Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

The average conformation at micromolar [Ca2+] of Ca2+-atpase with bound nucleotide differs from that adopted with the transition state analog ADP.AlFx or with AMPPCP under crystallization conditions at millimolar [Ca2+

Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP.fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1.AMPPCP and the E1.ADP.AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca2+ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca2+ known to be sufficient to saturate the transport sites. This concerns various properties, including ATPase susceptibility to proteolytic cleavage by proteinase K, ATPase reactivity toward SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1.ADP.AlFx complex for the first time), and also the rates of 45Ca2+-40Ca2+ exchange at site "II." These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca2+ ion in the E1.AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1.AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1.AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, toward the transition state leading to phosphorylation and Ca2+ occlusion.     

Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2

Deubiquitinating enzymes (DUBs) catalyze the removal of attached ubiquitin molecules from amino groups of target proteins. The large family of DUBs plays an important role in the regulation of the intracellular homeostasis of different proteins and influences therefore key events such as cell division, apoptosis, etc. The DUB family members UCH-L3 and USP2 are believed to inhibit the degradation of various tumor-growth-promoting proteins by removing the trigger for degradation. Inhibitors of these enzymes should therefore lead to enhanced degradation of oncoproteins and may thus stop tumor growth. To develop an enzymatic assay for the search of UCH-L3 and USP2 inhibitors, C-terminally labeled ubiquitin substrates were enzymatically synthesized. We have used the ubiquitin-activating enzyme E1 and one of the ubiquitin-conjugating enzymes E2 to attach a fluorescent lysine derivative to the C terminus of ubiquitin. Since only the epsilon-NH(2) group of the lysine derivatives was free and reactive, the conjugates closely mimic the isopeptide bond between the ubiquitin and the lysine side chains of the targeted proteins. Various substrates were synthesized by this approach and characterized enzymatically with the two DUBs. The variant consisting of the fusion protein between the large N-terminal NusA tag and the ubiquitin which was modified with alpha-NH(2)-tetramethylrhodamin-lysine, was found to give the highest dynamic range in a fluorescence polarization readout. Therefore we have chosen this substrate for the development of a miniaturized, fluorescence-polarization-based high-throughput screening assay.     

Structural studies of the neural-cell-adhesion molecule by X-ray and neutron reflectivity

The structures of adhesion proteins play an important role in the formation of intercellular junctions and the control of intermembrane spacing. This paper describes the combination of neutron and X-ray specular reflectivity measurements to investigate the structure of the ectodomain of the neural-cell-adhesion molecule (NCAM). The measurements with unmodified NCAM suggest the presence of a bend in the extracellular region. Measurements with the polysialic-acid-modified form of NCAM reveal that, at physiological ionic strength, the carbohydrate chains extend beyond the range of the unmodified protein. The excluded volume of the polymer is also ionic-strength-dependent, as expected for a polyelectrolyte. The structural characteristics obtained from these independent analyses of X-ray and neutron reflectivity data agree with each other, with prior reflectivity studies, and with molecular dimensions obtained from direct-force measurements. These results provide structural insights into the configuration of the NCAM ectodomain and the regulation of NCAM adhesion by post-translational modification.     

Triple helix formation with Drosophila satellite repeats. Unexpected stabilization by copper ions

The Drosophila melanogaster (AAGAGAG)(n) satellite repeat represents up to 1.5% of the entire fly genome and may adopt non-B DNA structures such as pyrimidine triple helices. UV melting and electrophoretic mobility shift assay experiments were used to monitor the stability of intermolecular triple helices as a function of size, pH, and backbone or base modification. Three to four repeats of the heptanucleotide motif were sufficient to allow the formation of a stable complex, especially when modified TFOs were used. Unexpectedly, low concentrations (40-100 microM) of Cu(2+) were found to favor strongly pyrimidine triplex formation under near-physiological conditions. In contrast, a much higher magnesium concentration was required to stabilize these triplexes significantly, suggesting that copper may be an essential stabilizing factor for pyrimidine triplexes.     

Climate change and size evolution in an island rodent species: new perspectives on the island rule

As stated by the island rule, small mammals evolve toward gigantism on islands. In addition they are known to evolve faster than their mainland counterparts. Body size in island mammals may also be influenced by geographical climatic gradients or climatic change through time. We tested the relative effects of climate change and isolation on the size of the Japanese rodent Apodemus speciosus and calculated evolutionary rates of body size change since the last glacial maximum (LGM). Currently A. speciosus populations conform both to Bergmann's rule, with an increase in body size with latitude, and to the island rule, with larger body sizes on small islands. We also found that fossil representatives of A. speciosus are larger than their extant relatives. Our estimated evolutionary rates since the LGM show that body size evolution on the smaller islands has been less than half as rapid as on Honshu, the mainland-type large island of Japan. We conclude that island populations exhibit larger body sizes today not because they have evolved toward gigantism, but because their evolution toward a smaller size, due to climate warming since the LGM, has been decelerated by the island effect. These combined results suggest that evolution in Quaternary island small mammals may not have been as fast as expected by the island effect because of the counteracting effect of climate change during this period.