Characterization of the enzymatic activity of the actin cross-linking domain from the Vibrio cholerae MARTX Vc toxin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX(Vc)), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX(Vc) (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324-1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding.     

Localization of a new alpha-actinin binding site in the COOH-terminal part of actin sequence

The interaction of filamentous actin with alpha-actinin, an actin cross-linking protein, is well established. On the other hand, monomeric actin-alpha-actinin interaction has been a subject of controversy. In this report, we have characterized the interaction of monomeric actin, coated on plastic plates under conditions of non-polymerization, with alpha-actinin in presence of magnesium. Using specific polyclonal anti-actin antibodies, with the whole molecule or purified peptides, we have localized two sites of interaction on action molecule: one near Thr-103 and a new one in the twenty last amino acids.     

神经钙蛋白δ的功能及其分子作用机制

神经钙蛋白δ(neurocalcinδ)作为神经钙敏感蛋白(neuronal calcium sensors,NCSs)家族的重要成员之一,具有分布广泛、结构较保守的特性.早期研究发现,神经钙蛋白δ具有两对EF手结构(EF1,EF2,EF3和EF4).EF1不能结合Ca2+,而EF2、EF3和EF4与Ca2+结合能促使其N端豆蔻酰暴露,进而实现其由细胞质到细胞质膜的转移定位以及与靶蛋白的结合,从而发挥重要效应.本综述根据神经钙蛋白δ的"Ca2+-豆蔻酰基开关"特性,一方面介绍其能与膜鸟苷酸环化酶反应,参与cGMP信号转导,进而影响视觉和嗅觉,甚至血压等生物学活动;另一方面,介绍神经钙蛋白δ通过与S100β、网格蛋白、肌动蛋白、微管等蛋白质之间的相互作用,并参与细胞内囊泡运输,从而影响细胞内大分子包装、运输等过程.本文还阐明了神经钙蛋白δ参与精子发生、细胞癌变、肾病发生等过程.由于神经钙蛋白δ对了解某些疾病的发生原理、信号转导过程、细胞内信息调控网络等具有重要意义,本综述将为研究相关疾病提供新的研究方向与理论基础.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin I by cross-linking actin filaments

The actin-activated Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I was previously shown to be cooperatively dependent on the myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This observation was rationalized by assuming that myosin I contains a high-affinity and a low-affinity F-actin-binding site and that binding at the low-affinity site is responsible for the actin-activated ATPase activity. Therefore, enzymatic activity would correlate with the cross-linking of actin filaments by myosin I, and the cooperative increase in specific activity at high myosin:actin ratios would result from the fact that cross-linking by one myosin molecule would increase the effective F-actin concentration for neighboring myosin molecules. This model predicts that high specific activity should occur at myosin:actin ratios below that required for cooperative interactions if the actin filaments are cross-linked by catalytically inert cross-linking proteins. This prediction has been confirmed by cross-linking actin filaments with either of three gelation factors isolated from Acanthamoeba, one of which has not been previously described, or by enzymatically inactive unphosphorylated Acanthamoeba myosin I.     

Structure and Function of Palladin's Actin Binding Domain

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.     

The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex.

It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Do?, A. (1991) Biochemistry 30, 5769-5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1-133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.     

WICH, a member of WASP-interacting protein family, cross-links actin filaments

In yeast, Verprolin plays an important role in rearrangement of the actin cytoskeleton. There are three mammalian homologues of Verprolin, WIP, CR16, and WICH, and all of them bind actin and Wiskott-Aldrich syndrome protein (WASP) and/or neural-WASP. Here, we describe a novel function of WICH. In vitro co-sedimentation analysis revealed that WICH not only binds to actin filaments but also cross-links them. Fluorescence and electron microscopy detected that this cross-linking results in straight bundled actin filaments. Overexpression of WICH alone in cultured fibroblast caused the formation of thick actin fibers. This ability of WICH depended on its own actin cross-linking activity. Importantly, the actin cross-linking activity of WICH was modified through a direct association with N-WASP. Taken together, these data suggest that WICH induces a bundled form of actin filament with actin cross-linking activity and the association with N-WASP suppresses that activity. WICH thus appears to be a novel actin bundling protein.     

A new model for the interaction of dystrophin with F-actin

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.     

p-NN''-phenylenebismaleimide, a specific cross-linking agent for F-actin.

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN'-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN'-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.     

Molecular cloning and functional characterization of mouse coactosin-like protein.

Coactosin was first isolated from Dictyostelium discoideum and, as reported, human coactosin-like protein (CLP) was identified in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. A mouse CLP (mCLP) cDNA clone was identified among EMBL/GenBank EST sequences. The derived amino acid sequence (142 residues) was 95.1% identical with human CLP. Here, we also show that mCLP interacts with actin and 5LO in the two-hybrid system. High-speed cosedimentation assays and GST-binding assays confirmed these protein interactions. In chemical cross-linking experiments, one molecule of mCLP was covalently linked to either one subunit of actin or one molecule of 5LO. The mCLP-F-actin and mCLP-5LO associations were pH-insensitive and Ca(2+)-independent. However, association with actin was best observed at low salt concentrations, while association with 5LO was favored by salt, indicating different binding characteristics.     

The rigor configuration of smooth muscle heavy meromyosin trapped by a zero-length cross-linker

When chicken gizzard heavy meromyosin (HMM) in its rigor complex with actin was reacted with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), HMM cross-linked with actin but also the two heads of the HMM molecule cross-linked to each other [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry 28, 1898-1904, 1905-1912]. By ultracentrifugal fractionation of the EDC-treated acto-HMM in the presence of Mg-ATP, we obtained a preparation enriched for gizzard HMM with cross-linked heads. When HMM molecules in this preparation were rotary-shadowed and observed in an electron microscope, many head pairs were in contact with each other. The amount of HMM with cross-linked heads determined by electron microscopy was equal to that of the cross-linked NH2-terminal 24K tryptic fragments of HMM heavy chains determined by NaDodSO4 gel electrophoresis, indicating that this cross-linking is primarily responsible for the contact observed between two HMM heads. Most pairs of the contacted heads originated in the same HMM molecule, although a few pairs belonged to different HMM molecules. Cross-linking between the two heads of the same HMM molecule appeared to occur within the distal, more globular half of each head. However, the cross-linking sites were located at different positions within the globular portion. The actin-activated Mg-ATPase activity of the HMM sample treated with EDC in the presence of actin increased in a biphasic manner, depending on the concentration of F-actin, with two apparent association constants: 2.9 x 10(4) M-1 and one much less than 1 x 10(4) M-1. Since the apparent association constant obtained with the HMM control was similar to the latter value, the association constant for HMM molecules with cross-linked heads was identified to be the former value. The binding of HMM to actin was thus strengthened at least by a factor of 3 by the cross-linking between two HMM heads. These results suggest that HMM heads are trapped by treatment with EDC in the rigor complex configuration and that this configuration is retained even after the HMM has been released from actin. The EDC reactivity of rabbit skeletal muscle HMM, however, was different from that of chicken gizzard HMM. The treatment of acto-HMM complexes with EDC did not generate cross-linking between two skeletal muscle HMM heads.     

UNC-87 is an actin-bundling protein

The Caenorhabditis elegans unc-87 gene product is essential for the maintenance of the nematode body wall muscle where it is found colocalized with actin in the I band. The molecular domain structure of the protein reveals similarity to the C-terminal repeat region of the smooth muscle actin-binding protein calponin. In this study we investigated the in vitro function of UNC-87 using both the full-length recombinant molecule and several truncated mutants. According to analytical ultracentrifugation UNC-87 occurs as a monomer in solution. UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but not with monomeric G-actin, and exhibited potent actin filament bundling activity. Actin binding was independent of the presence of tropomyosin and the actin cross-linking proteins filamin and alpha-actinin. Consistent with its actin bundling activity in vitro, UNC-87 tagged with green fluorescent protein associated with and promoted the formation of actin stress fiber bundles in living cells. These data identify UNC-87 as an actin-bundling protein and highlight the calponin-like repeats as a novel actin-binding module.     

The Actin cross-linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF(N)ACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF(N)ACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg(2+) and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.     

Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii

A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.     

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.     

Cooperative effects of cofilin (ADF) on actin structure suggest allosteric mechanism of cofilin function

Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules.     

Neurocalcin: a novel calcium-binding protein from bovine brain.

A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.     

Interaction of myosin with F-actin: time-dependent changes at the interface are not slow

The kinetics of formation of the actin-myosin complex have been reinvestigated on the minute and second time scales in sedimentation and chemical cross-linking experiments. With the sedimentation method, we found that the binding of the skeletal muscle myosin motor domain (S1) to actin filament always saturates at one S1 bound to one actin monomer (or two S1 per actin dimer), whether S1 was added slowly (17 min between additions) or rapidly (10 s between additions) to an excess of F-actin. The carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, EDC)-induced cross-linking of the actin-S1 complex was performed on the subsecond time scale by a new approach that combines a two-step cross-linking protocol with the rapid flow-quench technique. The results showed that the time courses of S1 cross-linking to either of the two actin monomers are identical: they are not dependent on the actin/S1 ratio in the 0.3-20-s time range. The overall data rule out a mechanism by which myosin rolls from one to the other actin monomer on the second or minute time scales. Rather, they suggest that more subtle changes occur at the actomyosin interface during the ATP cycle.     

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer.     

Interaction of actinogelin with actin. No nucleation but high gelation activity

Nucleation activity of actin polymerization of actinogelin, a calcium-sensitive F-actin cross-linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl-actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization. By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F-actin through its end(s), and cross-links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F-actin and actinogelin. Under non-gelling high Ca2+ conditions, binding of actinogelin along the side of F-actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination. Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20 degrees C was 6 micrograms/ml for 250 micrograms/ml of actin. Comparing this value with those of the other actin cross-linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; more than 50 micrograms/ml of the protein was required for gelation. At 37 degrees C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20 degrees C, but this was still higher than that of gizzard alpha-actinin at 20 degrees C. Thus, role of actinogelin as an efficient and Ca2+-regulated cross-linker of microfilaments was substantiated.