Biosynthese in vitro d'iodothyronines a partir du diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine

In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) (equimolecular in tyrosyl rings).Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction.Synthesis of thyroid hormones from Tyr(I)₂-Tyr(I)₂ is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)₂).A mechanism of iodothyronine formation via Tyr(I)₂ - Tyr(I)₂ and different from the one occuring for Tyr(I)₂ is suggested.     

Biosynthese in vitro d'iodothyronines a partir du diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine

In vitro biosynthesis of iodothyronines from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine A comparative study of two types of in vitro synthesis of iodothyronines has been done from 3,5-diiodotyrosine and from diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) (equimolecular in tyrosyl rings).Incubations are made with rat thyroid gland minces in Eagle's medium or with thyroid microsomal fraction.Synthesis of thyroid hormones from Tyr(I)₂-Tyr(I)₂ is faster and more important than from diiodo-3,5-L-tyrosine (Tyr(I)₂).A mechanism of iodothyronine formation via Tyr(I)₂ - Tyr(I)₂ and different from the one occuring for Tyr(I)₂ is suggested.

Résumé

Une étude comparative de deux types de synthèse in vitro d'iodothyronines a été faite à partir de la 3,5-diiodotyrosine Tyr(I)₂ et à partir d'un dipeptide iodé: le diiodo-3,5-L-tyrosyl-diiodo-3,5-L-tyrosine (Tyr(I)₂-Tyr(I)₂) dans des conditions équimoléculaires en noyaux tyrosyl.Les incubations sont effectuées en présence de coupes de thyroïdes de rat en milieu de survie ou en présence de fraction microsomale thyroïdienne.La synthèse d'hormones thyroïdienes à partir du Tyr(I)₂-Tyr(I)₂ est plus rapide et plus importante qu'à partir de la Tyr(I)₂.Un mécanisme de synthèse des iodothyronines à partir du Tyr(I)₂-Tyr(I)₂ différent de celui intervenant pour la Tyr(I)₂ est proposé.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: I. Manifestation of the elimination of the alanine side chain of the N-terminal diiodotyrosine

It was previously shown that 3,5-diido-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, acts as a precursor in the in vitro synthesis of thyroid hormones, and a mechanism of syntheis was proposed.We investigated this pathway by incubations of I₂Tyr-I₂Tyr with microsomal solubilized thyroid proteins. I₂Tyr-T₂Tyr was doubly labeled: iodinated with ¹³¹I on the ring and tritiated either on the alanine side-chain of the N- or C-terminal diidotyrosine. It is shown that only the C-terminal alanine participates in the synthesis, the N-terminal alanine being eliminated.The result proved that I₂Tyr-I₂Tyr acts as precursor through a mechanism which is different from the one involving I₂Tyr. This mechanism consists of: Schiff base formation with pyridoxal; free radical formation and cyclization; peptide bond cleavage and removal of the pyridoxal · alanine complex.     

The synthesis of [A 19-3-iodotyrosine] and [A 19-3,5-diiodotyrosine]insulin (porcine)

The chemical synthesis of [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), using the amino-acid derivatives 3-iodotyrosine and 3,5-diiodotyrosine is described. The synthesis of the iodinated A-chains were performed by segment condensation in solution using acid labile protecting groups. The hydroxyl groups of Tyr(I) and Tyr(I2) were unprotected. For the temporary protection of the alpha-amino groups of the A-chain segments containing iodinated tyrosines, the 1-(4-biphenylyl)-1-methylethoxycarbonyl group was selected. After deprotection and sulphitolysis the iodinated A-chain tetra-S-sulphonates were purified by ion exchange chromatography on DEAE cellulose at pH 5.6. Reduction to the sulphhydryl form and the combination with native porcine B-chain yielded [Tyr(I)A19] and [Tyr(I2)A19]insulin (porcine), respectively. Purification of the first product was achieved by gel filtration and of the later by ion exchange chromatography on CM-cellulose at pH 4.5 and gel filtration. The monoiodinated insulin had a biological activity of 24 +/- 2% and the diiodinated analogue 2.6 +/- 0.2% as determined in an in vitro lipogenesis assay with epididymal adipocytes.     

Halogenated aromatic amino acid 3,5-dibromo-<Emphasis Type="SmallCaps">d</Emphasis>-tyrosine produces beneficial effects in experimental stroke and seizures

The effects of the halogenated aromatic amino acid 3,5-dibromo-d-tyrosine (3,5-DBr-d-Tyr) were studied in rat models of stroke and epileptic seizures caused by middle cerebral artery occlusion (MCAo) through respective intracerebral injection of endothelin-1 (ET-1) and intraperitoneal (i.p.) injection of pentylenetetrazole (PTZ). 3,5-DBr-d-Tyr was administered as three bolus injections (30 or 90 mg/kg, i.p.) starting at 30, 90, and 180 min after ET-1 administration or as a single bolus (30 mg/kg, i.p.) 15 min prior to PTZ administration. Neurological deficits and infarct volume were estimated 3 days after ET-1 administration and seizure score was assessed during the first 20 min after PTZ administration. The safety of 3,5-DBr-d-Tyr was evaluated in control animals using telemetry to measure cardiovascular parameters and immunostaining to assess the level of activated caspase-3. 3,5-DBr-d-Tyr significantly improved neurological function and reduced infarct volume in the brain even when the treatment was initiated 3 h after the onset of MCAo. 3,5-DBr-d-Tyr significantly depressed PTZ-induced seizures. 3,5-DBr-d-Tyr did not cause significant changes in arterial blood pressure, heart rate and spontaneous locomotor activity, nor did it increase the number of activated caspase-3 positive cells in the brain. We conclude that 3,5-DBr-d-Tyr, by alleviating the deleterious effects of MCAo and PTZ in rats with no obvious intrinsic effects on cardiovascular parameters and neurodegeneration, exhibits promising potential as a novel therapeutic direction for stroke and seizures.     

Fmoc/solid-phase synthesis of Tyr(P)-containing peptides through t-butyl phosphate protection.

The synthesis of Tyr(P)-containing peptides by the use of Fmoc-Tyr(PO3Me2)-OH in Fmoc/solid phase synthesis is complicated since, firstly, piperidine causes cleavage of the methyl group from the -Tyr(PO3Me2)-residue during peptide synthesis and, secondly, harsh conditions are needed for its final cleavage. A very simple method for the synthesis of Tyr(P)-containing peptides using t-butyl phosphate protection is described. The protected phosphotyrosine derivative, Fmoc-Tyr(PO3tBu2)-OH was prepared in high yield from Fmoc-Tyr-OH by a one-step procedure which employed di-t-butyl N,N-diethyl-phosphoramidite as the phosphorylation reagent. The use of this derivative in Fmoc/solid phase peptide synthesis is demonstrated by the preparation of the Tyr(P)-containing peptides, Ala-Glu-Tyr(P)-Ser-Ala and Ser-Ser-Ser-Tyr(P)-Tyr(P).     

Low-temperature crystal structures of tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodico pper(II) and tetrakis-mu-3,5-diisopropylsalicylatobis-diethyletheratodicopp er(II) and their role in modulating polymorphonuclear leukocyte activity in overcoming seizures

Two binuclear copper(II) complexes of 3,5-diisopropylsalicylic acid were characterized by single crystal X-ray diffraction methods and examined for anti-inflammatory activity using activated polymorphonuclear leukocytes and for anticonvulsant activities using electroshock and metrazol models of seizures. These complexes were crystallized from dimethylformamide (DMF) or diethylether. Tetrakis-mu-3,5-diisopropylsalicylatobis-dimethylformamidodicop per(II) [Cu(II)2(3,5-DIPS)4(DMF)2] I is in space group P 1; a = 10.393 (2), b = 11.258 (2), c = 12.734 (2) A, alpha = 96.64 (2), beta = 92.95 (2), gamma = 94.90 (2) degrees; V = 1471.7 (4) A3; Z = 1. Tetrakis-mu-3,5-diisopropylsalicylatobis-etheratodicopper(II ) [Cu(II)2(3,5-DIPS)4(ether)2] II is in space group P 1; a = 10.409 (3), b = 11.901 (4), c = 12.687 (6) A, alpha = 91.12 (5), beta = 90.84 (5), gamma = 100.90 (4) degrees; V = 1542 (1) A3; Z = 1. The structure of I was determined at 140 K from 4361 unique reflections (I > 2sigma(1)) and refined on F2 to R1 = 0.04 and wR2 = 0.09. The structure of II was determined at 180 K from 4605 unique reflections (I > 2sigma(I)) and refined on F2 to R1 = 0.05 and wR2 = 0.13. Each compound is a crystallographically centrosymmetric binuclear complex with Cu atoms bridged by four 3,5-diisopropylsalicylate ligands related by a symmetry center [Cu-Cu(i): 2.6139 (9) A in I and 2.613 (1) in II]. The four nearest O atoms around each Cu atom form a nearly rectangular planar arrangement with the square pyramidal coordination completed by the dimethylformamide (or diethylether) oxygen atom occupying an apical position, at a distance of 2.129 (2) A in I and 2.230 (3) A in II. Each Cu atom is displaced towards the DMF (or diethylether) ligand, by 0.189 A in I and 0.184 A in II, from the plane of the four O atoms. The crystal structures of I and II are essentially similar to each other, except for the DMF or diethylether accommodation. Many disorder phenomena were found in the crystal structure of I. Copper(II)2(3,5-DIPS)4(DMF)2 inhibited polymorphonuclear leukocyte (PMNL) oxidative metabolism in vitro. This effect was concentration related and significant for concentrations higher than 10 microg or 0.68 nmol/ml. Copper(II)2(3,5-DIPS)4(DMF)2 was more active than the parent ligand, 3,5-DIPS, as has been demonstrated with copper complexes of other non-steroidal anti-inflammatory drugs. The DMF and diethylether ternary complexes of Cu(II)2(3,5-DIPS)4 were found to have anticonvulsant activity in the maximal electroshock model of grand mal epilepsy in doses ranging from 26 to 258 micromol/kg of body mass following intraperitoneal, subcutaneous, or oral treatment. The DMF ternary complex was also found to be effective in the subcutaneous injection of metrazol model of petit mal epilepsy. We conclude that both ternary copper complexes are lipophilic and bioavailable, capable of facilitating the inflammatory response to brain injury and causing the subsidence of this response in bringing about remission of these disease states.     

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

Urinary excretion of unconjugated and conjugated 3,5-diiodothyronine

A radioimmunoassay for the estimation of 3,5-diiodothyronine (3,5-T2) in human urine has been established. The urinary excretion of both glucuronide and sulfate conjugates of 3,5-T2 were estimated after enzymatic deconjugation. In 19 healthy controls the median excretion of unconjugated 3,5-T2 was 276 pmol/d, whereas the median excretion of glucuronidated and sulfated 3,5-T2 in 7 healthy subjects was 448 and 451 pmol/d, respectively. The median excretion of 154 pmol/d in 9 hypothyroid patients did not differ from that found in controls. In contrast 12 patients with hyperthyroidism had an enhanced excretion, 1312 pmol/d (P less than 0.01). Compared with previous data on the daily degradation of 3,5-T2, it is concluded that approximately one-sixth of degradated 3,5-T2 is excreted in the urine.     

Mechanism of an in vitro biosynthesis of thyroid hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine: II. Role of the pyridoxal phosphate and of the peroxidase

In this paper several details of the in vitro pathway of synthesis of hormones from 3,5-diiodo-l-tyrosyl-3,5-diiodo-l-tyrosine, I₂Tyr-I₂Tyr, have been investigated.

1. 1. We showed by incubations of I₂Tyr-I₂Tyr with a tautomerase that a phenylpyruvic form of the dipeptide did not occur.

2. 2. The reaction required pyridoxal phosphate: this coenzyme acts as an activator of the molecule.

3. 3. The peroxidase is involved in the reaction, since in the absence of a hydrogen peroxidase-generationg system there is no synthesis of iodothyronines when I₂Tyr-I₂Tyr is used as a precursor.

The significance of these findings is discussed and in particular it is concluded that such a mechanism cannot be transposed completely to conditions in vivo.     

Identifiying human MHC supertypes using bioinformatic methods

Classification of MHC molecules into supertypes in terms of peptide-binding specificities is an important issue, with direct implications for the development of epitope-based vaccines with wide population coverage. In view of extremely high MHC polymorphism (948 class I and 633 class II HLA alleles) the experimental solution of this task is presently impossible. In this study, we describe a bioinformatics strategy for classifying MHC molecules into supertypes using information drawn solely from three-dimensional protein structure. Two chemometric techniques-hierarchical clustering and principal component analysis-were used independently on a set of 783 HLA class I molecules to identify supertypes based on structural similarities and molecular interaction fields calculated for the peptide binding site. Eight supertypes were defined: A2, A3, A24, B7, B27, B44, C1, and C4. The two techniques gave 77% consensus, i.e., 605 HLA class I alleles were classified in the same supertype by both methods. The proposed strategy allowed "supertype fingerprints" to be identified. Thus, the A2 supertype fingerprint is Tyr(9)/Phe(9), Arg(97), and His(114) or Tyr(116); the A3-Tyr(9)/Phe(9)/Ser(9), Ile(97)/Met(97) and Glu(114) or Asp(116); the A24-Ser(9) and Met(97); the B7-Asn(63) and Leu(81); the B27-Glu(63) and Leu(81); for B44-Ala(81); the C1-Ser(77); and the C4-Asn(77).     

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.     

A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of 3H2O and adsorption of [3H]-tyrosine by charcoal

A rapid, simple and sensitive assay has been developed for tyrosine-3-monooxygenase, the enzyme catalyzing the rate-limiting step in catecholamine biosynthesis. The assay is based upon the release of 3H2O from 3H-[3,5]-L-tyrosine with adsorption of the isotopic substrate (and its metabolites) by an aqueous slurry of activated charcoal. This method routinely yields low blank values and is simpler than the procedure requiring the use of cation exchange columns to separate the isotopic substrate from the 3H2O formed during the hydroxylation reaction.     

Avidin binding of radiolabeled biotin derivatives

Three N-acyl derivatives of biotinylethylenediamine were prepared: I, biotinylamidoethyl-3-(3-[125I]iodo-4-hydroxyphenyl)propionamide; II, biotinylamidoethyl-[3H]acetamide; and III, biotinylamidoethyl-3-(3,5-[125I]diiodo-4-hydroxyphenyl)propionamid e. Each compound was combined with a large excess of avidin, yielding 1:1 molar complexes. Aside from a small fraction of each complex that dissociated more rapidly, the dissociation half-lives of these complexes were: I, 41 days; II, 4.4 days; and III, 148 days. The iodo- (mono or di) hydroxyphenylpropionyl moieties of I and III, therefore, contribute significantly to the binding strength of these compounds toward avidin. We also formed 4:1 complexes of I, II, and III with avidin (compound in excess), each of which exhibited biphasic dissociation, with initial half-lives of 4, 3.2, and 24 days, respectively. Thus, I or especially III potentially can be used as a sensitive tracer in quantitative studies with avidin.     

G(1)/S CDK is inhibited to restrain mitotic onset when DNA replication is blocked in fission yeast

Cyclin-dependent kinase (CDK) Tyr15 phosphorylation plays a major role in regulating G(2)/M CDKs, but the role of this phosphorylation in regulating G(1)/S CDKs is less clear. We have studied the regulation and function of Cdc2-Tyr15 phosphorylation in the fission yeast Schizosaccharomyces pombe G(1)/S CDK Cig2/Cdc2. This complex is subject to high level Cdc2-Tyr15 phosphorylation inhibiting its kinase activity in hydroxyurea-treated cells blocked in S-phase. We show that this Tyr15 phosphorylation is required to maintain efficient mitotic checkpoint arrest, because Cig2 accumulates during the block and this accumulation can advance mitotic onset. This mitotic induction operates, at least in part, through activation of the normal G(2)/M CDK complex Cdc13/Cdc2. Thus, Tyr15 phosphorylation of G(1)/S CDK complexes is important in the checkpoint control blocking mitotic onset when DNA replication is inhibited.     

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) potentiates cardiac contractility via activation of the ryanodine receptor

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.