Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid.

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.

The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

Deficit of human ornithine aminotransferase in gyrate atrophy: Molecular, cellular, and clinical aspects.

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA.

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics.

Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.

Mechanism of Inactivation of Ornithine Aminotransferase by (1S,3S)-3-Amino-4-(hexafluoropropan-2-ylidenyl)cyclopentane-1-carboxylic Acid.

The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1-carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1'-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.

R180T variant of δ-ornithine aminotransferase associated with gyrate atrophy: biochemical, computational, X-ray and NMR studies provide insight into its catalytic features.

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Çº reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased Km value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb.

An ornithine ω-aminotransferase required for growth in the absence of exogenous proline in the archaeon Thermococcus kodakarensis.

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus, but perhaps also throughout the Archaea.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase.

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent Tm values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, Tm values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.

Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.

Ornithine aminotransferase versus GABA aminotransferase: implications for the design of new anticancer drugs.

Ornithine aminotransferase (OAT) and γ-aminobutyric acid aminotransferase (GABA-AT) are classified under the same evolutionary subgroup and share a large portion of structural, functional, and mechanistic features. Therefore, it is not surprising that many molecules that bind to GABA-AT also bind well to OAT. Unlike GABA-AT, OAT had not been viewed as a potential therapeutic target until recently; consequently, the number of therapeutically viable molecules that target OAT is very limited. In this review the two enzymes are compared with respect to their active-site structures, catalytic and inactivation mechanisms, and selective inhibitors. Insight is offered that could aid in the design and development of new selective inhibitors of OAT for the treatment of cancer.

Subgrouping Automata: automatic sequence subgrouping using phylogenetic tree-based optimum subgrouping algorithm.

Sequence subgrouping for a given sequence set can enable various informative tasks such as the functional discrimination of sequence subsets and the functional inference of unknown sequences. Because an identity threshold for sequence subgrouping may vary according to the given sequence set, it is highly desirable to construct a robust subgrouping algorithm which automatically identifies an optimal identity threshold and generates subgroups for a given sequence set. To meet this end, an automatic sequence subgrouping method, named 'Subgrouping Automata' was constructed. Firstly, tree analysis module analyzes the structure of tree and calculates the all possible subgroups in each node. Sequence similarity analysis module calculates average sequence similarity for all subgroups in each node. Representative sequence generation module finds a representative sequence using profile analysis and self-scoring for each subgroup. For all nodes, average sequence similarities are calculated and 'Subgrouping Automata' searches a node showing statistically maximum sequence similarity increase using Student's t-value. A node showing the maximum t-value, which gives the most significant differences in average sequence similarity between two adjacent nodes, is determined as an optimum subgrouping node in the phylogenetic tree. Further analysis showed that the optimum subgrouping node from SA prevents under-subgrouping and over-subgrouping.

Functional analysis of missense mutations of OAT, causing gyrate atrophy of choroid and retina.

We studied eight kindreds with gyrate atrophy of choroid and retina (GA), a rare autosomal recessive disorder caused by mutations of the OAT gene, encoding the homoexameric enzyme ornithine-delta-aminotransferase. We identified four novel and five previously reported mutations. Missense alleles were expressed in yeast strain carrying a deletion of the orthologous of human OAT. All mutations markedly reduced enzymatic activity. However, the effect on the yeast growth was variable, suggesting that some mutations retain residual activity, below the threshold of the enzymatic assay. Mutant proteins were either highly unstable and rapidly degraded, or failed to assemble to form the active OAT hexamer. Where possible, fibroblast analysis confirmed these data. We found no correlation between the residual enzymatic activity and the age of onset, or the severity of symptoms. Moreover, the response to B6 was apparently not related to the specific mutations carried by patients. Overall these data suggest that other factors besides the specific OAT genotype modulate (GA) phenotype in patients. Finally, we found that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator known to increase mitochondrial biogenesis, markedly stimulates OAT expression, thus representing a possible treatment for a subset of GA patients with hypomorphic alleles.

Synthesis and characterization of a stimulus-responsive L-ornithine-degrading hydrogel.

Hydrogels provide a highly favorable matrix for immobilizing growth factors, enzymes or cells for biomedical applications like tissue engineering, drug delivery or the treatment of metabolic diseases. In this study we describe the synthesis and characterization of a hydrogel able to degrade L-ornithine, a metabolite that is highly elevated in congenital hyperornithinemia. The hydrogel was synthesized by embedding the L-ornithine-degrading enzymes L-ornithine aminotransferase (OAT) and L-ornithine decarboxylase (ODC) into a polymer network. The network was formed from linear polyacrylamide crosslinked by heterodimers of ODC and ornithine decarboxylase antizyme (OAz). The resulting hydrogel was shown to be stable under physiological conditions and to efficiently degrade L-ornithine. The hydrogel-stabilizing ODC-OAz interactions could subsequently be dissociated by the addition of antizyme inhibitor (AzI) which resulted in the inducible dissolution of the hydrogel. This L-ornithine-degrading hydrogel that can efficiently be eliminated when its functionality is no longer required might represent a first step towards an enzyme substitution approach against hyperornithinemia.

[Expression and bioinformatic analysis of ornithine aminotransferase 
in non-small cell lung cancer].

BACKGROUND: It has been proven that ornithine aminotransferase (OAT) might play an important role in the oncogenesis and progression of numerous malignant tumors. The aim of this study is to detect the mRNA and protein expression of OAT in non-small cell lung cancer (NSCLC), as well as to analyze the bioinformatic features and binary interactions. METHODS: OAT mRNA expression was detected in A549 and 16HBE cell lines by reverse transcription-polymerase chain reaction. OAT protein expression was determined in 55 cases of NSCLC and 17 cases of adjacent non-tumor lung tissues by immunohistochemical staining. The bioinformatic features and binary interactions of OAT were analyzed. Gene ontology annotation and signal pathway analysis were performed. RESULTS: OAT mRNA expression in A549 cells was 2.85-fold lower than that in 16HBE cells. OAT protein expression was significantly higher in NSCLC tissues than that in adjacent non-tumor lung tissues. A significant difference of OAT protein expression was existed between squamous cell lung cancer and adenocarcinoma (P < 0.05), but was not correlated with the gender, age, lymph node metastasis, tumor size, and TNM stages. Bioinformatic analysis suggested that OAT was a highly homologous and stable protein located in the mitochondria. An aminotran-3 domain and several sites of phosphorylation, which may function in signal transduction, gene transcription, and molecular transit, were found. In the 54 selected binary interactions of OAT, TNF and TRAF6 play roles in the NF-κB pathway. CONCLUSIONS: OAT may play an important role in the oncogenesis and progression of NSCLC. Thus, OAT may be a novel biomarker for the diagnosis of NSCLC or a new target for its treatment.

A blue native polyacrylamide gel electrophoretic technology to probe the functional proteomics mediating nitrogen homeostasis in Pseudomonas fluorescens.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

Protein S-glutathionylation in malaria parasites.

AIMS: Protein S-glutathionylation is a widely distributed post-translational modification of thiol groups with glutathione that can function as a redox-sensitive switch to mediate redox regulation and signal transduction. The malaria parasite Plasmodium falciparum is exposed to intense oxidative stress and possesses the enzymatic system required to regulate protein S-glutathionylation, but despite its potential importance, protein S-glutathionylation has not yet been studied in malaria parasites. In this work we applied a method based on enzymatic deglutathionylation, affinity purification of biotin-maleimide-tagged proteins, and proteomic analyses to characterize the Plasmodium glutathionylome. RESULTS: We identified 493 targets of protein S-glutathionylation in Plasmodium. Functional profiles revealed that the targets are components of central metabolic pathways, such as nitrogen compound metabolism and protein metabolism. Fifteen identified proteins with important functions in metabolic pathways (thioredoxin reductase, thioredoxin, thioredoxin peroxidase 1, glutathione reductase, glutathione S-transferase, plasmoredoxin, mitochondrial dihydrolipoamide dehydrogenase, glutamate dehydrogenase 1, glyoxalase I and II, ornithine δ-aminotransferase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase [PK], and phosphoglycerate mutase) were further analyzed to study their ability to form mixed disulfides with glutathione. We demonstrate that P. falciparum GAPDH, PK, and ornithine δ-aminotransferase are reversibly inhibited by S-glutathionylation. Further, we provide evidence that not only P. falciparum glutaredoxin 1, but also thioredoxin 1 and plasmoredoxin are able to efficiently catalyze protein deglutathionylation. INNOVATION: We used an affinity-purification based proteomic approach to characterize the Plasmodium glutathionylome. CONCLUSION: Our results indicate a wide regulative use of S-glutathionylation in the malaria parasite and contribute to our understanding of redox-regulatory processes in this pathogen.