JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

Expression of flotillins in the human placenta: potential implications for placental transcytosis

A proteomics survey of human placental syncytiotrophoblast (ST) apical plasma membranes revealed peptides corresponding to flotillin-1 (FLOT1) and flotillin-2 (FLOT2). The flotillins belong to a class of lipid microdomain-associated integral membrane proteins that have been implicated in clathrin- and caveolar-independent endocytosis. In the present study, we characterized the expression of the flotillin proteins within the human placenta. FLOT1 and FLOT2 were coexpressed in placental lysates and BeWo human trophoblast cells. Immunofluorescence microscopy of first-trimester and term placentas revealed that both proteins were more prominent in villous endothelial cells and cytotrophoblasts (CTs) than the ST. Correspondingly, forskolin-induced fusion in BeWo cells resulted in a decrease in FLOT1 and FLOT2, suggesting that flotillin protein expression is reduced following trophoblast syncytialization. The flotillin proteins co-localized with a marker of fluid-phase pinocytosis, and knockdown of FLOT1 and/or FLOT2 expression resulted in decreased endocytosis of cholera toxin B subunit. We conclude that FLOT1 and FLOT2 are abundantly coexpressed in term villous placental CTs and endothelial cells, and in comparison, expression of these proteins in the ST is reduced. These findings suggest that flotillin-dependent endocytosis is unlikely to be a major pathway in the ST, but may be important in the CT and endothelium.     

Synthesis of Several Fructo-oligosaccharides by Asparagus Fructosyltransferases

Nine fructo-oligosaccharides, synthesized in vitro from sucrose by an enzyme preparation from asparagus roots, were isolated and their structures were elucidated to be 1^F (1-β-fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose) and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n=1 (neokestose), 2 and 3] and 1^F (1-β-fructofuranosyl)_m-6^G (1-β-fructofuranosyl)_n sucrose [m=1, n=1; m=2, n =1; and m =1, n=2]. These saccharides are all known to occur naturally in asparagus roots, but 6^G (1-β-fructofuranosyl)₃ sucrose and 1^F (1-β-fructofuranosyl)_m-6^G-(1-β-fructofuranosyl)_n sucrose (m=1, n =1; and m=1, n=2) were the first saccharides enzymatically synthesized in vitro. Also three types of fructosyltransferases were presumed to be involved in the biosynthesis of these oligosaccharides in asparagus roots.     

N′-Isopropylnornicotine in Burley Tobacco (Nicotiana tabacum)

Allyl sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), typical flavor components of Allium vegetables, have been shown to inhibit benzo[a]pyrene (B[a]P)-induced carcinogenesis in animal models. As a possible mechanism of this inhibition, the effect of these volatile substances on cytochrome P450 (CYP)1 (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B[a]P was investigated using a human hepatoma cell model (HepG2). DADS and DATS inhibited the B[a]P-induced ethoxyresorufin O-deethylase (EROD) activity, a marker enzyme for CYP1, by 30-90% and 70-95% at 100-1,000 μM concentration, respectively. The cell viability, an indicator of the capacity to inhibit B[a]P bioactivation, was increased by treatments of 100-1,000 μM DADS and 10-100 μM DATS. Immunoblot results indicated that the B[a]P inducible CYP1A2 protein was suppressed by 100-1,000 μM of DADS and 10-100 μM of DATS, but CYP1A1 and 1B1 were not detectable in any microsomes. Analysis of B[a]P metabolites revealed that the level of 7,8-diol formed was significantly reduced in the DADS and DATS treated microsomes as compared to the control. The level of 9,10-diol and 4,5-diol formed was also lowered by the allyl sulfide treatments. These results suggest that the protective mechanism of allyl sulfides on B[a]P-induced carcinogenesis is possibly related with the modulation of CYP1-mediated bioactivation of B[a]P.     

Studies on BHC Isomers and their Related Compounds

β-BTC(3, 4/5, 6),¹⁾ γ-BTC(3, 4, 6/5), and ε-BTC(3, 4, 5/6) were synthesized from α-BTC (3, 6/4, 5) by stepwise routes.     

Syntheses of α and γ-BHC Alkylthio Analogs

Meso-(1245/36)-1,2,4,5,6-pentachloro-3-methylthiocyclohexane, and (124/356)-1,2,4,5,6-pentachloro-3-methylthio and ethylthiocyclohexanes were prepared from (1234/56)-1,4,5,6-tetrachloro-2,3-epoxycyclohexane (α-BTC cis-epoxide).     

Syntheses and Proton Magnetic Resonance Studies at 300 MHz of Two New Bromopentachlorocyclohexanes and Three New Bromotrichlorocyclohexenes

Two diastereomers of 6-bromo-1, 2, 3, 4, 5-pentachlorocyclohexane were synthesized from the dl (36/45)-diastereomer of 3, 4, 5, 6-tetrachlorocyclohexene (α-BTC) and the dl (346/5)-diastereomer (γ-BTC) by several stepwise routes. Both of these new products were shown to have the configuration of lindane by NMR studies at 300 MHz and by the synthetic routes. Three diastereomers of 6-bromo-3, 4, 5-trichlorocyclohexene were also prepared and the configurations determined partly by means of 300 MHz NMR.