The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis

Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.     

Phosphorylation of a neuronal-specific beta-tubulin isotype

Adult rats were intracraneally injected with [32P] phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.     

Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii.

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin. We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiibeta-T1 and beta5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.     

Tubulin isotype usage in vivo: a unique spatial distribution of the minor neuronal-specific beta-tubulin isotype in pheochromocytoma cells

The neuronal cells of vertebrates express two beta-tubulin isotypes, called Class II and Class III, that are neuronal specific. In order to determine the distribution of the minor Class III isotype, site-directed antibodies were raised to synthetic peptides representing the carboxyl terminal, isotype-defining domains of the tubulins. These antibodies were applied to PC12 cells at various stages of differentiation. The Class III isotype was found to be expressed in undifferentiated PC12 cells as well as in cells at every stage of differentiation. The concentration of the Class III isotype, relative to the total beta-tubulin complement, did not change significantly. Indirect double immunofluorescence microscopy demonstrated that the Class III isotype was found in the soma and the neurites of differentiated PC12 cells; this spatial pattern of Class III expression paralleled the total beta-tubulin pattern. Although the anti-Class III antiserum could stain in vitro assembled neuronal microtubules in a filamentous pattern, a close examination of the Class III staining pattern in flattened PC12 cells revealed that this isotype was not incorporated into the nonaxoplasmic array of microtubules. Rather, the Class III isotype was localized in a nonfilamentous, granular pattern that was not readily extracted with nonionic detergent. Cells treated with taxol and then flattened and stained showed that the Class III isotype could be induced to assemble into microtubule bundles by taxol. Thus, the minor neuronal beta-tubulin isotype appears to be spatially specialized in its pattern of expression.     

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.     

WCI,a novel wheat chymotrypsin inhibitor: purification,primary structure,inhibitory properties and heterologous expression

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical–chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7–89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.     

Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters beta-tubulin isotype expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.     

Expression of Tubb3, a beta-tubulin isotype, is regulated by androgens in mouse and rat Sertoli cells

Our previous analysis of Sertoli cell androgen receptor (AR) knockout (SCARKO) mice revealed that several cytoskeletal components are a potential target of androgen action. Here, we found that one of these components, the beta-tubulin isotype Tubb3, is differentially regulated in testes from SCARKO mice (relative to littermate controls) from Postnatal Day 10 to adulthood. The Tubb3 gene is unique in this respect, as at Day 10, no other beta-tubulin genes are significantly regulated by AR. We further characterized androgen regulation of Tubb3 in vivo and in vitro and demonstrated that it is a conserved feature in both mice and rats. To investigate whether androgens directly regulate Tubb3 expression, we screened for androgen response elements (AREs) in the Tubb3 gene. In silico analysis revealed the presence of four ARE motifs in Tubb3 intron 1, two of which bind to AR in vitro. Mutation of one of these (ARE1) strongly reduced androgen-dependent reporter gene expression. These results, coupled with the finding that the AR binds to the Tubb3 ARE region in vivo, suggest that Tubb3 is a direct target of AR. Our data strengthen the contention that androgens exert their effects on spermatogenesis, in part, through modulation of the Sertoli cell cytoskeleton. Androgen regulation of beta-tubulin has also been described in neurons, fortifying the already known similarity in microtubule organization in Sertoli cell processes and neurons, the only other cell type in which Tubb3 is known to be expressed.     

Inactivation,complementation, and heterologous expression of encP,a novel bacterial phenylalanine ammonia-lyase gene

The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of l-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.     

Tubulin folding: the special case of a beta-tubulin isotype from the Antarctic psychrophilic ciliate Euplotes focardii

Folding assistance is a fundamental requirement of certain proteins, and it may be subjected to physicochemical constraints in case of organisms adapted to polar temperatures. Limited information is available about protein folding in the polar environment. Folding of tubulin provides one of the few studied cases. Here, we report a pilot folding analysis of a divergent beta-tubulin isotype, named EFBT3, from the Antarctic psychrophilic ciliate Euplotes focardii. To attain its native monomeric structure, beta-tubulin needs the assistance of the eukaryotic class II chaperonin CCT and cofactor A (CofA). The in vitro folding reaction of EFBT3 with CCT and CofA purified from rabbit did not generate any folded product. In contrast, the reaction performed with the rabbit reticulocyte lysate, that contains all the chaperones required for efficient tubulin folding, was productive, suggesting that additional factors besides purified CCT and CofA are required for EFBT3 to attain its monomeric structure. We also demonstrated that the rare Cys281 of EFBT3 is critical for the folding reaction. Model predictions indicate that EFBT3 binds to CofA differently from yeast beta-tubulin, suggesting a diverse folding mechanism that may be correlated with microtubule cold adaptation.     

Three expressed sequences within the human beta-tubulin multigene family each define a distinct isotype

This paper describes the isolation and complete sequence of a novel expressed human beta-tubulin gene (beta 2). The sequence is compared with that of two other expressed human beta-tubulin genes (M40 and 5 beta). All are encoded by four exons. Though the boundaries of each exon are absolutely conserved among the three genes, the intervening sequences differ considerably in size and sequence content. Two of the genes (M40 and 5 beta) contain one (M40) or ten (5 beta) members of the middle repetitive Alu family sequences within one of their intervening sequences. Comparison of the amino acid sequences encoded by each gene reveals a high level of homology overall, though there is significant divergence between the carboxy termini of two of the genes. The pattern of expression of each beta-tubulin gene has been studied in several different human cell lines using unique non-crosshybridizing probes derived from the 3' untranslated regions. Two of the genes, M40 and beta 2, are expressed at varying levels in all of the cell lines examined, though the level of expression of one of these genes parallels the other in most cases. The third gene, 5 beta, is detectably expressed only in cells of neural origin. Thus, distinct human beta-tubulin isotypes are encoded by genes whose exon size and number has been conserved evolutionarily, but whose pattern of expression may be regulated either co-ordinately or uniquely. Of the approximately 15 sequences contained in the human beta-tubulin multigene family, nine have now been sequenced fully. The overall composition of the multigene family and the evolutionary relationships among its various members are discussed.     

Regulation of azophenylarsonate-specific repertoire expression. II. Fine specificity and isotype heterogeneity of anti-arsonate antibodies

This report investigates the isotope and binding site heterogeneity of IgG anti-azophenylarsonate (ARS) monoclonal antibodies derived from splenic fragment cultures of nonimmune and immune A/J mice. By using five arsanilic acid derivatives in a hapten inhibition assay to determine fine specificity, the secondary ARS-specific repertoire was found to be extremely heterogeneous, with 52 unique patterns among the 81 antibodies analyzed. CRIA+ and CRIA+/- clonotypes were as heterogeneous as CRIA- antibodies, but a majority of CRIA+/- clones maintained a characteristic relative affinity for two of the haptens, p-arsanilic acid (p-ARS) and p-azobenene arsonic acid-N-tyrosine (ABA-tyr), which was identical to the prototype CRIA+ hybridoma protein R16.7. We also observed that the average relative affinity of CRIA- antibodies from immunized donors was higher than nonimmune mice with respect to p-ARS and ABA-tyr, and that there was a further increase in avidity to ABA-tyr over time after immunization. In contrast, no affinity maturation occurred among the CRIA+ and CRIA+/- clones. Rare antibodies were also identified within the nonimmune repertoire that had higher relative affinities than R16.7. Despite this, a greater proportion of CRIA+ and CRIA+/- clones produced IgG antibody in vitro. Thus, the predominance of CRIA+ antibodies in the ARS-specific immune repertoire and the preferential differentiation of CRIA+ precursor cells to IgG secretors cannot be entirely explained by the avidity of this clonotype family for p-ARS or ABA-tyr.     

Three novel mammalian toll-like receptors: gene structure, expression, and evolution

We describe three novel genes, encoding members of the Toll-like receptor (Tlr) family (TLR7, TLR8, and TLR9). These Tlr family members, unlike others reported to date, were identified within a genomic database. TLR7 and TLR8 each have three exons, two of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene. The remaining gene (TLR9) resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least two splice forms, one of which is monoexonic and one of which is biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus. The novel Tlrs comprise a cluster as nearest phylogenetic neighbors. Combining all sequence data related to Toll-like receptors, we have drawn several inferences concerning the phylogeny of vertebrate and invertebrate Tlrs. According to our best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most three or four were transmitted with the primordial vertebrate line. Phylogenetic data that we have adduced in the course of this work also suggest the existence of a Drosophila equivalent of MyD88, and indicate that the plasma membrane protein SIGIRR is close functional relative of MyD88 in mammals. Finally, a single present-day representative of the Toll-like proteins in Drosophila has striking cytoplasmic domain homology to mammalian Tlrs within the cluster that embraces TLRs 1, 2, 4, and 6. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago.