Mutations in β‐actin: influence on polymer formation and on interactions with myosin and profilin

Aspenström, Pontus; Schutt, Clarence E.; Lindberg, Uno; Karlsson, Roger

doi:10.1016/0014-5793(93)80215-g

Cited by 38 publications

(41 citation statements)

References 49 publications

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“…The severe nucleation defect in a yeast Cys-374 to Ser actin mutant (38) and the lethality of a yeast C-terminal-truncated mutant (39) are therefore consistent with a significant role for the antiparallel dimer as a filament precursor for in vitro nucleation and in vivo function. The abnormalities are not otherwise likely explained by disruption of intrafilament contacts between actin subunits because the Lorenz model shows no actin-actin contacts at the C terminus (32).…”

Section: Discussionmentioning

confidence: 57%

Polylysine Induces an Antiparallel Actin Dimer That Nucleates Filament Assembly

Bubb¹,

Govindasamy²,

Yarmola³

et al. 2002

Journal of Biological Chemistry

111

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An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-Å resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.Actin filament nucleation occurs very slowly de novo, but it occurs rapidly as a necessary step in actin-based motility (1). The formation of a dimer from monomeric subunits is the most thermodynamically unfavorable nucleation step with an estimated equilibrium dissociation constant of 4.6 M (in contrast to 0.6 mM for conversion of dimer to trimer) in a recent molecular dynamic simulation of nucleation (2). The formation of an effective nucleus may be accelerated in vivo by an actin-binding protein such as gelsolin, which can stabilize dimeric actin, or by a protein complex such as Arp2/3 that is thought to contain two actin-like molecules constrained in an orientation that promotes nucleation (3, 4). Antiparallel actin dimers have been identified as a precursor to actin filament polymerization by covalent cross-linking during polymerization induced with divalent cations (5). A gelsolin-actin complex capable of nucleating filament growth at the slow growing, pointed end of filaments has also been shown by covalent cross-linking to contain two actin subunits in the antiparallel configuration (6). The assumption of an antiparallel configuration of subunits is based on evidence that Cys-374 in the C terminus of actin is the only residue involved in the cross-linking reaction. In contrast, when polymerization is complete, intrafilament cross-linking yields a parallel dimer. More recently, electron microscopy has revealed that newly formed actin filaments show evidence of incorporation of antiparallel dimers. This incorporation results in a branched filament network, implying that the dimers have nucleating activity (7). Interestingly, analysis of a Listeria model of cell motility using high-resolution laser tracking provides evidence that filaments elongate in 5.4 nm steps, consistent with in vivo incorporation of dimeric actin (8).In the current work, we provide evidence that polylysine nucleates actin polymerization by e...

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Section: Discussionmentioning

confidence: 57%

Polylysine Induces an Antiparallel Actin Dimer That Nucleates Filament Assembly

Bubb¹,

Govindasamy²,

Yarmola³

et al. 2002

Journal of Biological Chemistry

111

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“…A C374S mutation in avian nonmuscle ␤-actin causes adverse changes in critical concentration, the ability to translocate in an in vitro motility assay, and the ability to interact with profilin (8). Modification of Cys-374 with fluorescent reagents such as pyrene maleimide affects both the myosin S1 ATPase activity and sliding velocity using in vitro motility assays, although the specificity of the effect depends on the particular fluorescent probe utilized (7).…”

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confidence: 99%

A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype

McKane

Wen

Boldogh

et al. 2005

Journal of Biological Chemistry

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To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.The actin C-terminal region, consisting of residues 364 -375, is located in subdomain 1 on the opposite side of the protein from that of the N terminus and is believed to play an important role in the interaction of actin with different actin-binding proteins such as profilin (1, 2), cofilin (3), and gelsolin (4) in the determination of actin filament stability and in the allosteric behavior of the actin filament. The crystal structure of the filament shows that a direct interaction of the N and C termini is virtually impossible because of the intervening mass of the core of subdomain 1 (Fig. 1).The sequence of the C-terminal peptide is highly conserved throughout the actin family. The sequences for mammalian and avian muscle actins are identical and there are only two differences between yeast and higher eukaryotic actins, a substitution of His in yeast at residue 372 for Arg and a substitution of Ser at residue 365 for Arg just outside of the helix in this peptide. In the first case the His and the Arg carry at least a partial positive charge.Removal of the C-terminal three residues in yeast actin causes cell inviability, whereas removal of either or both of the first two residues, beginning with the C terminus, shows milder phenotypes including temperature sensitivity and increased cell size (5). The actin C-terminal dipeptide or tripe...

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“…Actin of rabbit skeletal muscle has five cysteine residues at different positions, but only CyS-374 in the C-terminal region is exposed and important for polymerization (Aspenström et al, 1993). The same residue is a decisive site for S-nitrosation (DalleDonne et al, 2000).…”

Section: α-Actinmentioning

confidence: 99%

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

Belcastro

Gaucher

Corti

et al. 2017

Biological Chemistry

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Decades of chemical, biochemical and pathophysiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called 'redox regulation' of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific aminoacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties (S-nitrosation, leading to formation of protein S-nitrosothiols) or binding of low molecular weight thiols (S-thionylation, i.e . in particular S-glutathionylation, S-cysteinylglycinylation and S-cysteinylation).A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S-nitrosations and S-thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases.

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Mutations in β‐actin: influence on polymer formation and on interactions with myosin and profilin

Cited by 38 publications

References 49 publications

Polylysine Induces an Antiparallel Actin Dimer That Nucleates Filament Assembly

Polylysine Induces an Antiparallel Actin Dimer That Nucleates Filament Assembly

A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology

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