Biomedical subjects

L Lafanechère

Publications and source records attributed to L Lafanechère.

8 recordsLinked to original sources

PubMed2004

Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin.

In most eukaryotic cells, the C-terminal amino acid of alpha-tubulin is aromatic (Tyr in mammals and Phe in Saccharomyces cerevisiae) and is preceded by two glutamate residues. In mammals, the C-terminal Tyr of alpha-tubulin is subject to cyclic removal from the peptide chain by a carboxypeptidase and readdition to the chain by a tubulin-Tyr ligase. There is evidence that tubulin-Tyr ligase suppression and the resulting accumulation of detyrosinated (Glu) tubulin favor tumor growth, both in animal models and in human cancers. However, the molecular basis for this apparent stimulatory effect of Glu tubulin accumulation on tumor progression is unknown. Here we have developed S. cerevisiae strains expressing only Glu tubulin and used them as a model to assess the consequences of Glu tubulin accumulation in cells. We find that Glu tubulin strains show defects in nuclear oscillations. These defects are linked to a markedly decreased association of the yeast ortholog of CLIP170, Bik1p, with microtubule plus-ends. These results indicate that the accumulation of Glu tubulin in cells affects microtubule tip complexes that are important for microtubule interactions with the cell cortex.

Amino Acid Substitution↗

PubMed2001

Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis.

Tubulin, the dimeric subunit of microtubules, is a major cell protein that is centrally involved in cell division. Tubulin is subject to specific enzymatic posttranslational modifications including cyclic tyrosine removal and addition at the COOH terminus of the alpha-subunit. Tubulin is normally extensively tyrosinated in cycling cells. However, we have previously shown that detyrosinated tubulin accumulates in cancer cells during tumor progression in nude mice. Tubulin detyrosination, resulting from suppression of tubulin tyrosine ligase and the resulting unbalanced activity of tubulin-carboxypeptidase, apparently represents a strong selective advantage for cancer cells. We have now analyzed the occurrence and significance of tubulin detyrosination in human breast tumors. We studied a total of 134 breast cancer tumors from patients with or without known complications over a follow-up period of 31 +/- 10 months. The mean age of the patients at the time of diagnosis was 57 years. For each patient, detailed data concerning the histology and extension of the tumor were available. Tumor cells containing detyrosinated tubulin were visualized by immunohistochemical staining of paraffin-embedded tissue sections. Cancer cells with detyrosinated tubulin were observed in 53% of the tumors and were predominant in 19.4% of the tumors. Tubulin detyrosination correlated to a high degree of significance (P < 0.001) with a high Scarf-Bloom-Richardson (SBR) grade, a known marker of tumor aggressiveness. Among SBR grade 1 tumors, 3.8% were strongly positive for tubulin detyrosination compared with 65.4% of the SBR grade 3 tumors. The SBR component showing the strongest correlation with tubulin detyrosination was the mitotic score. In the entire patient population, neither the SBR grade nor the detyrosination index had significant prognostic value (P = 0.11, P = 0.27, respectively), whereas a combined index was significantly correlated with the clinical outcome (P = 0.02). A preliminary subgroup analysis indicated that tubulin detyrosination may define high- and low- risk groups in breast cancer tumors with an SBR grade of 2. Our study shows that tubulin detyrosination is a frequent occurrence in breast cancer, easy to detect, and linked to tumor aggressiveness.

Adult↗

PubMed2001

Evidence for a role of the (alpha)-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes.

Microinjected mAb YL1/2, an (alpha)-tubulin antibody specific for the tyrosinated form of the protein, blocks the cell cycle in developing oocytes. Here, we have investigated the mechanism involved in the mAb effect. Both developing starfish and Xenopus oocytes were injected with two different (alpha)-tubulin C terminus antibodies. The injected antibodies blocked cell entry into mitosis through specific inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized microtubules and was mimicked by injected synthetic peptides corresponding to the tyrosinated (alpha)-tubulin C terminus, whereas peptides lacking the terminal tyrosine were ineffective. These results indicate that tyrosinated (alpha)-tubulin, or another protein sharing the same C-terminal epitope, is involved in specific regulation of cyclin B synthesis in developing oocytes.

Amino Acid Sequence↗

PubMed2000

Preliminary crystallographic study of a complex formed between the alpha/beta-tubulin heterodimer and the neuronal growth-associated protein SCG10.

Crystals of a complex formed between the alpha/beta-tubulin heterodimer and SCG10, a neuron-specific growth-associated protein, have been obtained by the hanging drop method. They belong to the space group P2(1)2(1)2(1), with unit cell parameters a = 56 A, b = 353 A, c = 466 A and four molecular complexes in the asymmetric unit. A complete X-ray diffraction data set to 6.1 A resolution has been collected using synchrotron radiation. This represents a challenging opportunity to study at a molecular level the structure-function relationships between a microtubule-destabilizing protein, SCG10, and tubulin.

Animals↗

PubMed2000

The third tubulin pool.

Tubulin normally undergoes a cycle of detyrosination/tyrosination on the carboxy terminus of its alpha-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 alpha-subunit (delta2-tubulin). Delta2-tubulin is generated from detyrosinated tubulin through an irreversible reaction. Delta2-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. Delta2-tubulin may be a useful marker of malignancy in human tumors.

Biomarkers, Tumor↗

PubMed1998

STOP proteins are responsible for the high degree of microtubule stabilization observed in neuronal cells.

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.

Amino Acid Sequence↗

PubMed1998

Separation of tubulin subunits under nondenaturing conditions.

The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.

Animals↗

PubMed1998

Suppression of tubulin tyrosine ligase during tumor growth.

The C terminus of the tubulin alpha-subunit of most eukaryotic cells undergoes a cycle of tyrosination and detyrosination using two specific enzymes, a tubulin tyrosine ligase (TTL) and a tubulin carboxypeptidase. Although this enzyme cycle is conserved in evolution and exhibits rapid turnover, the meaning of this modification has remained elusive. We have isolated several NIH-3T3 derived clonal cell lines that lack TTL (TTL-). TTL- cells contain a unique tubulin isotype (delta2-tubulin) that can be detected with specific antibodies. When injected into nude mice, both TTL- cells and TTL- cells stably transfected with TTL cDNA form sarcomas. But in tumors formed from TTL rescued cells, TTL is systematically lost during tumor growth. A strong selection process has thus acted during tumor growth to suppress TTL activity. In accord with this result, we find suppression of TTL activity in the majority of human tumors assayed with delta2-tubulin antibody. We conclude there is a widespread loss of TTL activity during tumor growth in situ, suggesting that TTL activity may play a role in tumor cell regulation.

3T3 Cells↗