Acetylation turns leucine into a drug by membrane transporter switching

AbstractSmall changes to molecules can have profound effects on their pharmacological activity as exemplified by the addition of the two-carbon acetyl group to make drugs more effective by enhancing their pharmacokinetic or pharmacodynamic properties. N-acetyl-d,l-leucine is approved in France for vertigo and its l-enantiomer is being developed as a drug for rare and common neurological disorders. However, the precise mechanistic details of how acetylation converts leucine into a drug are unknown. Here we show that acetylation of leucine switches its uptake into cells from the l-type amino acid transporter (LAT1) used by leucine to organic anion transporters (OAT1 and OAT3) and the monocarboxylate transporter type 1 (MCT1). Both the kinetics of MCT1 (lower affinity compared to LAT1) and the ubiquitous tissue expression of MCT1 make it well suited for uptake and distribution of N-acetyl-l-leucine. MCT1-mediated uptake of a N-acetyl-l-leucine as a prodrug of leucine bypasses LAT1, the rate-limiting step in activation of leucine-mediated signalling and metabolic process inside cells such as mTOR. Converting an amino acid into an anion through acetylation reveals a way for the rational design of drugs to target anion transporters.

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Synchronized Infection of Cell Cultures by Magnetically Controlled Virus

Journal of Virology ◽

10.1128/jvi.79.1.622-625.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 622-625 ◽

Cited By ~ 37

Author(s):

Hillel Haim ◽

Israel Steiner ◽

Amos Panet

Keyword(s):

Adsorption Process ◽

Diffusion Rate ◽

Cell Attachment ◽

Diffusion Limited ◽

Rate Limiting Step ◽

Immunodeficiency Virus ◽

Rate Limiting ◽

And Diffusion ◽

Entry Mechanism

ABSTRACT To override the diffusion-limited adsorption step of viral infection, we magnetically synchronized cell attachment. Human immunodeficiency virus type 1-based lentivirus preparations were rendered magnetically reactive by association with magnetite nanoparticles, 50 nm in diameter. Application of a magnetic field resulted in immediate redistribution of the viral inoculum to the cell-associated state and completion of the productive adsorption process within 1 min. Independent of adsorption time, viral concentration, and diffusion rate, infection subsequently progressed by the receptor-mediated entry mechanism. Synchronization of this rate-limiting step of infection may now be applied to analyze isolated events in the viral replication sequence.

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The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Journal of Virology ◽

10.1128/jvi.00316-09 ◽

2009 ◽

Vol 83 (19) ◽

pp. 10048-10057 ◽

Cited By ~ 16

Author(s):

Ruben M. Markosyan ◽

Michael Y. Leung ◽

Fredric S. Cohen

Keyword(s):

Human Immunodeficiency Virus ◽

Coiled Coil ◽

Fusion Pore ◽

Helix Bundle ◽

Rate Limiting Step ◽

Immunodeficiency Virus ◽

Rate Limiting ◽

Kinetics Of ◽

Pore Enlargement

ABSTRACT Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.

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Activity and genomic organization of human glucose transporter 9 (GLUT9), a novel member of the family of sugar-transport facilitators predominantly expressed in brain and leucocytes

Biochemical Journal ◽

10.1042/bj3500771 ◽

2000 ◽

Vol 350 (3) ◽

pp. 771-776 ◽

Cited By ~ 66

Author(s):

Holger DOEGE ◽

Andreas BOCIANSKI ◽

Hans-Georg JOOST ◽

Annette SCHÜRMANN

Keyword(s):

Amino Acid ◽

Glucose Transporter ◽

Sequence Similarity ◽

Organic Anion ◽

Sugar Transport ◽

Amino Acid Identity ◽

Transport Activity ◽

Anion Transporters ◽

The Family ◽

Glucose Transporter 9

The GLUT9 gene encodes a cDNA which exhibits significant sequence similarity with members of the glucose transporter (GLUT) family. The gene is located on chromosome 9q34 and consists of 10 exons separated by short introns. The amino acid sequence deduced from its cDNA predicts 12 putative membrane-spanning helices and all the motifs (sugar-transporter signatures) that have previously been shown to be essential for transport activity. A striking characteristic of GLUT9 is the presence of two arginines in the putative helices 7 and 8 at positions where the organic anion transporters harbour basic residues. The next relative of GLUT9 is the glucose transporter GLUT8/GLUTX1 (44.8% amino acid identity with GLUT9). A 2.6-kb transcript of GLUT9 was detected in spleen, peripheral leucocytes and brain. Transfection of COS-7 cells with GLUT9 produced expression of a 46-kDa membrane protein which exhibited reconstitutable glucose-transport activity and low-affinity cytochalasin-B binding. It is concluded that GLUT9 is a novel member of the family of sugar-transport facilitators with a tissue-specific function.

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A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus

Biochemical Journal ◽

10.1042/bj2520661 ◽

1988 ◽

Vol 252 (3) ◽

pp. 661-666 ◽

Cited By ~ 33

Author(s):

M C Sheehan ◽

C J Bailey ◽

B C A Dowds ◽

D J McConnell

Keyword(s):

Amino Acid ◽

Alcohol Dehydrogenase ◽

Bacillus Stearothermophilus ◽

Cross Reactivity ◽

Ph Optimum ◽

Western Blots ◽

Antigenic Activity ◽

Rate Limiting Step ◽

Steady State Kinetic ◽

Rate Limiting

An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay (‘e.l.i.s.a.’) procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.

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Reactions of 1,3-diacylthioureas with methoxide ion and with amines

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19870120 ◽

1987 ◽

Vol 52 (1) ◽

pp. 120-131 ◽

Cited By ~ 2

Author(s):

Jaromír Kaválek ◽

Josef Jirman ◽

Vojeslav Štěrba

Keyword(s):

Carbonyl Group ◽

Rate Constants ◽

Dissociation Constants ◽

Equilibrium Constants ◽

Rate Limiting Step ◽

Limiting Step ◽

Order Of Magnitude ◽

Acetyl Group ◽

Methoxide Ion ◽

Rate Limiting

Rate constants of base-catalyzed methanolysis and dissociation constants in methanol have been determined for benzoylthiourea (II), 1,3-diacetylthiourea (III), 1,3-dibenzoylthiourea (IV), and 1-acetyl-3-benzoylthiourea (V). With the diacyl derivatives III and IV, the reaction of methoxide ion with the neutral substrate is accompanied by that of methoxide with the substrate anion (at higher alkoxide concentrations). Above 0.1 mol l-1 CH3O(-), the rate constants are also affected by medium. The rate of the reaction of neutral diacyl derivative is decreased, and that of the reaction of methoxide with the substrate anion is rapidly increased. The dissociation constant of II is higher than that of acetylthiourea (I) by about one order of magnitude, but the attack of methoxide on the carbonyl group of II is about three times slower than that in I. The benzoyl group at the N1 nitrogen exhibits a greater activating influence (in both the rate and the equilibrium constants) on the other NHCOR group than the acetyl group does. With V the ratio of methanolysis rate constants is 9 : 1 in favour of the acetyl group. The reaction of diacetyl derivative III with 1-butanamine has been followed in butanamine buffers. At the lowest butanamine concentrations, the reaction is second order in the amine, and the rate-limiting step is the proton transfer from the intermediate to the second amine molecule. At the highest butanamine concentrations the reaction becomes first order in the amine, and the rate-limiting step changes to the attack of butanamine on the carbonyl group of diacetyl derivative III.

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Organic Anion Transporter 1B1: An Important Factor in Hepatic Thyroid Hormone and Estrogen Transport and Metabolism

Endocrinology ◽

10.1210/en.2008-0169 ◽

2008 ◽

Vol 149 (9) ◽

pp. 4695-4701 ◽

Cited By ~ 45

Author(s):

Wendy M. van der Deure ◽

Edith C. H. Friesema ◽

Frank Jan de Jong ◽

Yolanda B. de Rijke ◽

Frank H. de Jong ◽

...

Keyword(s):

Thyroid Hormone ◽

Organic Anion ◽

Organic Anion Transporter ◽

Hepatic Transport ◽

Transfected Cells ◽

Estrone Sulfate ◽

Anion Transporters ◽

Anion Transporter ◽

Metabolism Of Bilirubin

Sulfation is an important pathway in the metabolism of thyroid hormone and estrogens. Sulfation of estrogens is reversible by estrogen sulfatase, but sulfation of thyroid hormone accelerates its degradation by the type 1 deiodinase in liver. Organic anion transporters (OATPs) are capable of transporting iodothyronine sulfates such as T4 sulfate (T4S), T3S, and rT3S or estrogen sulfates like estrone sulfate (E1S), but the major hepatic transporter for these conjugates has not been identified. A possible candidate is OATP1B1 because model substrates for this transporter include the bilirubin mimic bromosulfophthalein (BSP) and E1S, and it is highly and specifically expressed in liver. Therefore, OATP1B1-transfected COS1 cells were studied by analysis of BSP, E1S, and iodothyronine sulfate uptake and metabolism. Two Caucasian populations (155 blood donors and 1012 participants of the Rotterdam Scan Study) were genotyped for the OATP1B1-Val174Ala polymorphism and associated with bilirubin, E1S, and T4S levels. OATP1B1-transfected cells strongly induced uptake of BSP, E1S, T4S, T3S, and rT3S compared with mock-transfected cells. Metabolism of iodothyronine sulfates by cotransfected type 1 deiodinase was greatly augmented in the presence of OATP1B1. OATP1B1-Val174 showed a 40% higher induction of transport and metabolism of these substrates than OATP1B1-Ala174. Carriers of the OATP1B1-Ala174 allele had higher serum bilirubin, E1S, and T4S levels. In conclusion, OATP1B1 is an important factor in hepatic transport and metabolism of bilirubin, E1S, and iodothyronine sulfates. OATP1B1-Ala174 displays decreased transport activity and thereby gives rise to higher bilirubin, E1S, and T4S levels in carriers of this polymorphism.

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Biology of a Novel Organic Solute and Steroid Transporter, OSTΑ-OSTβ

Experimental Biology and Medicine ◽

10.1177/153537020523001001 ◽

2005 ◽

Vol 230 (10) ◽

pp. 689-698 ◽

Cited By ~ 37

Author(s):

Nazzareno Ballatori

Keyword(s):

Amino Acid ◽

Bile Acids ◽

Mdck Cells ◽

Organic Anion ◽

Basolateral Membrane ◽

Comparative Approach ◽

Prostaglandin E ◽

Anion Transporters ◽

Organic Solute ◽

Tm Domain

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340–amino acid, seven-transmembrane (TM) domain protein (OSTΑ) and a putative 128–amino acid, single-TM domain ancillary polypeptide (OSTβ). When OSTΑ and OSTβ are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTΑ and OSTβ are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTΑ and OSTβ proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse OstΑ–Ostβ mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTΑ-OSTβ is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.

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Role of Asp104 in the SHV β-Lactamase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00848-06 ◽

2006 ◽

Vol 50 (12) ◽

pp. 4124-4131 ◽

Cited By ~ 20

Author(s):

Christopher R. Bethel ◽

Andrea M. Hujer ◽

Kristine M. Hujer ◽

Jodi M. Thomson ◽

Mark W. Ruszczycky ◽

...

Keyword(s):

Amino Acid ◽

Catalytic Efficiency ◽

Saturation Mutagenesis ◽

Amino Acid Residues ◽

Rate Limiting Step ◽

Limiting Step ◽

Threefold Increase ◽

Rate Limiting ◽

Hydrolysis Of

ABSTRACT Among the TEM-type extended-spectrum β-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV β-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in β-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys β-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser β-lactamases. Correspondingly, the overall catalytic efficiency (k cat/Km ) of hydrolysis for cefotaxime was increased from 0.60 ± 0.07 μM−1 s−1 (mean ± standard deviation) for Gly238Ser to 1.70 ± 0.01 μM−1 s−1 for the Asp104Lys and Gly238Ser β-lactamase (threefold increase). We also showed that (i) k 3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser β-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 β-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

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A novel missense mutation in the γ-glutamylcysteine synthetase catalytic subunit gene causes both decreased enzymatic activity and glutathione production

Blood ◽

10.1182/blood-2002-11-3622 ◽

2003 ◽

Vol 102 (2) ◽

pp. 725-730 ◽

Cited By ~ 41

Author(s):

David Hamilton ◽

Jian Hui Wu ◽

Moulay Alaoui-Jamali ◽

Gerald Batist

Keyword(s):

Amino Acid ◽

Enzymatic Activity ◽

Missense Mutation ◽

Binding Studies ◽

Rate Limiting Step ◽

Number Of Patients ◽

Evolutionarily Conserved ◽

Glutamylcysteine Synthetase ◽

Rate Limiting ◽

Conserved Residue

Abstractγ-Glutamylcysteine synthetase (γ-GCS) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis: the adenosine triphosphate (ATP)–dependent ligation of glutamate and cysteine. γ-GCS consists of a catalytic (γ-GCSH) and modifier (γ-GCSL) subunit. Hereditary deficiency of γ-GCS has been reported in a small number of patients and is associated with low erythrocyte levels of γ-GCS and GSH leading to hemolytic anemia. Here we report a novel γ-GCSH mutation, isolated from the cDNA of 2 related patients diagnosed with γ-GCS deficiency. Each was found to be homozygous for a C>T missense mutation at nucleotide 379, encoding for a predicted Arg127Cys amino acid change. Computerized structure modeling identified that the mutated amino acid lies within a cleft on the protein surface of γ-GCSH, and the border of this cleft was shown to contain Cys249, an evolutionarily conserved residue that has been proven to lie near the binding site of γ-GCSH. Transfection studies showed that the mutation is associated with decreased GSH production, and binding studies using purified recombinant protein showed that the mutant protein has markedly decreased enzymatic activity compared to wild type.

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Hydride Abstraction as the Rate-Limiting Step of the Irreversible Inhibition of Monoamine Oxidase B by Rasagiline and Selegiline: A Computational Empirical Valence Bond Study

International Journal of Molecular Sciences ◽

10.3390/ijms21176151 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6151

Author(s):

Tana Tandarić ◽

Alja Prah ◽

Jernej Stare ◽

Janez Mavri ◽

Robert Vianello

Keyword(s):

Rational Design ◽

Valence Bond ◽

Practical Significance ◽

Mao Inhibition ◽

Empirical Valence Bond ◽

Rate Limiting Step ◽

Limiting Step ◽

Hydride Abstraction ◽

Anion Transfer ◽

Rate Limiting

Monoamine oxidases (MAOs) catalyze the degradation of a very broad range of biogenic and dietary amines including many neurotransmitters in the brain, whose imbalance is extensively linked with the biochemical pathology of various neurological disorders, and are, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. Still, despite this practical significance, the precise molecular mechanism underlying the irreversible MAO inhibition with clinically used propargylamine inhibitors rasagiline and selegiline is still not unambiguously determined, which hinders the rational design of improved inhibitors devoid of side effects current drugs are experiencing. To address this challenge, we present empirical valence bond QM/MM simulations of the rate-limiting step of the MAO inhibition involving the hydride anion transfer from the inhibitor α-carbon onto the N5 atom of the flavin adenin dinucleotide (FAD) cofactor. The proposed mechanism is strongly supported by the obtained free energy profiles, which confirm a higher reactivity of selegiline over rasagiline, while the calculated difference in the activation Gibbs energies of ΔΔG‡ = 3.1 kcal mol−1 is found to be in very good agreement with that from the measured literature kinact values that predict a 1.7 kcal mol−1 higher selegiline reactivity. Given the similarity with the hydride transfer mechanism during the MAO catalytic activity, these results verify that both rasagiline and selegiline are mechanism-based irreversible inhibitors and offer guidelines in designing new and improved inhibitors, which are all clinically employed in treating a variety of neuropsychiatric and neurodegenerative conditions.

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