Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Ketogenic essential amino acids replacement diet ameliorated hepatosteatosis with altering autophagy-associated molecules

Ketogenic amino acid (KAA) replacement diet has been shown to cure hepatic steatosis, a serious liver disease associated with diverse metabolic defects. In this study, we investigated the effects of KAA replacement diet on nutrition sensing signaling pathway and analyzed whether induction of hepatic autophagy was involved. Mice are fed with high fat diet (HFD) or KAA replacement in high-fat diet (30% fat in food; HFD)-fed (HFD^KAAR) and sacrificed at 8, 12, 16 weeks after initiation of experimental food. Hepatic autophagy was analyzed in protein expression of several autophagy-associated molecules and in light chain-3 green fluorescent protein (LC-3 GFP) transgenic mice. HFD^KAAR showed increased AMP-activated protein kinase (AMPK) phosphorylation and enhanced liver kinase B1 (LKB1) expression compared to control HFD-fed mice. The KAA-HFD-induced activation of AMPK was associated with an increased protein expression of sirtuin 1 (Sirt1), decreased forkhead box protein O3a (Foxo3a) level, and suppression of mammalian target of rapamycin (mTOR) phosphorylation compared with the HFD-fed mice. The intervention study revealed that a KAA-replacement diet also ameliorated all the established metabolic and autophagy defects in the HFD-fed mice, suggesting that a KAA-replacement diet can be used therapeutically in established diseases. These results indicate that KAA replacement in food could be a novel strategy to combat hepatic steatosis and metabolic abnormalities likely involvement of an induction of autophagy.     

A new membrane-bound cytochrome c works as an electron donor to the photosynthetic reaction center complex in the purple bacterium, Rhodovulum sulfidophilum

A new type of membrane-bound cytochrome c was found in a marine purple photosynthetic bacterium, Rhodovulum sulfidophilum. This cytochrome c was significantly accumulated in cells growing under anaerobic photosynthetic conditions and showed an apparent molecular mass of approximately 100 kDa when purified and analyzed by SDS-PAGE. The midpoint potential of this cytochrome c was 369 mV. Flash-induced kinetic measurements showed that this new cytochrome c can work as an electron donor to the photosynthetic reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular mass was nearly equal to 50 kDa. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochrome c(2), although its predicted secondary structure resembles that of cytochrome c(y). Phylogenetic analyses suggested that this new cytochrome c has evolved from cytochrome c(2). We, thus, propose its designation as cytochrome c(2m). Mutants lacking this cytochrome or cytochrome c(2) showed the same growth rate as the wild type. However, a double mutant lacking both cytochrome c(2) and c(2m) showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c(2m) or the water-soluble cytochrome c(2) work as a physiological electron carrier in the photosynthetic electron transfer pathway of Rvu. sulfidophilum.     

Polyglutamine expansion mutation yields a pathological epitope linked to nucleation of protein aggregate: determinant of Huntington's disease onset

Polyglutamine (polyQ) expansion mutation causes conformational, neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. These diseases are characterized by the aggregation of misfolded proteins, such as amyloid fibrils, which are toxic to cells. Amyloid fibrils are formed by a nucleated growth polymerization reaction. Unexpectedly, the critical nucleus of polyQ aggregation was found to be a monomer, suggesting that the rate-limiting nucleation process of polyQ aggregation involves the folding of mutated protein monomers. The monoclonal antibody 1C2 selectively recognizes expanded pathogenic and aggregate-prone glutamine repeats in polyQ diseases, including Huntington's disease (HD), as well as binding to polyleucine. We have therefore assayed the in vitro and in vivo aggregation kinetics of these monomeric proteins. We found that the repeat-length-dependent differences in aggregation lag times of variable lengths of polyQ and polyleucine tracts were consistently related to the integration of the length-dependent intensity of anti-1C2 signal on soluble monomers of these proteins. Surprisingly, the correlation between the aggregation lag times of polyQ tracts and the intensity of anti-1C2 signal on soluble monomers of huntingtin precisely reflected the repeat-length dependent age-of-onset of HD patients. These data suggest that the alterations in protein surface structure due to polyQ expansion mutation in soluble monomers of the mutated proteins act as an amyloid-precursor epitope. This, in turn, leads to nucleation, a key process in protein aggregation, thereby determining HD onset. These findings provide new insight into the gain-of-function mechanisms of polyQ diseases, in which polyQ expansion leads to nucleation rather than having toxic effects on the cells.     

Myristoylation of human immunodeficiency virus type 1 gag protein is required for efficient env protein transportation to the surface of cells

Highly conserved amino acids in the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) Pr55(gag) are recognized to be critical for the attachment of myristic acid. We previously reported that the env protein was not detected on the cell surface by blocking of N-myristoylation of Pr55(gag) with N-myristoyl glycinal diethylacetal. Here, we constructed a mutant by substituting the N-terminal glycine of Pr55(gag) with alanine to demonstrate that N-myristoylation of Pr55(gag) is required for efficient env protein transportation to the cell surface. The expression level of the env protein on the surface of Jurkat cells transfected with the myristoylation-defective phenotype was observed to be significantly reduced by electron microscopic analyses with a gold-labeled monoclonal antibody against the env protein. In addition, Jurkat cells transfected with the myristoylation-defective phenotype lost the ability of envelope-mediated cell-to-cell fusion. The results suggest that N-myristoylation of the HIV-1 gag protein is necessary for efficient env protein transportation to the cell surface.     

Specific Frequency Distribution of Erythrocytic X-Protein Alleles in Indigenous Sheep Populations in East Asia

The nonhemoglobin erythrocytic X-protein polymorphism consisting of two phenotypes called X-positive [X(+)] and X-negative [X(-)] was determined in 576 unrelated healthy native sheep of East Asia, using one-dimensional and horizontal starch gel electrophoresis. A striking difference in the frequency of the X allele coding dominantly for the X(+) type between the northern and southern populations of native East Asian sheep divided by the boundary of the Himalaya Mountains was seen (P < 0.0001). The X allele frequency ranged from 0 to 0.0438 with an average of 0.0323 in the northern population examined, consisting of the Bhyanglung, Baruwal, Yunnan, and Khalkhas sheep belonging to the Tibetan and Mongolian sheep groups. In contrast, the frequency of the same allele was in the range of 0.2037-0.4655 and the mean frequency was 0.2998 in the southern population tested, consisting of the Bengal, Kagi, Lampuchhre, Vietnamese, and Myanmar sheep, which belong to the Indian sheep group. This finding suggests that the X allele appears to be an Indian sheep marker and is potentially important in phylogenetic studies on native sheep populations, especially in East Asia.     

Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin

We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.     

Blockage of HIV-1 production through inhibition of proviral DNA synthesis by N,O-didecanoyl serinal dimethylacetal

Six serinal derivatives were synthesized and tested for their anti-human immunodeficiency virus type-1 (HIV-1) activity against HIV-1-infected cells. Of the 6 serinal derivatives tested, only N,O-didecanoyl serinal dimethylacetal (DDSD) was found to strongly suppress progeny virus production from acute HIV-1-infected CEM cells, while not suppressing the HIV-1 p24 production from latent HIV-1-infected ACH-2 cells after stimulation with phorbol 12-myristate 13-acetate. DDSD also inhibited the synthesis of HIV-1 proviral DNA at 20-50 microM, not only 1 h but also 24 h after HIV-1 infection. Taken together, DDSD is a potent inhibitor of HIV-1 production, and may become a unique leading compound for chemotherapy of acquired immunodeficiency syndrome.