A triangle lattice model that predicts transmembrane helix configuration using a polar jigsaw puzzle

We developed a method of predicting the tertiary structures of seven transmembrane helical proteins in triangle lattice models, assuming that the configuration of helices is stabilized by polar interactions. Triangle lattice models having 12 or 11 nearest neighbor pairs were used as general templates of a seven-helix system, then the orientation angles of all helices were varied at intervals of 15 degrees. The polar interaction energy for all possible positions of each helix was estimated using the calculated polar indices of transmembrane helices. An automated system was constructed and applied to bacteriorhodopsin, a typical membrane protein with seven transmembrane helices. The predicted optimal and actual structures were similar. The top 100 predicted helical configurations indicated that the helix-triangle, CFG, occurred at the highest frequency. In fact, this helix-triangle of bacteriorhodopsin forms an active proton-pumping site, suggesting that the present method can identify functionally important helices in membrane proteins. The possibility of studying the structure change of bacteriorhodopsin during the functional process by this method is discussed, and may serve to explain the experimental structures of photointermediate states.     

Microtubule bundling by tau proteins in vivo: analysis of functional domains.

Tau varies both in the N-terminal region (three types) and in the C-terminal repeated microtubule binding domain (two types), generating six isoforms through alternative splicing. To understand the differences between the isoforms and to determine which domains are important for microtubule bundling, we performed transfection studies on fibroblasts using tau isoforms and deletion mutants to quantify their ability to bundle microtubules. By comparing the isoforms, we found that a longer N-terminal region induced microtubule bundling more efficiently, but changes in the microtubule binding domain did not. Mutants lacking the proline rich region or the repeated domain did not bind to microtubules. Although all the other mutants could bind to and bundle microtubules, deletion in the N-terminal neutral region or the first half of the C-terminal tail caused a significant decrease in microtubule bundling, indicating the importance of these regions in microtubule bundling.     

Gem GTPase and tau: morphological changes induced by gem GTPase in cho cells are antagonized by tau

A series of observations have indicated that tau, one of the major microtubule-associated proteins, is involved in neuronal cell morphogenesis and axonal maintenance. Tau is also the major component of paired helical filaments found in brains affected by Alzheimer's disease. To explore an as yet unidentified role of tau in vivo, approximately 11,000 mRNAs were profiled from tau-deficient mouse brains and compared with those from control brains at the same ages. The expression of Gem GTPase, a small GTP-binding protein of the ras superfamily, was significantly increased in the brains of tau-deficient mice at 8 weeks of age. Because Gem GTPase is a negative regulator of the Rho-Rho kinase pathway for cytoskeletal organization, this protein was transiently overexpressed in Chinese hamster ovary cells that do not express tau. Overexpression of Gem GTPase induced a marked elongation of Chinese hamster ovary cells, and simultaneous expression of tau eliminated this effect, although tau did not bind directly to Gem GTPase. This anti-elongation activity of tau was attributed to its microtubule-binding domain, and homologous domains of microtubule-associated proteins 2 and 4 exhibited similar antagonistic activities. Taken together, the present results indicate that the level of Gem GTPase and its cell elongation activity are modulated by tau and suggest that tau may be involved in a Gem GTPase-mediated signal transduction pathway.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (～5 nm wide, 100–200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deepetch technique, these ～5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of ～5 nm fibrils that are morphologically distinct from the ～8 nm myosin-containing fibrils which were identified by antimyosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actinvesicle, actin-intermediate filament and vesicle-plasma membrane linkages.     

Length of Deoxyribonucleic Acid of PBSX-like Particles of Bacillus subtilis Induced by 4-Nitroquinoline-1-Oxide

The formation of particles resembling PBSX phages was induced by 4-nitroquinoline-1-oxide in a Marburg strain of Bacillus subtilis. All particles were homogeneous in their morphology. Physical and biological analyses revealed that the deoxyribonucleic acid (DNA) carried by these particles are fragments of host-cell DNA. The contour length of the DNA is 4.25 mum, corresponding to a molecular weight of 8.1 x 10(6) daltons.     

Synthetic Nucleosides and Nucleotides. 43. Inhibition of Vertebrate Telomerases by Carbocyclic Oxetanocin G (C.OXT-G) Triphosphate Analogues and Influence of C.OXT-G Treatment on Telomere Length in Human HL60 Cells

Telomerase, responsible for telomere synthesis, is expressed in ～ 90% of human tumor cells but seldom in normal somatic cells. In this study, inhibition by carbocyclic oxetanocin G triphosphate (C.OXT-GTP) and its analogues was investigated in order to clarify the susceptibility of telomerase to various nucleotide analogues. C.OXT-GTP competitively inhibited telomerase activity with respect to dGTP. However, C.OXT-GTP had a potent inhibitory effect on DNA polymerase α. It was examined whether the nucleoside (C.OXT-G) was able to alter telomere length in cultured human HL60 cells. Contrary to expectation, long-term treatment with 10 μM C.OXT-G was found to cause telomere lengthening.     

Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.     

Design, synthesis, and biological activity of non-amidine factor Xa inhibitors containing pyridine N-oxide and 2-carbamoylthiazole units

Based on the both of results for X-ray studies of tetrahydrothiazolopyridine derivative 1c and FXV673, we synthesized a series of thiazol-5-ylpyridine derivatives containing pyridine N-oxide and 2-carbamoylthiazole units to optimize the S4 binding element. N-Oxidation of thiazol-5-ylpyridine increased the anti-fXa activity more than 10-fold independent on the position of N-oxide. The 4-pyridine N-oxide derivatives 3a and 3d excelled over the tetrahydrothiazolopyridine 1b in potency. 2-Methylpyridine N-oxide 3d exhibited 49-fold selectivity over thrombin. Our modeling study proposed a binding mode that the pyridine N-oxide ring of 3a stuck into the "cation hole" , and the oxide anion of 3a occupied in the almost same space to that of FXV673. From observations of the SAR and modeling studies, we suggested the possibilities that the formation of hydrogen bond with the oxide anion in the "cation hole" and the affinity of cationic pyridine ring to S4 subsite were responsible for increase in anti-fXa activity.     

Three Types of Acidic Polysaccharides Associated with Coccolith of Pleurochrysis haptonemofera: Comparison with Pleurochrysis carterae and Analysis Using Fluorescein-Isothiocyanate-Labeled Lectins

In the coccolithophorid microalgae acidic polysaccharides are considered to be involved in the formation of the calcified scale, coccolith. Characteristics of the acidic polysaccharides extracted from the cell surface of the coccolithophorid Pleurochrysis haptonemofera were analyzed. The acidic polysaccharides on the cell surface can be detected by measuring fluorescence of cells after fluorescein-isothiocyanate-labeled lectin staining by flow cytometry. Flow cytometric analyses revealed that the acidic polysaccharides remained on the cell surface even after CaCO₃ in the coccolith was dissolved by lowering pH, but they were extracted by subsequent EDTA or EGTA treatment, suggesting that they are bound not into the CaCO₃ crystals of the coccolith, but onto the surface via Ca²⁺. Analyses of the acidic polysaccharides by anion exchange chromatography, colloidal precipitation with divalent cations, and polyacrylamide gel electrophoresis (PAGE) revealed that P. haptonemofera has 3 types of acidic polysaccharides (Ph-PS-l, -2, and -3). The PAGE patterns suggested that Ph-PS-2 has a repeated structure with a broad range of molecular weight, as in Pleurochrysis carterae, while Ph-PS-1 and -3 contain several minor components in addition to a major component, respectively. The minor components in Ph-PS-1 and -3 that have not been found in P. carterae might be characteristic of P. haptonemofera. Analyses of both the cell surface treated by various concentrations of EDTA and EGTA and the extracts suggested that Ph-PS-2, which is distinguishable by a higher affinity to concanavalin A, is bound onto the coccolith surface more intensely than the other two types of acidic polysaccharides.