Alzheimer's amyloid beta-peptide (1-42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing role for methionine 35

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.     

Protection of GroEL by its methionine residues against oxidation by hydrogen peroxide

GroEL undergoes an important functional and structural transition when oxidized with hydrogen peroxide (H2O2) concentrations between 15 and 20mM. When GroEL was incubated for 3h with 15 mM H2O2, it retained its quaternary structure, chaperone and ATPase activities. Under these conditions, GroEL's cysteine and tyrosine residues remained intact. However, all the methionine residues of the molecular chaperone were oxidized to the corresponding methionine-sulfoxides under these conditions. The oxidation of the methionine residues was verified by the inability of cyanogen bromide to cleave at the carboxyl side of the modified methionine residues. The role for the proportionately large number (23) of methionine residues in GroEL has not been identified. Methionine residues have been reported to have an antioxidant activity in proteins against a variety of oxidants produced in biological systems including H2O2. The carboxyl-terminal domain of GroEL is rich in methionine residues and we hypothesized that these residues are involved in the protection of GroEL's functional structure by scavenging H2O2. When GroEL was further incubated for the same time, but with increasing concentrations of H2O2 (>15 mM), the oxidation of GroEL's cysteine residues and a significant decrease of the tyrosine fluorescence due to the formation of dityrosines were observed. Also, at these higher concentrations of H2O2, the inability of GroEL to hydrolyze ATP and to assist the refolding of urea-unfolded rhodanese was observed.     

Oxidative and nitrative modifications of enkephalins by reactive nitrogen species

The interaction of Leucine-enkephalin (Leu-enkephalin) with reactive nitrogen species has been investigated. Reactive nitrogen species are capable of nitrating and oxidizing Leu-enkephalin. HPLC analysis shows the formation of two major enkephalin derivatives by peroxynitrite. The tyrosine amino-terminal residue of Leu-enkephalin is converted either to 3-nitrotyrosine thus producing nitroenkephalin and to dityrosine by dimerization with the production of an enkephalin dimer. The evidence of the formation of the nitroenkephalin and of the enkephalin dimer—dienkephalin—was achieved by electrospray ionisation mass spectrometry. In addition to peroxynitrite, the methylene blue photosensitized oxidation of enkephalin in the presence of nitrite leads to the formation of the nitrated peptide. Moreover, the nitropeptide can be also obtained by peroxidase-generated nitrogen reactive species.     

Methionine sulfoxide reductases preferentially reduce unfolded oxidized proteins and protect cells from oxidative protein unfolding

Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the K(m) lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins.     

Characterization and solution structure of mouse myristoylated methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. The cytosolic form of the enzyme is myristoylated, but it is not known to translocate to membranes, and the function of myristoylation is not established. We compared the biochemical and biophysical properties of myristoylated and nonmyristoylated mouse methionine sulfoxide reductase A. These were almost identical for both forms of the enzyme, except that the myristoylated form reduced methionine sulfoxide in protein much faster than the nonmyristoylated form. We determined the solution structure of the myristoylated protein and found that the myristoyl group lies in a relatively surface exposed "myristoyl nest." We propose that this structure functions to enhance protein-protein interaction.     

Probing the metal ion selectivity in methionine aminopeptidase via changes in the luminescence properties of the enzyme bound europium ion

We report herein, for the first time, that Europium ion (Eu³⁺) binds to the “apo” form of Escherichia coli methionine aminopeptidase (EcMetAP), and such binding results in the activation of the enzyme as well as enhancement in the luminescence intensity of the metal ion. Due to competitive displacement of the enzyme-bound Eu³⁺ by different metal ions, we could determine the binding affinities of both “activating” and “non-activating” metal ions for the enzyme via fluorescence spectroscopy. The experimental data revealed that among all metal ions, Fe²⁺ exhibited the highest binding affinity for the enzyme, supporting the notion that it serves as the physiological metal ion for the enzyme. However, the enzyme-metal binding data did not adhere to the Irving-William series. On accounting for the binding affinity vis a vis the catalytic efficiency of the enzyme for different metal ions, it appears evident that that the “coordination states” and the relative softness” of metal ions are the major determinants in facilitating the EcMetAP catalyzed reaction.     

Possible autocrine enkephalin regulation of catecholamine release in human pheochromocytoma cells

AIMS: Pheochromocytomas are catecholamine-secreting tumors that also synthesize and secrete several neuropeptides, including opioids. A negative regulation of catecholamine secretion by opioids has been postulated in chromaffin cells. However, results obtained so far are contradictory when referred to human pheochromocytomas. The aim of this study was to define the role of locally produced enkephalins on catecholamine release in human pheochromocytoma cells. MAIN METHODS: Cells obtained from eleven human pheochromocytomas of different genetic origins were cultured for 5 days. Cultures were maintained under basal condition or under enkephalin, dexamethasone and naloxone alone or in combination with enkephalin or dexamethasone-stimulated conditions. Catecholamine and enkephalin levels in the culture medium were measured by HPLC-ED and RIA respectively. KEY FINDINGS: Enkephalin induced a decrease in norepinephrine levels in all tumor cultures. Dexamethasone treatment, which increased enkephalin levels, also decreased catecholamine levels. On the other hand, the addition of naloxone to the cultures reverted to normal the inhibitory action exerted by enkephalin and dexamethasone treatments. SIGNIFICANCE: These results suggest the existence of an autocrine negative regulatory loop exerted by enkephalin on norepinephrine release in human pheochromocytoma cells.     

蛋氨酸脑啡肽对免疫系统恢复作用的研究

采用固相合成法合成蛋氨酸脑啡肽,制备成注射液。对经放化疗后免疫系统受到伤害的晚期肿瘤志愿者进行治疗,经静脉滴注15 d。患者免疫系统指标全面迅速恢复,淋巴细胞亚群间比例趋于合理,证明蛋氨酸脑啡肽可以用于癌症患者的免疫系统的恢复治疗。     

Reaction of [Pt(Gly-Gly-N,N',O)I]- with the N-acetylated dipeptide L-methionyl-L-histidine: selective platination of the histidine side chain by intramolecular migration of the platinum(II) complex

The reaction of the monofunctional [Pt(Gly-Gly-N,N′,O)I]⁻ complex, in which Gly-Gly is the dipeptide glycyl-glycine coordinated through two nitrogen and oxygen atoms, with the N-acetylated dipeptide l-methionyl-l-histidine (MeCOMet-His) studied by ¹H NMR spectroscopy. All reactions were carried out in 50 mM phosphate buffer at pD 7.4 and at 25 °C. In the initial stage of the reaction, the platinum(II) complex forms the kinetically favored [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-S)]⁻ complex, with unidentate coordination of the MeCOMet-His dipeptide through the sulfur atom of the methionine residue. In the second stage of the reaction, complete intramolecular migration of the [Pt(Gly-Gly-N,N′,O)] unit from the sulfur to the N3 nitrogen atom of imidazole was observed and a new platinum(II)-peptide complex, [Pt(Gly-Gly-N,N′,O)(MeCOMet-His-N3)]⁻ was formed. In comparison with previous results obtained for the reaction of [Pt(dien)Cl]⁺ with different methionine- and histidine-containing peptides, this migration reaction was sufficiently fast and strongly selective to the N3 atom of the imidazole ring of the histidine side chain. This study is an important step in the development of new platinum(II) complexes for selective covalent modification of peptides and proteins.     

1H, 15N and 13C NMR assignments of mouse methionine sulfoxide reductase B2

A recombinant mouse methionine-r-sulfoxide reductase 2 (MsrB2ΔS) isotopically labeled with ¹⁵N and ¹⁵N/¹³C was generated. We report here the ¹H, ¹⁵N, and ¹³C NMR assignments of the reduced form of this protein. An erratum to this article can be found at