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M R Bubb

Publications and source records attributed to M R Bubb.

32 records · Page 2Linked to original sources
PubMed1996

Self-assembly of the brain MAP-2 microtubule-binding region into polymeric structures resembling Alzheimer filaments.

The neuronal microtubule-associated protein known as MAP-2 has not been considered to be a subunit of paired helical filaments (PHFs) in neurofibrillary tangles seen in Alzheimer's Disease. We now describe the assembly of paired helical filament-like structures from MAP-2's 203-residue microtubule-binding region (MTBR). SDS gel electrophoresis and equilibrium ultracentrifugation suggest that a dimeric form, cross-linked by an interchain disulfide, is involved in polymerization. MAP-2 MTBR polymers bind thioflavin-S, a dye used to histochemically localize Alzheimer neurofibrillary tangles. Our finding that PHF-like structures assemble from a MAP-2 fragment raises new questions about MAP-2's role in the etiology of Alzheimer's Disease.

Alzheimer Disease↗
PubMed1995

Kinetic model for the inhibition of actin polymerization by actobindin.

Although Acanthamoeba actobindin binds actin monomers, its inhibition of actin polymerization differs from that of a simple monomer-sequestering protein in that actobindin inhibits nucleation very much more than elongation [Lambooy, P. K., & Korn, E. D. (1988) J. Biol. Chem. 263, 12836-12843] and can induce the accumulation of actin dimers in stoichiometric excess of the actobindin concentration [Bubb, M. R., Knutson, J. R., Porter, D. K., & Korn, E. D. (1994) J. Biol. Chem. 269, 25592-25597]. We now describe a "catalytic" model for the interaction of actobindin with actin monomer that quantitatively accounts for the effects of actobindin on the kinetics of actin polymerization de novo and the elongation of actin filaments. We propose that, in a polymerizing buffer, actobindin binds to two actin subunits forming an heterotrimeric complex that is incompetent for nucleation, self-association, and elongation. Actobindin can, however, dissociate from this complex, leaving a novel actin dimer that can participate in elongation but remains incompetent for nucleation and self-association. Under appropriate conditions, the concentration of this novel actin dimer can exceed the actobindin concentration; thus, the model is catalytic rather than stoichiometric. The experimentally observed time course of actin polymerization de novo, the rate of elongation of filaments, and the amount of actin dimer formed as a function of actobindin concentration are all consistent with the catalytic model and inconsistent with the stoichiometric model. The rate of actobindin-induced actin dimer formation is consistent with the hypothesis that the rate-limiting step is this pathway is the formation of a precursor heterotrimeric complex.

Acanthamoeba↗
PubMed1995

Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

3T3 Cells↗
PubMed1994

Actobindin binds with high affinity to a covalently cross-linked actin dimer.

Actobindin, a 9.8-kDa protein purified from Acanthamoeba castellanii, contains two actin-binding sites that can simultaneously bind two actin monomers. However, actobindin inhibits actin polymerization to a greater extent than can be explained by its affinity for actin monomers (site-specific KD = 3.3 microM). This paradox would be resolved if actobindin could interfere with the nucleation phase of polymerization by using both binding sites to bind simultaneously to an actin oligomer because the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-linked actin dimer prepared from cross-linked F-actin binds to actobindin with high affinity (apparent KD = 11 nM) in accordance with theoretical predictions for simultaneous binding of two actin subunits per single actobindin and consistent with the hypothesis that actobindin might bind to native actin oligomers and prevent them from nucleating polymerization. Furthermore, the interaction with cross-linked dimer exhibits specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, only this isomeric dimer is produced when cross-linking reagents are added to actin undergoing polymerization in the presence of actobindin. Therefore, if actobindin inhibits polymerization by interacting with a native dimer whose conformation is similar to that of the cross-linked dimer with slower electrophoretic mobility, then actobindin must either block the cross-linking sites or convert the dimer to a different conformation.

Acanthamoeba↗
PubMed1994

Actobindin induces the accumulation of actin dimers that neither nucleate polymerization nor self-associate.

Actobindin purified from Acanthamoeba castellanii inhibits the nucleation, but not the elongation, phase of actin polymerization. Previously, we had speculated that actobindin, which can simultaneously bind two actin monomers (Bubb, M.R., Lewis, M.S., and Korn, E.D. (1991) J. Biol. Chem. 266, 3820-3826), might preferentially interact with small oligomers and inhibit their ability to elongate (Lambooy, P.K., and Korn, E.D. (1988) J. Biol. Chem. 263, 12836-12843). In the accompanying paper (Bubb, M.R., Lewis, M.S., and Korn, E.D. (1994) J. Biol. Chem. 269, 25587-25591), we show that under non-polymerizing conditions, actobindin binds to covalently cross-linked actin dimers with higher affinity than to two actin monomers. The sedimentation velocity and fluorescence anisotropy experiments described in this paper show that actobindin prevents the formation of actin oligomers larger than an actin dimer under conditions in which, in the absence of actobindin, actin rapidly polymerizes to F-actin with no detectable small oligomers. Moreover, the molar concentration of actin dimer formed in the presence of actobindin can exceed the total actobindin concentration. These results indicate that actobindin does not form a stable complex with native actin dimer but, rather, causes the accumulation of dimers that are unable to nucleate polymerization or self-associate.

Acanthamoeba↗
PubMed1994

Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin.

Jasplakinolide, a naturally occurring cyclic peptide from the marine sponge, Jaspis johnstoni, has both fungicidal and antiproliferative activity. We now report that this peptide is a potent inducer of actin polymerization in vitro. The peptide has a much greater effect on Mg(2+)-actin than on Ca(2+)-actin. Competitive binding studies using rhodamine-phalloidin suggest that jasplakinolide binds to F-actin competitively with phalloidin with a dissociation constant of approximately 15 nM. This compares favorably to the previously reported IC50 of 35 nM for the antiproliferative effect of jasplakinolide on PC3 prostate carcinoma cells. The binding curve suggests that nearest neighbor positive cooperativity influences the binding of jasplakinolide (and perhaps also phalloidin) to F-actin. These results imply that jasplakinolide may exert its cytotoxic effect in vivo by inducing actin polymerization and/or stabilizing pre-existing actin filaments.

Actins↗
PubMed1993

Rabbit skeletal muscle actin behaves differently than Acanthamoeba actin when added to soluble extracts of Acanthamoeba castellanii.

Cold extracts of Acanthamoeba castellanii in polymerizing buffer contain 32 microM unpolymerized actin of which about 20% polymerizes (as measured by ultracentrifugation) when the extract is warmed to 22 degrees C. As quantified by the increase in fluorescence of pyrene-labeled actin, 16% of muscle G-actin and 46% of Acanthamoeba G-actin polymerized when 0.8 microM of each was added to warm extracts of Acanthamoeba. Added muscle F-actin (1.2 microM) rapidly and totally depolymerized and then partially repolymerized whereas 1.2 microM added Acanthamoeba F-actin was stable indefinitely. Furthermore, muscle actin subunits were completely removed from copolymers of muscle and Acanthamoeba F-actin while all the amoeba actin remained polymerized when the copolymers contained at least 50% amoeba actin. These results suggest that exogenous tracer actin may not be an accurate indicator of the dynamics of endogenous actin in extracts and cells.

Acanthamoeba↗
PubMed1992

Profilin-actin complexes directly elongate actin filaments at the barbed end.

We demonstrate that the profilin-G-actin complex can elongate actin filaments directly at the barbed end but cannot bind to the pointed end. During elongation, the profilin-actin complex binds to the barbed filament end, whereupon profilin is released, leaving the actin molecule behind. This was first proposed by Tilney [Tilney, L. G., et al. (1983) J. Cell Biol. 97, 112-124] and demonstrated by Pollard and Cooper [(1984) Biochemistry 23, 6631-6641] by electron microscopy. We show that a model without any outside energy supply, in contrast to the mechanism proposed by Pollard and Cooper, can be fitted to our and their [Kaiser et al. (1986) J. Cell Biol. 102, 221-226] findings. Input of outside energy is necessary only if profilin-mediated elongation continues after free G-actin has been lowered to or below the critical concentration observed at the barbed end in the absence of profilin.

Acanthamoeba↗
PubMed1991

The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif.

Actobindin is an 88-amino acid polypeptide, containing two almost identical repeated domains of 33 and 34 residues. Depending on the molar ratios in which they are mixed, actobindin binds either one or two actin molecules. We cross-linked actobindin and actin in the 1:1 complex, using the zero-length cross-linker 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. The cross-linked peptides were purified after consecutive CNBr cleavage and trypsin and Staphylococcus protease V8 digestions, and the cross-linked side chains were identified by amino acid sequencing. Isopeptide linkages were formed between residues Glu-100 of actin and Lys-16 of actobindin. In addition, we found a connection between one or more of the acidic residues 1,2, or 3 of actin and Lys-16 and Lys-52 of actobindin. The cross-linked regions in actobindin contain Leu-Lys-His-Ala-Glu-Thr motifs, similar to sequences observed in several other actin-binding proteins.

Acanthamoeba↗
PubMed1991

The interaction of monomeric actin with two binding sites on Acanthamoeba actobindin.

Actobindin was previously shown to be an 88-residue polypeptide (Mr 9761) with an internal tandem repeat of 33-34 amino acids. Sedimentation equilibrium experiments have confirmed this Mr for native actobindin. Pyreneglyoxal-labeled actobindin had a similar Mr by sedimentation equilibrium analysis and bound to actin in a manner qualitatively similar to unmodified actobindin as determined by gel electrophoretic analysis of covalently cross-linked products. The stoichiometry of the actin-actobindin interaction was determined from the change in apparent Mr of pyrene-glyoxal-labeled actobindin in the presence of actin, as determined by scanning the ultracentrifuge cell at a wavelength that detected only the labeled protein. These data were consistent with the formation of a complex containing two actin and one actobindin molecules. The overall KD describing the binding of the first actin to either of the two sites on actobindin was 3.3 microM. The binding constant for the second actin suggested either negative cooperativity or inequality of the two actin-binding sites. Similar binding constants were obtained by analysis of the fluorescence enhancement that occurred when actobindin bound to actin labeled with either pyrene iodoacetamide or 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3-diazole. Cross-linking experiments with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxy-sulfosuccinimide qualitatively agreed with predictions made from a two-binding site model. Additionally, both the fluorescence and cross-linking experiments suggested that the interaction of the two actin molecules may contribute to the stability of the heterotrimeric complex.

Acanthamoeba↗
PubMed1990

The covalent structure of Acanthamoeba actobindin.

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.

Acanthamoeba↗
PubMedDate not supplied

Inclusion body myositis long after dermatomyositis: a report of two cases.

Dermatomyositis, polymyositis, and inclusion body myositis are rare illnesses which appear to be distinct in clinical and pathologic features, pathogenesis, natural history, and response to therapy. We report two patients who first developed dermatomyositis, and then, after a disease-free interval of many years, developed inclusion body myositis. This may have useful therapeutic implications for patients with dermatomyositis whose illness bocomes refractory to treatment.

Adult↗