Crystal structure of p14TCL1, an oncogene product involved in T-cell prolymphocytic leukemia, reveals a novel beta-barrel topology.

BACKGROUND: Chromosome rearrangements are frequently involved in the generation of hematopoietic tumors. One type of T-cell leukemia, T-cell prolymphocytic leukemia, is consistently associated with chromosome rearrangements characterized by the juxtaposition of the TCRA locus on chromosome 14q11 and either the TCL1 gene on 14q32.1 or the MTCP1 gene on Xq28. The TCL1 gene is preferentially expressed in cells of early lymphoid lineage; its product is a 14 kDa protein (p14TCL1), expressed in the cytoplasm. p14TCL1 has strong sequence similarity with one product of the MTCP1 gene, p13MTCP1 (41% identical and 61% similar). The functions of the TCL1 and MTCP1 genes are not known yet. They have no sequence similarity to any other published sequence, including those of well-documented oncogene families responsible for leukemia. In order to gain a more fundamental insight into the role of this particular class of oncogenes, we have determined the three-dimensional structure of p14TCL1. RESULTS: The crystal structure of p14TCL1 has been determined at 2.5 A resolution. The structure was solved by molecular replacement using the solution structure of p13MTCP1, revealing p14TCL1 to be an all-beta protein consisting of an eight-stranded antiparallel beta barrel with a novel topology. The barrel consists of two four-stranded beta-meander motifs, related by a twofold axis and connected by a long loop. This internal pseudo-twofold symmetry was not expected on basis of the sequence alone, but structure-based sequence analysis of the two motifs shows that they are related. The structures of p13MTCP1 and p14TCL1 are very similar, diverging only in regions that are either flexible and/or involved in crystal packing. p14TCL1 forms a tight crystallographic dimer, probably corresponding to the 28 kDa species identified in solution by gel filtration experiments. CONCLUSIONS: Structural similarities between p14TCL1 and p13MTCP1 suggest that their (unknown) function may be analogous. This is confirmed by the fact that these proteins are implicated in analogous diseases. Their structure does not show similarity to other oncoproteins of known structure, confirming their classification as a novel class of oncoproteins.

pH dependence of 5-fluorouracil uptake observed by in vivo 31P and 19F nuclear magnetic resonance spectroscopy.

Multinuclear nuclear magnetic resonance spectroscopy was used to follow the metabolism and kinetics of 5-fluorouracil (5FU) after i.v. administration at a dose of 100 mg/kg on Wistar rats. 31P spectra allow one to determine both the energetic status and the pH of the tissues under investigation, while serial 19F spectra reveal the drug clearance. Analyses of 5FU kinetics show that the half-life of 5FU elimination is about 35 min in tissue with a pH of 7.3. However, this half-life increases 2.5-fold when the local pH decreases below 6.9. Thus, acidification seems to induce a local retention of 5FU, which tends to prove the existence of active transport. This retention of the drug may have significant clinical implications for assessing and improving chemotherapy alone or in combination.

ESR study of free and immobilized elastase.

Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.

Comparative study of the iron-binding properties of human transferrins. II. Electron paramagnetic resonance of mixed metal complexes of human lactotransferrin.

Human lactotransferrin is able to bind two vanadyl(IV) ions in specific metal-binding sites. The EPR signals of the two vanadyl bound ions, however, appear as one. This result suggests that the environments of the binding sites of human lactotransferrin are similar. The binding activity is promoted to pH 4 using carbonate or bicarbonate as synergistic anion. This unusual stability of the anion-binding site, which is destroyed below pH 6 for other transferrins, can explain in part the great stability of the metallic complexes of human lactotransferrin. However, the different sensitivities of the two metal-binding sites towards protonation permit the preparation of mixed vanadyl(IV), iron(III) complexes with VO2+ bound either on the N-terminal (acid-labile or B site) or on the C-terminal (acid-stable or A site) site. Analysis of the spectra of such mixed complexes shows the presence of a third nonspecific VO2+-binding site termed A'. The nonspecific A' site seems to be located on the outer surface of the protein close to the C-terminal site.

Solution structure of human p8MTCP1, a cysteine-rich protein encoded by the MTCP1 oncogene, reveals a new alpha-helical assembly motif.

MTCP1 (for Mature-T-Cell Proliferation) is the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8(MTCP1) protein encoded by the MTCP1 oncogene was determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After sequence specific assignments, a total of 931 distance restraints and 57 dihedral restraints were collected. The location of the three previously unassigned disulfide bridges was determined from preliminary DIANA structures, using a statistical analysis of intercystinyl distances. The solution structure of p8(MTCP1) is presented as a set of 30 DIANA structures, further refined by restrained molecular dynamics using a simulated annealing protocol with the AMBER force field. The r.m.s.d. values with respect to the mean structure for the backbone and all heavy atoms for a family of 30 structures are 0.73(+/-0.28) and 1.17(+/-0.23) A, when the structured core of the protein (residues 5 to 63) is considered. The solution structure of p8(MTCP1) reveals an original scaffold consisting of three alpha helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-hairpin which resembles an antiparallel coiled-coil. The third helix is oriented roughly parallel to the plane defined by the alpha-antiparallel motif and its axis forms an angle of approximately 60 degrees with respect to the main axis of this motif.

Metabolic effects of R-phenylisopropyladenosine during reversible forebrain ischemia studied by in vivo 31P nuclear magnetic resonance spectroscopy.

The metabolic effects of R-phenylisopropyladenosine (R-PIA), an agonist of adenosine A1 receptors, were studied by in vivo 31P NMR spectroscopy before, during, and after 30 min of reversible forebrain ischemia in the rat. R-PIA had no effect on cerebral metabolism before ischemia. During a 30-min ischemia, R-PIA reduced the decrease in phosphocreatine (43 +/- 11% of the control level at the end of ischemia vs. 27 +/- 9% in the reference group) and ATP (58 +/- 12% vs. 40 +/- 23%) and the increase in inorganic phosphate (672 +/- 210% vs. 905 +/- 229%). The intracellular acidosis elicited by ischemia was also less in the treated group (pH of 6.40 +/- 0.10 vs. 6.30 +/- 0.10). Recirculation was associated with a faster recovery of PCr, ATP, Pi, and pHi to control levels in the treated group than in the reference group. It is concluded that adenosine protects against ischemic injury by mechanisms that include metabolic protection.

NMR investigation of the electric properties and magnetic states of hemes and hemoproteins : the S = 2 state in ferrous porphyrins.

The proton NMR data of a synthetic model of deoxy-myoglobin and hemoglobin show one that:(i) the ground state of the high spin iron (II) complex must be described by at least two electronic levels separated by ca.200 cm-1 and resulting from the removal of the degeneracy of the 5E level; (ii) this in-plane rhombic distorsion is directed towards the methine bridges of the porphyrin ring. It is proposed that the rhombicity results from specific interactions of the lambda orbitals of the axial base with the iron d lambda orbitals.

Conformational analysis of pGlu-His-Amph and pGlu-His-Pro-Amph related to their central nervous system activity.

The compounds pGlu-His-Pro-Amph and pGlu-His-Amph obtained from the condensation of TRH or a fragment of TRH with amphetamine show activities which are different regarding the parent compounds. Although the two derivatives exhibit about the same low toxicity they differ in several pharmacological properties. Physicochemical analysis by 1H-NMR and CD spectroscopy was carried out in order to detect in the two compounds conformational differences that might explain their different activities. The results show that in the proline containing peptide the amphetamine has a hindered rotation in comparison with the compounds devoid of proline. This, together with the occurrence of a cis conformer having different properties than the trans conformer could be the origin of the biological difference observed between the two hybrid compounds.

The seven-stranded beta-barrel structure of apo-neocarzinostatin as compared to the immunoglobulin domain.

The three-dimensional structure of apo-NCS, as revealed by proton NMR, is based on an antiparallel seven-stranded beta-barrel. This fold is frequently encountered in protein structures, especially for immunoglobulin domains. The strands forming the barrel are joined by flexible loops of which three are implicated in the ligand binding site of these proteins. In this paper a preliminary comparison is given with respect to the static and dynamic properties of both the constant beta-barrel and the active loops for apo-NCS and the variable VH domain of an immunoglobulin Fab' fragment.

Synthesis and carcinogenic activity of oxidized benzacridines: potential metabolites of the strong carcinogen 7-methylbenz[c]acridine and of the inactive isomer 12-methylbenz[a]acridine.

The synthesis of 15 compounds related either to the benz[c]acridine or to the benz[a]acridine series is reported. Spectral data, i.e., NMR and EI fragmentation, are given. These compounds were tested for carcinogenic activity in mice of the XVIInc/Z strain by subcutaneous injection. Only three weak carcinogens were detected, 5,6-dihydro-5,6-dihydroxy-12-methylbenz[a]acridine, 3-methoxy-7-methylbenz[c]acridine, and 4-acetoxy-7-methylbenz[c]acridine. These results are discussed with consideration to the data previously obtained with other benzacridines and condensed quinolines.

Antitumor amino-substituted pyrido[3',4':4,5]pyrrolo[2,3-g]isoquinolines and pyrido[4,3-b]carbazole derivatives: synthesis and evaluation of compounds resulting from new side chain and heterocycle modifications.

New modifications of 10-[[3-(diethylamino)propyl]amino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-g]i sisoquinoline (1b) and 1-[[3-(diethylamino)propyl]amino]-9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carba zole (4b), which display important antitumor properties, were performed either on the side chain or on the intercalating heterocycle. Side chains were introduced by direct substitution of the corresponding chloro derivatives and 6-N-methyl-9-hydroxypyrido[4,3-b]carbazoles analogues were prepared via 9-O-benzoyl-1-chloroellipticines. Evaluation of all new compounds shows no significant increase of in vitro cytotoxicity and percent ILS on the L1210 leukemia system by comparison with the model compounds 1b and 4b.

Synthesis and antitumor activity of 1-[[(dialkylamino)alkyl]amino]-4-methyl-5H-pyrido[4,3-b]benzo[e]- and -benzo[g])indoles. A new class of antineoplastic agents.

The thermal Fischer indolization of hydrazones resulting from 4-hydrazino-5-methyl-1H-pyridin-2-one and various beta- and alpha-tetralones led to 4-methyl-6,7-dihydro-2H,5H-pyrido[4,3- b]benzo[e]indol-1-ones and 4-methyl-11-dihydro-2H,5H-pyrido[4,3- b]benzo[g]indol-1-ones, respectively. After aromatization, these compounds were transformed by phosphorus oxychloride, giving 1-chloro-4-methyl-5H-pyrido[4,3- b]benzo[e]- and -benzo[g]indoles which were substituted by [(dialkylamino)alkyl]amines. The resulting 1-[[(dialkylamino)alkyl]amino]-4-methyl-5H-pyrido- [4,3-b]benzo[e]- and -benzo[g]indoles, as well as hydroxy derivatives obtained by demethylation of methoxylated compounds with hydrobromic acid, were tested for antitumor activity in vitro (leukemic and solid tumor cells) and in vivo on various experimental tumor models using the standard NCI protocols. 1-[[3-(Dialkylamino)propyl]-amino]-4-methyl-9-hydroxy-5H-pyrido[4,3- b]benzo[e]indoles appeared as a promising new class of antineoplastic agents.

Follow-up by 31P NMR spectroscopy of the energy metabolism of malignant tumor in rats during treatment.

The energy metabolism of tumors in rats was investigated by in vivo 31P-NMR spectroscopy. The effects of radiotherapy, chemotherapy or radiotherapy combined with 5-fluorouracil (5-FU) chemotherapy were evaluated by observing the changes of these spectra in chemically induced subcutaneous fibrosarcoma in rats. Two milligrams of DMBA in solution in olive oil were administered subcutaneously in the flank of 20 Wistar rats and 17 fibrosarcoma occurred. 31P NMR spectra were recorded with a Brüker Medspec 30/47 spectrometer using a surface coil positioned over the tumor. We did not observe significant changes in the spectra during tumor growth. Radiotherapy and 5-FU chemotherapy alone did not induce major changes in the 31P spectra. But the situation was completely different for animals receiving the therapeutic combination. A clear increase in the ratio of inorganic phosphate to total phosphorus signal was observed 48 h after the first irradiation session. The pH shifted concurrently to the acidic range. No effect on tumor regression was observed in the rats from the chemotherapy group, while regression was less than 50% in rats treated by irradiation only, and at least 80% in the combined group.

Magnetic resonance imaging studies on nude mice grafted with colorectal adenocarcinoma using gadolinium-labeled monoclonal antibody.

A monoclonal antibody (Ab) 19.9 specific for colorectal carcinoma was labeled with a high number of gadolinium (Gd) atoms for its potential application as a contrast agent in magnetic resonance imaging (MRI). The DTPA was conjugated to 19.9 Ab via the bicyclic DTPA anhydride method (c. DTPA) using c. DTPA/Ab molar ratios between 5 and 150. The aggregates present in great amount at high c. DTPA/Ab ratios were systematically removed. Then the exact number of DTPA effectively conjugated, the immunoreactivity of the resulting 111In-DTPA-Ab were measured. The number of DTPA conjugated per antibody can be increased 20 to 25 with only a little loss of immunoreactivity. The 19.9 antibody conjugated with 16 and 25 DTPA was labeled with 153GdCl3 for pharmacokinetic studies on xenografted nude mice and with nonradioactive gadolinium to measure ex vivo the effect on the relaxation time T1 of the tumor. We found a 15 to 20% decrease of T1 on the tumor. In vivo experiments using a Bruker system and the same animal model showed a difference in the tumor contrast after the injection of 2 mg of Gd-labeled Ab.

Metabolic effects of kynurenate during reversible forebrain ischemia studied by in vivo 31P-nuclear magnetic resonance spectroscopy.

The metabolic effects of kynurenate, an endogenous excitatory amino acid antagonist, were studied by in vivo 31P-NMR spectroscopy before, during and after reversible forebrain ischemia in the rat. Kynurenate had no effect on cerebral metabolism before ischemia. During a 30-min ischemia, kynurenate protected against the decrease in phosphocreatine (up to -55 +/- 3% vs -73 +/- 3% in the reference group) and the increase in inorganic phosphate (up to +479 +/- 39% vs +805 +/- 66%), whereas there was no statistical difference in the decrease in intracellular pH (up to 6.37 +/- 0.05 vs 6.30 +/- 0.03) and ATP (up to -60 +/- 3% vs -60 +/- 7%). The recovery of PCr, Pi, and pHi to control levels during recirculation was faster in the treated group than in the reference group, whereas the time course of ATP recovery was similar in both groups. We conclude that kynurenate protects against neuronal loss, as previously reported, by mechanisms other than metabolic protection.

Cerebral metabolic changes induced by MK-801: a 1D (phosphorus and proton) and 2D (proton) in vivo NMR spectroscopy study.

The dynamic effects of the non-competitive NMDA receptor antagonist, MK-801 on brain metabolism were investigated over 105 minutes in unanesthetized rats by proton and phosphorus NMR spectroscopy. MK-801 (0.5 and 5 mg/kg, i.p) induced no changes in intracellular pH, and in phosphocreatine, ATP, and inorganic phosphate levels, indicating that the drug preserved energy and intracellular pH homeostasis. There were transient increases in lactate after both doses of MK-801, suggesting early activation of glycolysis, which was not immediately matched by enhanced oxidative metabolism or by enhanced blood flow. Thereafter, lactate control level was not restored after 0.5 mg/kg whereas it was restored after 5 mg/kg in spite of a sustained metabolic activation. The low dose of MK-801 also caused a continuous decrease in cerebral aspartate level (-38%) which is thought to match the enhanced energy demand, whereas the high dose caused shorter and smaller changes. The intracerebral glucose level rose after MK-801 injection, indicating that brain tissue had an adequate or even excessive supply of glucose. Glucose time course seemed to closely match the changes in blood flow elicited by MK-801. This is the first study giving the metabolic pattern of a pharmacological activation. We demonstrate an excess of glycolysis over oxidative metabolism in the early time similar to that following physiological and pathophysiological states such as photic stimulation and seizures. The difference between the effects of the two doses of MK-801 suggests that the adjustment of cerebral metabolism to MK-801 activation is faster and greater with the high dose than with the low dose.