acid, the optimal xylose concentration for BC production was 20 g l⁻¹. In the medium containing 20 g l⁻¹ xylose, the xylose utilization and BC production by G. xylinus were stimulated by acetic acid within certain concentration. The highest BC yield (1.35 ± 0.06 g l⁻¹) was obtained in the medium containing 20 g l⁻¹ xylose and 3 g l⁻¹ acetic acid after 14 days. This value was 6.17-fold higher than the yield (0.21 ± 0.01 g l⁻¹) in the medium only containing 20 g l⁻¹ xylose. The results analyzed by FE-SEM, FTIR, and XRD showed that acetic acid affected little on the microscopic morphology and physicochemical characteristics of BC. Base on the phenomenon observed, lignocellulosic acid hydrolysates (xylose and acetic acid are main carbon sources present in it) could be considered as one potential substrate for BC production.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

Pharmacological PPARα Activation Markedly Alters Plasma Turnover of the Amino Acids Glycine,Serine and Arginine in the Rat

The current study extends previously reported PPARα agonist WY 14,643 (30 µmol/kg/day for 4 weeks) effects on circulating amino acid concentrations in rats fed a 48% saturated fat diet. Steady-state tracer experiments were used to examine in vivo kinetic mechanisms underlying altered plasma serine, glycine and arginine levels. Urinary urea and creatinine excretion were measured to assess whole-body amino acid catabolism. WY 14,643 treated animals demonstrated reduced efficiency to convert food consumed to body weight gain while liver weight was increased compared to controls. WY 14,643 raised total amino acid concentration (38%), largely explained by glycine, serine and threonine increases. ³H-glycine, ¹⁴C-serine and ¹⁴C-arginine tracer studies revealed elevated rates of appearance (R_a) for glycine (45.5±5.8 versus 17.4±2.7 µmol/kg/min) and serine (21.0±1.4 versus 12.0±1.0) in WY 14,643 versus control. Arginine was substantially decreased (−62%) in plasma with estimated R_a reduced from 3.1±0.3 to 1.2±0.2 µmol/kg/min in control versus WY 14,643. Nitrogen excretion over 24 hours was unaltered. Hepatic arginase activity was substantially decreased by WY 14,643 treatment. In conclusion, PPARα agonism potently alters metabolism of several specific amino acids in the rat. The changes in circulating levels of serine, glycine and arginine reflected altered fluxes into the plasma rather than changes in clearance or catabolism. This suggests that PPARα has an important role in modulating serine, glycine and arginine de novo synthesis.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm×75 μm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10⁻¹⁰ M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-μl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, γ-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Cytosolic Phospholipase A(2) alpha/Arachidonic Acid Signaling Mediates Depolarization-Induced Suppression of Excitation in the Cerebellum

Background

Depolarization-induced suppression of excitation (DSE) at parallel fiber-Purkinje cell synapse is an endocannabinoid-mediated short-term retrograde plasticity. Intracellular Ca²⁺ elevation is critical for the endocannabinoid production and DSE. Nevertheless, how elevated Ca²⁺ leads to DSE is unclear.

Methodology/Principal Findings

We utilized cytosolic phospholipase A₂ alpha (cPLA₂α) knock-out mice and whole-cell patch clamp in cerebellar slices to observed the action of cPLA₂α/arachidonic acid signaling on DSE at parallel fiber-Purkinje cell synapse. Our data showed that DSE was significantly inhibited in cPLA₂α knock-out mice, which was rescued by arachidonic acid. The degradation enzyme of 2-arachidonoylglycerol (2-AG), monoacylglycerol lipase (MAGL), blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine (AEA), fatty acid amide hydrolase (FAAH), did not affect DSE. These results suggested that 2-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K⁺, indicating that large conductance Ca²⁺-activated potassium channel (BK) is sufficient to inhibit cPLA₂α/arachidonic acid-mediated DSE. In addition, we showed that the release of 2-AG was independent of soluble NSF attachment protein receptor (SNARE), protein kinase C and protein kinase A.

Conclusions/Significance

Our data first showed that cPLA₂α/arachidonic acid/2-AG signaling pathway mediates DSE at parallel fiber-Purkinje cell synapse.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

Transfer RNA changes in rat granulation tissue possibly related to collagen synthesis

Transfer RNAs for glycine, proline, lysine, serine and leucine were compared in developing rat granulation tissue 6 and 15 days after sterile subcutaneous implantation of pieces of cellulose sponge. The acceptance of glycine, proline and lysine by unfractionated tRNAs were ca. 30 per cent greater in tRNA derived from 15-day granulation tissue, whereas those of serine and leucine were unaltered. Cochromatography on benzoylated DEAE-cellulose of the ³H- and ¹⁴C-labeled aminoacyl-tRNAs from the two sources revealed a significant increase in the relative amount of one of the three glycyl-tRNA fractions in the 15-day granulation tissue, whereas the elution profiles for prolyl-, lysyl-, seryl-, and leucyl-tRNAs were unaltered. The changes observed suggest a causal relation to the enhanced synthesis of collagen in the late-stage granulation tissue.