Biomedical subjects
Publications and source records attributed to J Avila.
Protein 4.1 binds to tubulin, as determined by sedimentation and immunoelectron-microscopy analyses, at a molar ratio similar to that described for brain microtubule-associated proteins. The binding site appears to be located at the C-terminal region of tubulin. Experiments performed in situ by adding exogenous protein 4.1 to permeabilized 3T3 cells show that this protein binds to microtubule and nuclear components.
Tau factor, upon urea treatment, is able to polymerize in vitro. These polymers are composed of tau factor as shown by immunogold staining. The structure of tau polymers is very similar to that of paired helical filaments (PHFs) of Alzheimer's disease in their dimensions as well as in their periodicity. Metal shadowing of both polymers shows a similar twisting. Also, similar peptide maps were found for tau factor and a 33 kDa protein that is the main component of our PHF preparations.
Paired helical filaments, which are present in the brain of Alzheimer's disease patients have been isolated and characterized. Treatment of the filaments with reducing agents and detergents extracts several proteins from these structures. The remaining filaments are composed mainly of a protein with molecular weight of 33 kDa suggesting that this protein is the core component of these filaments. Peptide mapping using trypsin and endoproteinase Arg-C revealed that the 33 kDa protein, was a modified form of tau protein present in normal human brain.
The capacity for self-polymerization and shape of the tubulin polymers assembled after digestion with trypsin, Pronase, chymotrypsin, subtilisin, Staphylococcus aureus proteinase V8 and proteinase K were investigated. Digestion with trypsin, Pronase or chymotrypsin resulted in a decrease in the ability of tubulin for self-assembly, whereas limited proteolysis with subtilisin, S. aureus proteinase V8 or proteinase K resulted in an increase in such ability. The shape of the assembled polymers varied from typical microtubules (after the treatment with trypsin or Pronase) to sheets (after the treatment with chymotrypsin) and from hooked microtubules with a constant polarity (after the treatment with subtilisin) to the disappearance of a defined polarity of such polymers (after the treatment with S. aureus V8 proteinase or proteinase K). These results indicate that the tubulin C-terminal regions are involved in the regulation of microtubule polymerization, shape, directional growth and lateral interactions between tubulin protofilaments.
Calcium has been suggested to be an important element in the regulation of microtubule dynamics 'in vivo'. In this report we have analyzed the possibility that microtubule-associated protein 2 (MAP2) binds calcium. MAP2 was blue-stained with the cationic carbocyanine dye 'stains-all' in a similar way to that of calcium-binding proteins and bound 45Ca as estimated from dot-blotting experiments. The calcium-binding characteristics of MAP2, determined by equilibrium dialysis, indicated that MAP2 bound about 3 mol (n = 2.9 +/- 0.4) of calcium per mol of protein (Kd = (0.9 +/- 0.2).10(-5) M). Analysis of the Scatchard plots from equilibrium dialysis and dot-blot assays indicated that MAP2 also presented low-affinity calcium-binding sites (Kd = (0.3 +/- 0.2).10(-4) M). Incubation of nitrocellulose blots of proteolytically digested MAP2 with 45Ca indicated that the calcium-binding sites were located in the region that is not involved in the interaction with tubulin (projection region).
Neuroblastoma (N2A) cells were found to develop axon-like neurite extensions when grown in the presence of triiodothyronine (T3), while C6 cells (of glial origin) did not. Analysis of radiolabelled protein synthesis showed that, in N2A only, T3 increased the synthesis of a polypeptide corresponding in electrophoretic mobility to the microtubule-associated protein MAP 1B. Immunoblotting of total cell proteins with a monoclonal antibody confirmed that this polypeptide was immunologically related to MAP 1B. Further studies using indirect immunofluorescence with monoclonal antibodies against both tubulin and MAP 1B showed that both antigens were present in neurites. Taken together, these results suggest that T3 may control maturation of neural tissue via effects on the microtubule-associated proteins in cells of neuronal origin.
Purified brain microtubule protein is phosphorylated by endogenous protein kinase activities in the presence of [gamma-32P] ATP or [gamma-32P] GTP. Here we show that certain microtubule-associated proteins are phosphorylated differently by GTP or ATP as direct phosphoryl donors, suggesting the presence of distinct kinase activities, with different specificities, associated with microtubule protein.
The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid, sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with 65Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first 150 amino acids of both alpha- and beta-tubulin subunits.
A procedure is described for radioiodination to a high specific activity of proteins immobilized on nitrocellulose membranes. After radioiodination, the proteins can be removed from the nitrocellulose to perform such structural analyses as proteolytic mapping. This method allows the detection of small amounts of protein recognized by immunoblotting and may be used to compare the structure of immunologically related proteins.
A rabbit serum raised against sheep erythrocyte spectrin stained mainly the flagella of epimastigote forms of the parasitic protozoan Trypanosoma cruzi. In Western blots of T. cruzi proteins solubilized with Nonidet P-40 it recognized mainly a polypeptide doublet with an apparent molecular weight of about 240 kDa, while a pre-immune rabbit serum and a tubulin monoclonal antibody did not. The results are consistent with the idea that a spectrin-like protein may be involved in cytoskeleton-membrane interactions in flagella of T. cruzi.
A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.
Anticentromere antibodies (ACA) present in a high percentage of patients with complete or incomplete CREST scleroderma, and which are presently used in the diagnosis of this disease, also appear in some primary Raynaud's phenomenon patients. Three principal centromeric antigens, CENP-A, CENP-B and CENP-C, have been described as reacting with the sera of these individuals. We attempt to determine whether or not a correlation between the presence of ACA and serum reactivity against one or more of these peptides could be established, and have observed that CENP-A, but not CENP-B or CENP-C, is specifically recognized by all patients sera tested. The fact that this reactivity is clearly detectable at very high serum dilutions, thus eliminating other non-specific interference, suggests that anti-CENP-A activity might be useful in the diagnosis of patients with CREST-associated Raynaud's phenomenon.
The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.
Insulin receptor kinase phosphorylated tubulin in an insulin-dependent fashion. Two different populations of phosphotubulin were found. In tubulin dimers containing tyrosine at the carboxyl-terminal of their alpha subunit, phosphate was incorporated in that residue, and the phosphorylated protein did not assemble into polymers. In tubulin dimers lacking this tyrosine residue, phosphate was incorporated into different tyrosine residues located in other parts of the molecule, and the phosphoprotein retained its capacity to polymerize.
2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.
Microtubule-associated proteins (MAPs) have been isolated from different development stages of Drosophila melanogaster and characterized by their association to tubulin, but not to tubulin lacking its 4-kD carboxy terminal region (S-tubulin), and by their ability to promote tubulin polymerization. Following these criteria some peptides of Mr 255, 205, and 180 kD were identified as MAPs. By means of immunological analogy we have identified a peptide related to mammalian brain MAP known as tau factor.
Limited proteolysis of tubulin with subtilisin results in the cleavage of both tubulin subunits yielding S-tubulin heterodimer and 4 kDa peptide fragments containing the carboxyl-terminal domains of alpha- and beta-polypeptide chains. S-tubulin binds colchicine and the characterization of the binding of colchicine to S-tubulin molecules showed a decreased rate of decay of colchicine binding activity as compared to that of undigested tubulin. However, S-tubulin exhibited a lower colchicine binding constant than tubulin. Peptide fragments resulting from the controlled tryptic proteolysis of both pure tubulin and S-tubulin were purified by filtration chromatography and presented a strong colchicine binding activity with association constants of 4.5 X 10(6) and 2.7 X 10(6) M-1, respectively. Furthermore, these studies support our initial findings on the localization of the tubulin site for colchicine (Serrano L, Avila J, Maccioni RB: J Biol Chem 259:6607-6611, 1984) and define the colchicine binding domain in a domain of alpha-subunit from the point of limited tryptic cleavage to the site of subtilisin controlled proteolysis of that tubulin subunit. On the basis of these alterations in the interaction of colchicine upon removal of the C-terminal moiety of tubulin and since no change in the number of binding sites was found after subtilisin digestion, we suggest that the carboxyl-terminal region of tubulin subunits modulates the binding of colchicine.