Suppression of Adipocyte Differentiation by Aldo-keto Reductase 1B3 Acting as Prostaglandin F2�� Synthase

Prostaglandin (PG) F_2α suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor γ. However, PGF_2α synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF_2α, was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF_2α production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF_2α. These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF_2α suppressed adipocyte differentiation by acting through FP receptors.     

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3β (GSK-3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Interaction between glycogenin and glycogen synthase

Glycogen synthase plays a key role in regulating glycogen metabolism. In a search for regulators of glycogen synthase, a yeast two-hybrid study was performed. Two glycogen synthase-interacting proteins were identified in human skeletal muscle, glycogenin-1, and nebulin. The interaction with glycogenin was found to be mediated by the region of glycogenin which contains the 33 COOH-terminal amino acid residues. The regions in glycogen synthase containing both NH2- and COOH-terminal phosphorylation sites are not involved in the interaction. The core segment of glycogen synthase from Glu21 to Gly503 does not bind COOH-terminal fragment of glycogenin. However, this region of glycogen synthase binds full-length glycogenin indicating that glycogenin contains at least one additional interacting site for glycogen synthase besides the COOH-terminus. We demonstrate that the COOH-terminal fragment of glycogenin can be used as an effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver.     

Frizzled7 as an emerging target for cancer therapy

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

The central shaft of the catalytic core of ATP synthase, the γ subunit consists of a coiled-coil structure of N- and C-terminal α-helices, and a globular domain. The γ subunit of cyanobacterial and chloroplast ATP synthase has a unique 30–40-amino acid insertion within the globular domain. We recently prepared the insertion-removed α₃β₃γ complex of cyanobacterial ATP synthase (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865). Although the insertion is thought to be located in the periphery of the complex and far from catalytic sites, the mutant complex shows a remarkable increase in ATP hydrolysis activity due to a reduced tendency to lapse into ADP inhibition. We postulated that removal of the insertion affects the activity via a conformational change of two central α-helices in γ. To examine this hypothesis, we prepared a mutant complex that can lock the relative position of two central α-helices to each other by way of a disulfide bond formation. The mutant obtained showed a significant change in ATP hydrolysis activity caused by this restriction. The highly active locked complex was insensitive to N-dimethyldodecylamine-N-oxide, suggesting that the complex is resistant to ADP inhibition. In addition, the lock affected ϵ inhibition. In contrast, the change in activity caused by removal of the γ insertion was independent from the conformational restriction of the central axis component. These results imply that the global conformational change of the γ subunit indirectly regulates complex activity by changing both ADP inhibition and ϵ inhibition.     

Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is responsible for proinflammatory reactions in various infectious and non-infectious diseases. We have investigated the mechanism of anti-inflammatory activity of epoxyazadiradione, a limonoid purified from neem (Azadirachta indica) fruits, against MIF. Epoxyazadiradione inhibited the tautomerase activity of MIF of both human (huMIF) and malaria parasites (Plasmodium falciparum (PfMIF) and Plasmodium yoelii (PyMIF)) non-competitively in a reversible fashion (K(i), 2.11-5.23 μm). Epoxyazadiradione also significantly inhibited MIF (huMIF, PyMIF, and PfMIF)-mediated proinflammatory activities in RAW 264.7 cells. It prevented MIF-induced macrophage chemotactic migration, NF-κB translocation to the nucleus, up-regulation of inducible nitric-oxide synthase, and nitric oxide production in RAW 264.7 cells. Epoxyazadiradione not only exhibited anti-inflammatory activity in vitro but also in vivo. We tested the anti-inflammatory activity of epoxyazadiradione in vivo after co-administering LPS and MIF in mice to mimic the disease state of sepsis or bacterial infection. Epoxyazadiradione prevented the release of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and TNF-α when LPS and PyMIF were co-administered to BALB/c mice. The molecular basis of interaction of epoxyazadiradione with MIFs was explored with the help of computational chemistry tools and a biological knowledgebase. Docking simulation indicated that the binding was highly specific and allosteric in nature. The well known MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) inhibited huMIF but not MIF of parasitic origin. In contrast, epoxyazadiradione inhibited both huMIF and plasmodial MIF, thus bearing an immense therapeutic potential against proinflammatory reactions induced by MIF of both malaria parasites and human.