N-terminal aliphatic residues dictate the structure, stability, assembly, and small molecule binding of the coiled-coil region of cartilage oligomeric matrix protein.

The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function.

Submembraneous microtubule cytoskeleton: regulation of ATPases by interaction with acetylated tubulin.

The ATP-hydrolysing enzymes (Na(+),K(+))-, H(+)- and Ca(2+)-ATPase are integral membrane proteins that play important roles in the exchange of ions and nutrients between the exterior and interior of cells, and are involved in signal transduction pathways. Activity of these ATPases is regulated by several specific effectors. Here, we review the regulation of these P-type ATPases by a common effector, acetylated tubulin, which interacts with them and inhibits their enzyme activity.

Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex.

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex.

Tubulin must be acetylated in order to form a complex with membrane Na(+),K (+)-ATPase and to inhibit its enzyme activity.

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na(+),K(+)-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes.

Activation of the plasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H(+)-ATPase-acetylated tubulin complex.

In the yeast Saccharomyces cerevisiae, plasma membrane H(+)-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H(+)-ATPase, and therefore partitions into a detergent phase.

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules.

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells.

Re-examination of the post-translational arginylated protein of 125-kD initially identified as N-STOP.

Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP).

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs.

Post-translational incorporation of the antiproliferative agent azatyrosine into the C-terminus of alpha-tubulin.

Detyrosination/tyrosination of tubulin is a post-translational modification that occurs at the C-terminus of the alpha-subunit, giving rise to microtubules rich in either tyrosinated or detyrosinated tubulin which coexist in the cell. We hereby report that the tyrosine analogue, azatyrosine, can be incorporated into the C-terminus of alpha-tubulin instead of tyrosine. Azatyrosine is structurally identical to tyrosine except that a nitrogen atom replaces carbon-2 of the phenolic group.

Involvement of acetylated tubulin in the regulation of Na+,K+ -ATPase activity in cultured astrocytes.

The results presented support the view that the modulation of Na(+),K(+)-ATPase activity in living cells involves the association/dissociation of acetylated tubulin with the enzyme. We found that the stimulation of Na(+),K(+)-ATPase activity by L-glutamate correlates with decreased acetylated tubulin quantity associated with the enzyme. The effect of L-glutamate was abolished by the glutamate transporter inhibitor DL-threo-beta-hydroxyaspartate but was not affected by either specific agonists or antagonists.

Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture.

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro.

Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.

The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells.

Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein.

The microtubule-associated protein tau is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in neurons undergoing neurodegeneration in the brains of patients with Alzheimer disease (AD). We investigated the isoform-specific interaction of normal tau with AD hyperphosphorylated tau (AD P-tau). We found that the binding of AD P-tau to normal human recombinant tau was tau4L > tau4S > tau4 and tau3L > tau3S > tau3, and that its binding to tau4L was greater than to tau3L.

Brain plasma membrane Na+,K+-ATPase is inhibited by acetylated tubulin.

Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the alpha-subunit of Na+,K+-ATPase. In the present work we show that the interaction of tubulin with Na+K+-ATPase inhibits the enzyme activity.

Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane.

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification).

Posttranslational arginylation of soluble rat brain proteins after whole body hyperthermia.

We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway.

Association of tubulin carboxypeptidase with microtubules in living cells.

Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction.

Na+,K+-ATPase was found to be the membrane component responsible for the hydrophobic behavior of the brain membrane tubulin.

We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing.

Comparing efficacies of neurocognitive treatment and homework assistance programs for children with learning difficulties.

The purpose of the study was to analyze the relative efficacies of two treatments for children with learning difficulties. The first treatment consisted of multiple training components targeting specific cognitive and behavioral factors; the second treatment provided emotional support and supervision of school tasks. The participants were 94 Chilean schoolchildren (6 to 11 years of age).

A brain protein (P30) that immunoreacts with a polyclonal anti-pancreatic carboxypeptidase A antibody shows properties that are shared with tubulin carboxypeptidase.

A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 +/- 0.

Conversion of hydrophilic tubulin into a hydrophobic compound. Evidence for the involvement of membrane proteins.

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes.

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes.

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the alpha-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75-78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.

Isolation of alpha and beta brain tubulin subunits after alkaline treatment of the protein.

We demonstrate here that brain purified tubulin can be dissociated into alpha and beta subunits at pH > 10 and that the subunits can be separated by using the Triton X-114 phase separation system. After phase partition at pH > 10, alpha tubulin but not beta tubulin behaves as a hydrophobic compound appearing in the detergent rich phase. After three extractions of the alkaline aqueous phase with Triton X-114, about 90% of the alpha tubulin was recovered in the detergent rich phase.

Tubulin tyrosine ligase: protein and mRNA expression in developing rat skeletal muscle.

Alpha tubulin can be post-translationally tyrosinated at the carboxy-terminus by a specific enzyme: tubulin tyrosine ligase. The expression of tubulin tyrosine ligase mRNA and protein during the development of rat skeletal muscle was examined in the present study. A portion of the coding region of the rat ligase cDNA was isolated and sequenced.