Phosphorylation of tubulin tyrosine ligase: a potential mechanism for regulation of alpha-tubulin tyrosination

The tubulin tyrosination/detyrosination cycle is a well-established posttranslational modification, which is carried out by two enzymes: Tubulin Tyrosine Ligase (TTL) and Tubulin Tyrosine Carboxypeptidase (TTCP). In this paper, I present evidence suggesting that the cycle itself is under the hierarchical control of reversible phosphorylation and that PKC mediated phosphorylation of TTL inhibits its activity, thereby preventing tubulin tyrosination. Phosphorylation of TTL is predicted to occur in a postulated Mg(++)/-ATP binding fold, leading to inhibition of Mg(++)/ATP binding and TTL mediated catalysis. The implications of such control are also discussed.     

Structural basis of tubulin tyrosination by tubulin tyrosine ligase

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL–tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.     

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. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.     

Tubulin:tyrosine ligase in oocytes and embryos of Xenopus laevis

Tubulin:tyrosine ligase (TTL), which catalyzes the post-translational addition of tyrosine to the α chain of tubulin, exists in a wide variety of embryonic and adult vertebrate tissues. In the present study, we report that TTL exists in amphibian oocytes at a time when tubulin is a poor substrate for tyrosination, and when, in immature oocytes, tubulin is not polymerizable. Ligase activity detected at several stages of oogenesis and embryogenesis in Xenopus is compatible with mammalian brain tubulin in the tyrosination reaction. Within 3–5 hr after fertilization, [³H] tyrosine incorporated/μg endogenous tubulin increases approximately 3.5-fold over that in extracts prepared from the largest oocytes obtained. This increase cannot be accounted for by increasing levels of TTL. Ligase activity remains fairly constant throughout oogenesis and early embryogensis and rises significantly (2-fold) only 35–50 hr after fertilization. The late rise in embryonic ligase activity is not accompanied by a change in apparent k_m for tubulin.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.     

The Third Tubulin Pool

Tubulin normally undergoes a cycle of detyrosination/tyrosination on the carboxy terminus of its -subunit and this results in subpopulations of tyrosinated tubulin and detyrosinated tubulin. Brain tubulin preparations also contain a third major tubulin subpopulation which is non-tyrosinatable. This review describes the purification and the structural characterization of non-tyrosinatable tubulin. This tubulin variant lacks a carboxyterminal glutamyl-tyrosine group on its -subunit (2-tubulin). 2-tubulin is generated from detyrosinated tubulin through an irreversible reaction. 2-tubulin accumulates in neurons and in stable microtubule assemblies. It also accumulates in some tumor cells due to the frequent loss of tubulin tyrosine ligase in such cells. 2-tubulin may be a useful marker of malignancy in human tumors.     

Tubulin Tyrosine Ligase Like 12, a TTLL Family Member with SET- and TTL-Like Domains and Roles in Histone and Tubulin Modifications and Mitosis

hTTLL12 is a member of the tubulin tyrosine ligase (TTL) family that is highly conserved in phylogeny. It has both SET-like and TTL-like domains, suggesting that it could have histone methylation and tubulin tyrosine ligase activities. Altered expression of hTTLL12 in human cells leads to specific changes in H4K20 trimethylation, and tubulin detyrosination, hTTLL12 does not catalyse histone methylation or tubulin tyrosination in vitro, as might be expected from the lack of critical amino acids in its SET-like and TTLL-like domains. hTTLL12 misexpression increases mitotic duration and chromosome numbers. These results suggest that hTTLL12 has non-catalytic functions related to tubulin and histone modification, which could be linked to its effects on mitosis and chromosome number stability.     

Mathematical modeling of the microtubule detyrosination/tyrosination cycle for cell-based drug screening design

Microtubules and their post-translational modifications are involved in major cellular processes. In severe diseases such as neurodegenerative disorders, tyrosinated tubulin and tyrosinated microtubules are in lower concentration. We present here a mechanistic mathematical model of the microtubule tyrosination cycle combining computational modeling and high-content image analyses to understand the key kinetic parameters governing the tyrosination status in different cellular models. That mathematical model is parameterized, firstly, for neuronal cells using kinetic values taken from the literature, and, secondly, for proliferative cells, by a change of two parameter values obtained, and shown minimal, by a continuous optimization procedure based on temporal logic constraints to formalize experimental high-content imaging data. In both cases, the mathematical models explain the inability to increase the tyrosination status by activating the Tubulin Tyrosine Ligase enzyme. The tyrosinated tubulin is indeed the product of a chain of two reactions in the cycle: the detyrosinated microtubule depolymerization followed by its tyrosination. The tyrosination status at equilibrium is thus limited by both reaction rates and activating the tyrosination reaction alone is not effective. Our computational model also predicts the effect of inhibiting the Tubulin Carboxy Peptidase enzyme which we have experimentally validated in MEF cellular model. Furthermore, the model predicts that the activation of two particular kinetic parameters, the tyrosination and detyrosinated microtubule depolymerization rate constants, in synergy, should suffice to enable an increase of the tyrosination status in living cells.     

Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods

Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha-tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.     

Antigens and chemical composition of Blastomyces dermatitidis

Without these tools (skin-test and complement fixation antigens for epidemiological, diagnostic, and prognostic use) we are at least 50 years behind in our defining of the disease blastomycosis (63). This statement by Rippon et al. emphasizes the need for a well defined antigen or group of antigens from Blastomyces dermatitidis, not only for the two tests named above, but for any serological or immunological test. Several different preparations have been reviewed which are beginning to approach the quality necessary for such a sensitive and specific antigenic tool. All of these require further characterization and, in most instances, purification.One purified fraction isolated from blastomycin, the F fraction, has shown exceptional promise as a skin-test agent in guinea pigs but has not been further studied. A 10–30K molecular weight fraction of the yeast cytoplasm has shown good reactivity, and contains a protein shown by PAGE to be common to several skin-reactive preparations. This fraction has not been further purified.An ASWS preparation has been partially characterized and shown to be especially sensitive and specific in animals, though not yet in humans. Purification of this fraction by PAGE has uncovered a highly reactive protein which may be related to one isolated from the cytoplasm. Investigations using this purified component have not been attempted in humans.The A antigen has been well characterized regarding its applicability to human diagnosis. It has been partially purified and two of its components associated with its reactivity. It also has probably the best possibility as an immediate serologic tool. Even here, though, current preparations contain much extraneous material which could conceivably create cross-reactivity problems in the future.The ethanol-precipitate antigens which have shown such superior results in the past have not been employed recently, nor have they been extensively characterized. This fraction may contain the reactive A antigen or other antigens deserving of study. Hopefully, the use of this technique can be encouraged as one starting point for isolation procedures.The importance and isolation of enzymes and mannose containing antigens have probably not received adequate attention. An almost uniform identification of mannose in reactive preparations argues for purification procedures based on its presence.Finally, use of hybridoma technology to produce antibodies specific for antigens of B. dermatitidis promises to improve our understanding of the organism and to help isolate purer antigenic fractions.The search for antigens of importance in the immunology of B. dermatitidis should not be confined to any one or all of the antigens discussed above. A variety of components will likely be necessary for complete understanding of the disease and for diagnostic use. It has been observed that information of clinical value can be obtained from immunological reactions to several different antigens (35), thereby encouraging continuous efforts to obtain as many as possible.     

Gametoclonal variation detected in the nuclear ribosomal DNA from doubled haploid lines of a spring wheat (Triticum aestivum L., cv. ‘César’)

Summary The organization of the nuclear ribosomal DNA from a parental line of wheat (Triticum aestivum L., cv. César) and its anther-derived first cycle and second cycle doubled haploid lines has been analyzed by DNA-DNA molecular hybridization. Restricted DNA has been probed by three subclones of wheat nuclear ribosomal DNA covering the entire repeat unit. No significant difference was detected in the extent of methylation of ribosomal DNA of the doubled haploid lines with respect to the parental line. On the other hand, a variation has been found in the organization of the nontranscribed spacer region of ribosomal DNA of the first cycle doubled haploid line. This variation remains stable after a second cycle of in vitro androgenesis. However, one out of five second cycle doubled haploid lines so far tested showed an additional hybridization band present in the parental line but lacking in the first cycle doubled haploid line.     

Regulation of the central metabolism in relation to citric acid production in Saccharomycopsis lipolytica

The mechanism of the massive extracellular production of citric and isocitric acids by Saccharomycopsis lipolytica grown on n-paraffins has been studied. When growth stops, because of nitrogen limitation, the intracellular concentration of ATP sharply rises whereas that of AMP and ADP decreases to a low level. At the same time production of acids begins. The activity of the NAD-dependent isocitrate dehydrogenase which requires AMP for activity becomes very low and prevents the oxidative function of the citric acid cycle whereas isocitrate lyase is not inhibited. As citrate synthase inhibition by ATP appears to be insufficient to stop n-paraffin degradation, citric and isocitric acids accumulation can take place. Massive excretion of these acids, however, probably still involves other physiological changes brought about by nitrogen limitation, possibly some permeabilization of the cell to these acids.This work is a part of a Doctorat de Spécialité Thesis submitted by R. Marchal to the University of Nancy (1975)     

Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates

The physiological role of monocarboxylate transport in brownadipose tissue mitochondria has been reevaluated. We studied pyruvate,-ketoisovalerate, -ketoisocaproate, and phenylpyruvate uniportvia the uncoupling protein (UCP1) as a GDP-sensitive swelling inK⁺ salts induced by valinomycin orby monensin and carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone in Na⁺ salts. We have demonstratedthat this uniport is inhibited by fatty acids. GDP inhibition inK⁺ salts was not abolished by anuncoupler, indicating a negligible monocarboxylic acid penetration viathe lipid bilayer. In contrast, the electroneutral pyruvate uptake(swelling in ammonium pyruvate or potassium pyruvate induced by changein pH) mediated by the pyruvate carrier was inhibited by its specificinhibitor -cyano-4-hydroxycinnamate but not by fatty acids.Moreover, -cyano-4-hydroxycinnamate enhanced the energization ofbrown adipose tissue mitochondria, which was monitored fluorometricallyby 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cyclingof unipolar ketocarboxylates under certain physiological conditionswhile expelling these anions from the matrix. The cycle is completed ontheir return via the pyruvate carrier in anH⁺ symport mode.

     

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake

Background

Vaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97?kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13?MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories.

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Purification of UDP-galactose: diacylglycerol galactosyltransferase from chloroplast envelopes of spinach (Spinacia oleracea L.)

Uridine 5-diphosphate(UDP)-galactose: 1,2-diacylglycerol 3-O--d-galactopyranosyltransferase (EC 2.4.1.46) is an integral protein of chloroplast envelope membranes from which it has been partially purified (Covès et al., 1986, FEBS Lett. 208, 401–406). We have worked out a purification procedure which after removal of peripheral membrane proteins, solubilization and two chromotographic steps allowed us to identify a 22-kDa protein as the galactosyltransferase. Enrichment of enzymatic activity was paralleled by an enrichment of this protein and its radioactive derivative obtained by photoaffinity labelling with [-^–32P]UDP which is a potent inhibitor of the enzyme. The purification factor of about 350 is substantially higher than achieved previously and indicates that the enzyme represents less than 0.3% of the envelope proteins. The purified enzyme has a Km of 87 M for UDP-galactose with dioleoylglycerol as acceptor and could not be activated by addition of other lipids.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate - DTE dithioerythritol - MGD monogalactosyl diacylglycerol - PMSF phenylmethanesulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the Deutsche Forschungsgemeinschaft.     

The pyridine-nucleotide cycle in tobacco Enzyme activities for the de-novo synthesis of NAD

The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMNNMNNAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.Abbreviations HPLC high-performance liquid chromatography - QA quinolinic acid - NaMN nicotinic acid mononucleotide - NaAD nicotinic acid adenine dinucleotide - NMN nicotinamide mononucleotide     

Varroa destructor reproduction during the winter in Apis mellifera colonies in UK

The reproductive behaviour of female Varroa destructor mites invading worker brood cells during the winter months (January to mid-March) was investigated in four Apis mellifera colonies in UK. The number of viable offspring produced during a reproductive cycle, per mite, was only 0.5 during winter compared with 1.0 during the summer. This was mainly due to a large increase in the population of non-reproductive mites (winter 20%, summer 8%). This increase can be explained by the high level of male offspring mortality observed in winter (42% vs. 18% in summer), which results in nearly half of the newly reared female mites being unfertilised. Since mites that do reproduce lay a similar number of eggs in winter ( ) as in summer ( ), and the level of mortality suffered by the first female offspring is similar in winter (7%) as in summer (6%), it is probably not the internal physiological state of the host which causes the high level of winter non-reproduction, as has been previously suspected.