Mitochondrial GTP-AMP phosphotransferase. 1. Purification and properties.

GTP-AMP phosphotransferase has been purified 116-fold with a yield of 24% from beef heart mitochondria using freeze-thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G-100 and blue-dextran--Sepharose. It has crystallized from poly-(ethylene glycol) and is essential homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no --SS-- bonds or free --SH. Guanosine(5')pentaphospho(5')adenosine is a very strong inhibitor similar to adenosine(5')pentaphospho(5')adenosine as an inhibitor of cytosolic adenylate kinase.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.     

Enantiomeric cuparene-type sesquiterpenoids from Bazzania pompeana

Two enantiomeric cuparene-type sesquiterpenoids, (R)-(−)-cuparene (1) and (R)-(−)-δ-cuparenol (2), have been isolated from the liverwort, Bazzania pompeana. The structures and absolute configurations of the two compounds have been determined.     

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin

The enzyme GnT-III (beta 1,4-N-acetylglucosaminyltransferase III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) residue on glycoproteins. Our previous study described that the transfection of GnT-lll into mouse melanoma cells results in the enhanced expression of E-cadherin, which in turn leads to the suppression of lung metastasis. It has recently been proposed that the phosphorylation of a tyrosine residue of beta-catenin is associated with cell migration. The present study reports on the importance of bisecting GlcNAc residues by GnT-lll on tyrosine phosphorylation of beta-catenin using three types of cancer cell lines. An addition of bisecting GlcNAc residues to E-cadherin leads to an alteration in cell morphology and the localization of beta-catenin after epidermal growth factor stimulation. These changes are the result of a down-regulation in the tyrosine phosphorylation of beta-catenin. In addition, tyrosine phosphorylation of beta-catenin by transfection of constitutively active c-src was suppressed in GnT-III transfectants as well as in the case of epidermal growth factor stimulation. Treatment with tunicamycin abolished any differences in beta-catenin phosphorylation for the mock vis à vis the GnT-lll transfectants. Thus, the addition of a specific N-glycan structure, the bisecting GlcNAc to E-cadherin-beta-catenin complex, down-regulates the intracellular signaling pathway, suggesting its implication in cell motility and the suppression of cancer metastasis.     

Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.     

Chemical modulators of autophagy as biological probes and potential therapeutics

Autophagy is an evolutionarily conserved mechanism for protein degradation that is critical for the maintenance of homeostasis in man. Autophagy has unexpected pleiotropic functions that favor survival of the cell, including nutrient supply under starvation, cleaning of the cellular interior, defense against infection and antigen presentation. Moreover, defective autophagy is associated with a diverse range of disease states, including neurodegeneration, cancer and Crohn's disease. Here we discuss the roles of mammalian autophagy in health and disease and highlight recent advances in pharmacological manipulation of autophagic pathways as a therapeutic strategy for a variety of pathological conditions.