Regulation of nuclear translocation of extracellular signal-regulated kinase 5 by active nuclear import and export mechanisms

Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.     

Deletion of vitamin E enhances phenotype of Alzheimer disease model mouse

Increased oxidative damage is a prominent and early feature in Alzheimer disease (AD). However, whether it is a primary cause or merely a downstream consequence in AD pathology is still unknown. We previously generated alpha-tocopherol transfer protein knockout (Ttpa-/-) mice, in which lipid peroxidation in the brain was significantly increased by complete depletion of alpha-tocopherol (alpha-Toc). Here we crossed AD transgenic (APPsw) model mice (Tg2576) with Ttpa-/- mice. The resulting double-mutant (Ttpa-/- APPsw) mice showed earlier and more severe cognitive dysfunction in the Morris water maze, novel-object recognition, and contextual fear conditioning tests. They also showed increased amyloid beta-peptide (Abeta) deposits in the brain by immunohistochemical analysis, which was ameliorated with alpha-Toc supplementation. In this report we provide clear evidence indicating that chronic lipid peroxidation due to alpha-Toc depletion enhances AD phenotype in a mouse model.     

Structure, function and evolution of the mitochondrial division apparatus

Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses.     

Campest-5-en-3-one, an oxidized derivative of campesterol, activates PPARalpha, promotes energy consumption and reduces visceral fat deposition in rats

Dietary campest-5-en-3-one (campestenone), an oxidized derivative of campesterol, significantly reduced visceral fat weight and the concentration of triacylglycerol in serum and liver of rats. Dietary campestenone dramatically increased the activities and the mRNA expressions of mitochondrial and peroxisomal enzymes involved in beta-oxidation in the liver. Campestenone activated human peroxisome proliferator-activated receptor (PPAR) alpha as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. In contrast, dietary campestenone reduced the activities and the mRNA expressions of enzymes involved in fatty acid synthesis, except for the malic enzyme. Dietary campestenone decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level. Energy expenditure was significantly higher in the feeding of campestenone in rats. Dietary campestenone reduced hepatic cholesterol concentration and increased fecal excretion of neutral steroids originated from cholesterol. Lymphatic absorption of cholesterol was reduced by the coadministration of campestenone in rats cannulated in the thoracic duct. These observations suggest a possibility that campestenone has an ability to prevent coronary heart disease by improving obesity and abnormality of lipid metabolism.     

Syntheses, structures, and mesomorphism of a series of Ni(II) salen complexes with 4-substituted long alkoxy chains

A series of nickel(II) salen complexes containing 4-substituted alkoxy chains of aromatic rings, [Ni((4-C_nH_2n + 1O)₂salen)] (n = 3 (1), 4 (2), 6 (3), 8 (4), 10 (5), 12 (6), 14 (7), 16 (8), 18 (9), and 20 (10)), and their parent complex, [Ni((4-HO)₂salen)] (11) (salen = N,N′-ethylenebis(salicylideneiminato)), have been prepared and mesomorphic properties have been investigated. An X-ray crystallographic analysis revealed that complex 11 · 2DMF has one-dimensional stacking structure supported by the π-π interaction between the six-membered chelate and aromatic rings with the NiNi distances of alternatively 3.3957 and 3.7224 Å and that complex 3 is formed by one-dimensional stacking by weak CH?O type hydrogen bonded interaction between the five-membered chelate ring and phenoxo atoms of the dramatically distorted salen moieties with the NiNi distance of 5.994 Å. Complexes 1-6 did not exhibit any mesophases. On the other hand, complexes 7-10 with longer alkoxy chains of n = 14-20 showed an unusual metallomesogen of a lamello-columnar mesophase within the smectic layers with an interlamellar distance of 31.1 Å (7), 33.6 Å (8), 37.1 Å (9), and 39.5 Å (10) and nearly constant stacking distance of 6.19-6.24 Å between the inter-dimers, irrespective of the variation of the alkoxy chain lengths by the X-ray diffraction measurements of the liquid crystal. The relationship between molecular assemblies and mesomorphic properties is discussed.     

Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the (13)C resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that (13)C NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.     

Role of 1-hydroxybenzotriazole in oxidation by laccase from Trametes versicolor. Kinetic analysis of the laccase-1-hydroxybenzotriazole couple

In the current studies, we used Lineweaver-Burke analysis to examine the role of 1-hydroxybenzotriazole (HBT) in the oxidation of various compounds by laccase from Trametes versicolor. At low concentrations, HBT was a competitive inhibitor of the oxidation, but at high concentrations, it was a noncompetitive inhibitor. Analysis of the oxidation of ferrocytochrome c by the laccase-HBT couple showed that increasing the concentration of ferrocytochrome c did not affect the V(max) but reduced the apparent K(m). In addition, in the manganese peroxidase-Mn(II) reaction, which is a typical oxidation system by mediator, the apparent K(m) and V(max) increased as the concentration of the substrate 2,6-dimethoxyphenol was increased. These results indicate that HBT is involved in the binding of laccase and substrates that laccase cannot oxidize alone.