Potential Endogenous Substrates Research Articles

Ex vivo lipidomics reveal monoacylglycerols as substrates for a fatty acid amide hydrolase in the legume Medicago truncatula.

Fatty acid amide hydrolase (FAAH) is a conserved hydrolase in eukaryotes with promiscuous activity toward a range of acylamide substrates. The native substrate repertoire for FAAH has just begun to be explored in plant systemsoutside the model Arabidopsis thaliana. Here, we used ex vivo lipidomics to identify potential endogenous substrates for Medicago truncatula FAAH1 (MtFAAH1). We incubated recombinant MtFAAH1 with lipid mixtures extracted from M. truncatula and resolved their profiles via gas chromatography-mass spectrometry (GC-MS). Data revealed that besides N-acylethanolamines (NAEs), sn-1 or sn-2 isomers of monoacylglycerols (MAGs) were substrates for MtFAAH1. Combined with in vitro and computational approaches, our data support both amidase and esterase activities for MtFAAH1. MAG-mediated hydrolysis via MtFAAH1 may be linked to biological roles that are yet to be discovered.

Hernandezine acts as a CDK4 suppressor inhibiting tumor growth by the CDK4/PKM2/NRF2 axis in colon cancer

BackgroundThe cyclin-dependent kinase 4 (CDK4) interacts with its canonical and non-canonical substrates modulating the cell cycle in tumor cells. However, the potential substrates and the beyond-cell-cycle-regulated functions of CDK4 in colon cancer (CC) are still unknown. Hernandezine (HER) is previously verified to induce G0/G1 phase arrest and autophagic cell death in human cancer cells, which implies that HER might target G0/G1 phase-related proteins, including CDK4. PurposeThe present study tried to investigate the glycolytic metabolism and oxidative stress functions of CDK4 in colon cancer. Furthermore, the inhibitory effects and potential binding sites of HER on CDK4, as well as its anti-tumor activity were investigated in CC cells. MethodsThe mass spectrometry assay was performed to identify potential endogenous substrates of CDK4 and the correlation between glycolytic metabolic rate and CDK4 level in COAD patient tissues. Meanwhile, after inhibiting the activity or the expression of CDK4, the binding capacity of CDK4 to PKM2 and NRF2 and the latter two protein distributions in cytoplasm and nucleus were detected in CC cells. In vitro, the regulatory effects of the CDK4-PKM2-NRF2 axis on glycolysis and oxidative stress were performed by ECAR, OCR, and ROS assay. The inhibitory effect of HER on CDK4 activity was explored in CC cells and the potential binding sites were predicted and testified in vitro. Furthermore, tumor growth inhibition of HER by suppressing the CDK4-PKM2-NRF2 axis was also investigated in vitro and in vivo. ResultsPKM2 and NRF2 were identified as endogenous substrates of CDK4 and, high-expressed CDK4 was associated with low-level glycolysis in COAD. In vitro, inactivated CDK4 facilitated CDK4-PKM2-NRF2 complex formation which resulted in 1) inhibited PKM2 activity and retarded the glycolytic rate; 2) cytoplasm-detained NRF2 failed to transcript anti-oxidative gene expressions and induced oxidant stress. Additionally, as a CDK4 inhibitor, HER developed triple anti-tumor effects including induced G0/G1 phase arrest, suppressed glycolysis, and disrupted the anti-oxidative capacity of CC cells. Conclusion: The results first time revealed that CDK4 modulated glycolytic and anti-oxidative capacity of CC cells via bound to its endogenous substrates, PKM2 and NRF2. Additionally, 140Asp145Asn amino acid sites of CDK4 were potential targets of HER. HER exerts anti-tumor activity by inhibited the activity of CDK4, promoted the CDK4-PKM2-NRF2 complex formation in the CC cells.

Abstract 4834: Mitochondrial ClpXP degrades serine phosphorylated protein aggregates

Abstract ClpXP is a serine protease localized to the mitochondrial matrix and maintains mitochondrial proteostasis by degrading damaged/aggregated respiratory chain complex proteins. Both inhibition and hyperactivation of ClpP lead to impaired OXPHOS function and kill leukemic cells in vitro and in vivo. Yet, the marks that tag proteins for degradation by ClpXP remain unknown. The bacterial homologue of ClpXP degrades damaged proteins with arginine phosphorylation. Therefore, we assessed how phosphorylation affects ClpXP protease activity. Recombinant ClpXP was incubated with its unnatural substrate, FITC-tagged casein, in the presence of increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). In a dose-dependent manner, pSer and pThr free amino acids and short peptides inhibited casein degradation by ClpXP. In contrast, free Ser, Thr, phosphate, pArg and pTyr did not inhibit ClpXP protease activity. In addition, we characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green ATPase assay. Neither pSer nor pThr inhibited ClpX ATPase activity or ClpP peptidase activity. Thus, pSer and pThr only blocked ClpXP from degrading full-length protein without altering the enzymatic activity of the complex. Next, we characterized pSer and pThr binding to ClpXP by thermal shift binding assay. pSer and pThr free amino acids and short peptides bound to ClpX but not ClpP. All the dephosphorylated counterparts did not bind to ClpX. Therefore, pSer and pThr interact with ClpX outside the ATPase site. cyroEM studies of ClpX and pSer peptides indicated a putative pSer docking site in the central pore of ClpX. Previously, we identified OXPHOS complex II subunit SDHA as a potential ClpXP endogenous substrate. Therefore, we used SDHA as a model to assess whether serine phosphorylation influenced ClpXP protein degradation in intact cells. Using pSer immunoprecipitation, we observed increased serine phosphorylated SDHA in AML cells after ClpP and ClpX knockdown. The increased serine phosphorylated SDHA was specifically found in the insoluble fraction of the mitochondrial proteins. Supporting that serine phosphorylated SDHA represents aggregated protein, treatment of cells with ROS-inducing antimycin or 42°C heat shock also increased levels of serine phosphorylated SDHA in the fraction of insoluble mitochondrial proteins. Finally, adding recombinant ClpXP to mitochondrial lysates specifically degraded SDHA with serine phosphorylation. In summary, mitochondrial ClpXP degrades aggregated mitochondrial proteins marked by serine phosphorylated. Thus, this work identifies new degradation machinery for mitochondrial protease, advances the function of mitochondrial ClpXP, and highlights a new therapeutic strategy to target this protease. Citation Format: Yue Feng, Yulia Jitkova, Alexander Keszei, Jonathan St-Germain, Yongran Yan, Brian Raught, Mohammad Mazhab-Jafari, Aaron Schimmer. Mitochondrial ClpXP degrades serine phosphorylated protein aggregates. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4834.

Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice.

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

Screening for endogenous substrates reveals that CYP2D6 is a 5-methoxyindolethylamine O-demethylase.

The objective of this investigation was to screen for potential endogenous substrates for CYP2D6. Using recombinant CYP2D6, together with hepatic microsomes from CYP2D6-transgenic mice, human liver microsomes, and a specific anti-CYP2D6 monoclonal antibody, it was ascertained that CYP2D6 does not significantly metabolize the endogenous phenylethylamines 2-phenylethylamine, octopamine, synephrine, 3-methoxy-p-tyramine, 4-methoxy-m-tyramine, metanephrine, and normetanephrine, nor the indolethylamines tryptamine, serotonin, 6-methoxytryptamine, and melatonin, nor the beta-carbolines harman, norharman and tryptoline. However, the indolethylamines 5-methoxy-N,N-dimethyltryptamine (5-MDMT) and pinoline (6-methoxy-1,2,3,4-tetrahydro-beta-carboline) showed relatively high affinity for CYP2D6 in a spectral binding assay (K(s) 28 +/- 5, and 0.5 +/- 0.3 microm (mean +/- SEM), respectively) and were O-demethylated only by CYP2D6 in a panel of 15 recombinant common human P450s. Pinoline and 5-MDMT O-demethylase activities were 35- and 11-fold greater in liver microsomes from CYP2D6-humanized mice, respectively, than those in liver microsomes from control mice. Moreover, the increased activities were completely inhibited by an anti-CYP2D6 monoclonal antibody. Kinetic analysis with recombinant CYP2D6 gave K(m) and k(cat) values for 5-MDMT and pinoline O-demethylations of 12 +/- 1 microm and 65 +/- 1 min(-1) and 1.8 +/- 0.3 microm and 26 +/- 1 min(-1), respectively. These two substrates can be added to 5-methoxytryptamine, which we have recently reported to be an endogenous CYP2D6 substrate. CYP2D6 is therefore a relatively highly specific, high-affinity, high-capacity 5-methoxyindolethylamine O-demethylase. Polymorphic cytochrome CYP2D6 may therefore exert an influence on mood and behavior by the O-demethylation of these 5-methoxyindolethylamines found in the brain and pineal gland. These processes may also impact on mental and neurological health. The findings may open new vistas for the determination of CYP2D6 phenotype.

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Abstract Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l -cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K + , other metal ions such as Zn 2+ , Cu 2+ , Fe 3+ , Ca 2+ , Fe 2+ and Na + accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K m and V max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho -diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.

The high turnover Drosophila multidrug resistance-associated protein shares the biochemical features of its human orthologues

DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the “long” human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like β-estradiol 17-β- d-glucuronide, leukotriene C 4, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68–75 nmol P i/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.

Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

Divergence of Cofactor Recognition across Evolution: Coenzyme A Binding in a Prokaryotic Arylamine N-Acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.

A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery.

Ubiquitination of membrane-associated proteins can direct their proteasome-mediated degradation or activation at the endoplasmic reticulum (ER), as well as their endocytosis and intracellular sorting. However, the full spectrum of ubiquitinated membrane proteins has not been determined. Here we combined proteomic analysis with yeast genetics to identify 211 ubiquitinated membrane-associated proteins in Saccharomyces cerevisiae and map >30 precise sites of ubiquitination. Major classes of identified ubiquitinated proteins include ER-resident membrane proteins, plasma membrane-localized permeases, receptors, and enzymes, and surprisingly, components of the actin cytoskeleton. By determining the differential abundance of ubiquitinated proteins in yeast mutated for NPL4 and UBC7, which are major components of ER-associated degradation (ERAD), we furthermore were able to classify 83 of these identified ubiquitinated membrane proteins as potential endogenous substrates of the ERAD pathway. These substrates are highly enriched for proteins that localize to or transit through the ER. Interestingly, we also identified novel membrane-bound transcription factors that may be subject to ubiquitin/proteasome-mediated cleavage and activation at the ER membrane.

The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity.

The gene EPXH2 encodes for the soluble epoxide hydrolase (sEH), an enzyme involved in the regulation of cardiovascular and renal physiology containing two distinct domains connected via a proline-rich linker. The C-terminal domain containing the EH catalytic activity has been well studied. In contrast, a function for the N-terminal domain, which has high homology to the haloacid dehalogenase family of phosphatases, has not been definitively reported. In this study we describe the N-terminal domain as a functional phosphatase unaffected by a number of classic phosphatase inhibitors. Assuming a functional association between these catalytic activities, dihydroxy lipid phosphates were rationalized as potential endogenous substrates. A series of phosphorylated hydroxy lipids were therefore synthesized and found to be excellent substrates for the human sEH. The best substrate tested was the monophosphate of dihydroxy stearic acid (threo-910-phosphonoxy-hydroxy-octadecanoic acid) with K(m) = 21 +/- 0.3 microM, V(Max) = 338 +/- 12 nmol x min(-1) x mg(-1), and k(cat) = 0.35 +/- 0.01 s(-1). Therefore dihydroxy lipid phosphates are possible candidates for the endogenous substrates of the sEH N-terminal domain, which would represent a novel branch of fatty acid metabolism with potential signaling functions.

Glutathione Stimulates Sulfated Estrogen Transport by Multidrug Resistance Protein 1

Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that transports a range of hydrophobic xenobiotics, as well as relatively hydrophilic organic anion conjugates. The protein is present at high levels in testicular Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1 may protect germ cells from exposure to some cytotoxic xenobiotics, but potential endobiotic substrates in this organ have not been identified. Previously, we have shown certain D-ring, but not A-ring, estrogen glucuronides can act as competitive inhibitors of MRP1 mediated transport, suggesting that they are potential substrates for the protein. In the case of 17 beta-estradiol-17 beta-d-glucuronide, this has been confirmed by direct transport studies. The Leydig cell is the major site of estrogen conjugation in the testis. However, the principal products of conjugation are A-ring estrogen sulfates, which are then effluxed from the cell by an unknown transporter. To determine whether MRP1/mrp1 could fulfill this function, we used membrane vesicles from MRP1-transfected HeLa cells to assess this possibility. We found that estradiol and estrone 3-sulfate alone were poor competitors of MRP1-mediated transport of the cysteinyl leukotriene, leukotriene C(4). However, in the presence of reduced glutathione (GSH), their inhibitory potency was markedly increased. Direct transport studies using [(3)H]estrone 3-sulfate confirmed that the conjugated estrogen could be efficiently transported (K(m) = 0.73 microm, V(max) = 440 pmol mg(-)1 protein min(-)1), but only in the presence of either GSH or the nonreducing alkyl derivative, S-methyl GSH. In contrast to previous studies using vincristine as a substrate, we detected no reciprocal increase in MRP1-mediated GSH transport. These results provide the first example of GSH-stimulated, MRP1-mediated transport of a potential endogenous substrate and expand the range of MRP1 substrates whose transport is stimulated by GSH to include certain hydrophilic conjugated endobiotics, in addition to previously identified hydrophobic xenobiotics.

UGT2B23, a Novel Uridine Diphosphate-Glucuronosyltransferase Enzyme Expressed in Steroid Target Tissues That Conjugates Androgen and Estrogen Metabolites

Glucuronidation is widely accepted as a mechanism involved in the catabolism and elimination of steroid hormones from the body. However, relatively little is known about the enzymes involved, their specificity for the different steroids, and their site of expression and action. To characterize the pathway of steroid glucuronidation, a novel uridine diphosphate glucuronosyltransferase (UGT) enzyme was cloned and characterized. A 1768-bp complementary DNA, encoding UGT2B23 was isolated from a monkey liver library. Stable expression of UGT2B23 in human HK293 cells and Western blot analysis demonstrated the presence of a 51-kDa protein. The UGT2B23 transferase activity was tested with 62 potential endogenous substrates and was demonstrated to be active on 6 steroids and the bile acid, hyodeoxycholic acid. Kinetic analysis yielded apparent Michaelis constant (Km) values of 0.9, 13.5, 1.6, and 5.7 microM for the conjugation of androsterone (ADT), 3alpha-Diol, estriol, and 4-hydroxyestrone, respectively. RT-PCR analysis revealed that UGT2B23 transcript is expressed in several tissues, including the prostate, mammary gland, epididymis, testis, and ovary. Primary structure analysis shows that UGT2B23 is in the same family of enzymes as the previously characterized monkey isoforms UGT2B9 and UGT2B18, which are active on hydroxyandrogens. The characterization of UGT2B23 as a functional enzyme active on 3alpha-hydroxysteroids, and its expression in extrahepatic tissues, indicate that it may potentially play an important role in estrogen and androgen catabolism in peripheral steroid target tissues.

Lipoxygenase-2 oxygenates storage lipids in embryos of germinating barley.

Besides the pre-existing lipoxygenase (LOX-1) present in quiescent grains, a new lipoxygenase (LOX-2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. & Douma, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 form different products after incubation with linoleic acid, the (9S)- and (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991) FEBS Lett. 280, 159-162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. & Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-1 oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. Km values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2-8-fold for LOX-2). Interestingly, LOX-1 showed significantly higher Km values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di- and tri-linoleoylglycerol; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-1, with a corresponding larger extent of oxygenation. In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for beta-oxidation and therefore as a carbon source for the growing barley embryo.

Rapid nuclear translocation and increased activity of cyclin-dependent kinase 6 after T cell activation.

To elucidate the roles of cyclin-dependent kinase 6 (cdk6) in T cells, we examined its intracellular localization, kinase activity, and associated proteins in the Jurkat T lymphoblastoid cell line. Jurkat cells had a high level of cdk6, which was associated with cyclin D3, but not cyclin D2, the member of the cyclin D family. When stimulated by a combination of PHA and anti-CD28 mAb, cdk6 activity was up-regulated, as measured by an in vitro kinase assay using recombinant, truncated retinoblastoma tumor suppressor gene protein (Rb protein) as substrate. Activation was most prominent when cells were stimulated with the combination of PHA and anti-CD28, although significant increases were detected after stimulation with PHA alone. The combination also resulted in maximal activation of c-Jun kinase and IL-2 production. Costimulation resulted in a rapid translocation of cdk6 to the nucleus, as demonstrated by both confocal immunofluorescence microscopy and biochemical fractionation techniques. Cdk6 activation and nuclear translocation were also observed after stimulation of Jurkat cells using the anti-CD28 Ab in combination with a mAb to CD3 (OKT3). Furthermore, nuclear translocation was observed in normal human T lymphocytes isolated from peripheral blood and stimulated in vitro with PHA. Two potential endogenous cdk6 substrates (with apparent molecular masses of 75-80 and 55-60 kDa), which were immunoprecipitated with cdk6 and phosphorylated in the in vitro kinase assay, were also identified. These data demonstrate the rapid activation and intracellular translocation of cdk6, implicating this kinase in early signal transduction events in T cells.

Xenobiotic biotransformation in unicellular green algae. Involvement of cytochrome P450 in the activation and selectivity of the pyridazinone pro-herbicide metflurazon.

The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent K(m) values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)-4-hydroxy-5,5-dimethylhexane++ +, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae.

Genetic polymorphism of thiopurine S-methyltransferase: clinical importance and molecular mechanisms.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurines such as mercaptopurine and thioguanine. TPMT activity exhibits genetic polymorphism, with about 1 in 300 inheriting TPMT-deficiency as an autosomal recessive trait. If treated with standard dosages of thiopurines. TPMT-deficient patients accumulate excessive thioguanine nucleotides (TGN) in hematopoietic tissues, leading to severe hematopoietic toxicity that can be fatal. However, TPMT-deficient patients can be successfully treated with a 10-15-fold lower dosage of these medications. The human gene encoding polymorphic TPMT has been cloned and characterized, and two mutant alleles have recently been isolated from TPMT-deficient and heterozygous patients (TPMT*2, TPMT*3), permitting development of PCR-based methods to identify TPMT-deficient and heterozygous patients prior to therapy. TPMT*3 is the predominant mutant allele in American whites, accounting for about 75% of mutations in this population. Ongoing studies aim to better define the influence of TPMT activity on thiopurine efficacy, to identify additional mutant alleles and determine their frequency in different ethnic groups, to elucidate the mechanism(s) for loss of function of mutant proteins, to identify potential endogenous substrates and to define the molecular mechanisms of TPMT regulation. Together, these advances bold the promise of improving the safety and efficacy of thiopurine therapy.

Substrate specificity of human aldose reductase: identification of 4-hydroxynonenal as an endogenous substrate

Aldose reductase, which catalyzes the reduction of glucose to sorbitol as part of the polyol pathway, has been implicated in the development of diabetic complications and is a prime target for drug development. However, aldose reductase exhibits broad specificity for both hydrophilic and hydrophobic aldehydes, which suggests that aldose reductase may also be a detoxification enzyme. Several series of structurally related aldehydes were compared as substrates in order to deduce the structural features that result in low Michaelis constants. Aldehydes that contain an aromatic ring are generally excellent substrates, consistent with crystallographic data which suggest that aldose reductase possesses a large hydrophobic substrate binding site. However, there is little discrimination among different aromatic aldehydes. In addition, small hydrophilic aldehydes exhibit low K m values if the alpha-carbon is oxidized. Analysis of the binding of NADPH by fluorescence quenching techniques indicates that aldose reductase exhibits higher affinity for NADPH than NADP, suggesting that this enzyme is normally primed for reductive metabolism. Thus aldose reductase appears to have evolved to catalyze the reduction of a very broad range of aldehydes. Structural features of substrates that bind to aldose reductase with low K m values were used to identify potential endogenous substrates. 4-Hydroxynonenal, a reactive alpha-beta unsaturated aldehyde produced during oxidative stress, is an excellent substrate ( K m = 22 μM, k cat/ K m = 4.6 · 106 M −1 min −1). Reductive metabolism of endogenous aldehydes in addition to glucose, catalyzed by aldose reductase, may play an important role in the development of diabetic complications.

Acetylation of p-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells.

N-acetyl-p-aminobenzoylglutamate is a major urinary metabolite of folic acid. It is formed by acetylation of p-aminobenzoylglutamate following cleavage of the C9-N10 bond of folic acid. Using recombinant human type 1 (NAT1) and type 2 (NAT2) arylamine N-acetyltransferase, we have shown that p-aminobenzoylglutamate is a specific NAT1 substrate. At an acetyl-CoA concentration of 50 microM, the Km for p-aminobenzoylglutamate (pABG) acetylation by recombinant NAT1 was 130 +/- 13 microM. For the human pro-monocytic cell-line U937, the apparent Km was slightly higher (333 +/- 17 microM). Inhibitor studies supported NAT1-dependent acetylation of pABG by U937 cell cytosols. These studies are the first to identify a potential endogenous substrate for human NAT1 and suggest that this enzyme may be important in the cellular clearance of pABG.

Glucuronidation of thyroid hormone by human bilirubin and phenol UDP-glucuronyltransferase isoenzymes

The glucuronidation of thyroid hormone by UDP-glucuronyltransferases (UGTs) stably transfected in Chinese hamster V79 lung fibroblasts was investigated. Human bilirubin UGT (HP3) and phenol UGT (HP4) both catalysed the glucuronidation of T4 and rT3, whereas glucuronidation of T3 was not significant. rT3 was the preferred substrate for both isoenzymes, glucuronidation rates being 1.6- and 6.4-times higher than conjugation of T4 by HP3 and HP4 clones, respectively. This is the first identification of thyroid hormone as potential alternative endogenous substrate for bilirubin UGT.