扇贝毒素pectenotoxins(PTXs)研究进展

扇贝毒素(pectenotoxins,PTXs)是一类聚醚大环内酯结构的脂溶性海洋生物毒素,是由海洋甲藻中的鳍藻属Dinophysisspp.的几个种产生的,1984年首次从日本的养殖扇贝Patinopecten yessoensis中发现鉴定,具有很高的小鼠腹腔注射致死毒性。近年发现的地理区域不断扩大,我国尚属空白。就这一毒素的结构、来源生物、毒性、携带生物、地理分布、降解代谢及风险评估等研究现状作一系统综述,并分析展望了今后我国藻毒素研究的重点方向。     

Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins.

gCap39 is an actin filament end-capping protein which has a threefold repeated domain structure similar to the N-terminal half of gelsolin. However, unlike gelsolin, gCap39 does not sever actin filaments and dissociates completely from filament ends after calcium removal. We have capitalized on these differences to explore the structural basis for actin filament capping, severing, and their regulation. Using truncated gCap39, generated by limited proteolysis or deletion mutagenesis, we found that actin filament capping requires multiple gCap domains, and almost the entire molecule is necessary for optimal activity. gCap39 domain I, like the equivalent domain in gelsolin, contains an actin monomer binding site. gCap39 domains II-III are, however, different from gelsolin in that they do not bind to the side of actin filaments. Since filament side binding is hypothesized to be the first step in severing, lack of side binding may explain why gCap39 does not sever. This is confirmed directly by swapping gCap39 domains II-III for the side-binding gelsolin domains to generate a chimera which severs actin filaments. The chimera is Ca2+ independent in actin filament severing and capping, although gCap39 domain I itself is regulated by Ca2+.     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

Depolymerization of actin filaments by profilin. Effects of profilin on capping protein function

Profilin interacts with the barbed ends of actin filaments and is thought to facilitate in vivo actin polymerization. This conclusion is based primarily on in vitro kinetic experiments using relatively low concentrations of profilin (1-5 microm). However, the cell contains actin regulatory proteins with multiple profilin binding sites that potentially can attract millimolar concentrations of profilin to areas requiring rapid actin filament turnover. We have studied the effects of higher concentrations of profilin (10-100 microm) on actin monomer kinetics at the barbed end. Prior work indicated that profilin might augment actin filament depolymerization in this range of profilin concentration. At barbed-end saturating concentrations (final concentration, approximately 40 microm), profilin accelerated the off-rate of actin monomers by a factor of four to six. Comparable concentrations of latrunculin had no detectable effect on the depolymerization rate, indicating that profilin-mediated acceleration was independent of monomer sequestration. Furthermore, we have found that high concentrations of profilin can successfully compete with CapG for the barbed end and uncap actin filaments, and a simple equilibrium model of competitive binding could explain these effects. In contrast, neither gelsolin nor CapZ could be dissociated from actin filaments under the same conditions. These differences in the ability of profilin to dissociate capping proteins may explain earlier in vivo data showing selective depolymerization of actin filaments after microinjection of profilin. The finding that profilin can uncap actin filaments was not previously appreciated, and this newly discovered function may have important implications for filament elongation as well as depolymerization.     

Tropomodulin 3 binds to actin monomers

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.     

Crystal structures of monomeric actin bound to cytochalasin D

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin have not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD: one obtained by soaking preformed actin crystals with CD, and the other obtained by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play equally important roles in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. An ∼ 6° rotation of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure instead of being disordered, as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.     

Gelsolin as a calcium-regulated actin filament-capping protein.

Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.     

Actin filament capping protein from bovine brain.

An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F-actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the 'barbed' end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+-independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.     

Mammalian twinfilin sequesters ADP-G-actin and caps filament barbed ends: implications in motility

Twinfilins are conserved actin-binding proteins composed of two actin depolymerizing factor homology (ADF-H) domains. Twinfilins are involved in diverse morphological and motile processes, but their mechanism of action has not been elucidated. Here, we show that mammalian twinfilin both sequesters ADP-G-actin and caps filament barbed ends with preferential affinity for ADP-bound ends. Twinfilin replaces capping protein and promotes motility of N-WASP functionalized beads in a biomimetic motility assay, indicating that the capping activity supports twinfilin's function in motility. Consistently, in vivo twinfilin localizes to actin tails of propelling endosomes. The ADP-actin-sequestering activity cooperates with the filament capping activity of twinfilin to finely regulate motility due to processive filament assembly catalyzed by formin-functionalized beads. The isolated ADF-H domains do not cap barbed ends nor promote motility, but sequester ADP-actin, the C-terminal domain showing the highest affinity. A structural model for binding of twinfilin to barbed ends is proposed based on the similar foldings of twinfilin ADF-H domains and gelsolin segments.     

Interaction in vivo and in vitro between the yeast fimbrin, SAC6P, and a polymerization-defective yeast actin (V266G and L267G)

A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability. This actin will not polymerize in vitro but is compatible with cell viability. We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C. The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4. At this ratio, every actin monomer contacts a Sac6p actin binding domain. Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei. Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful. The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene. Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell.     

Actin cytoskeleton and small heat shock proteins: how do they interact?

Actin and small heat shock proteins (sHsps) are ubiquitous and multifaceted proteins that exist in 2 reversible forms, monomers and multimers, ie, the microfilament of the cytoskeleton and oligomers of the sHsps, generally, supposed to be in a spherical and hollow form. Two situations are described in the literature, where the properties of actin are modulated by sHsps; the actin polymerization is inhibited in vitro by some sHsps acting as capping proteins, and the actin cytoskeleton is protected by some sHsps against the disruption induced by various stressful conditions. We propose that a direct actin-sHsp interaction occurs to inhibit actin polymerization and to participate in the in vivo regulation of actin filament dynamics. Protection of the actin cytoskeleton would result from an F-actin-sHsp interaction in which microfilaments would be coated by small oligomers of phosphorylated sHsps. Both proteins share common structural motives suggesting direct binding sites, but they remain to be demonstrated. Some sHsps would behave with the actin cytoskeleton as actin-binding proteins capable of either capping a microfilament when present as a nonphosphorylated monomer or stabilizing and protecting the microfilament when organized in small, phosphorylated oligomers.     

Mechanism of action of cytochalasin B on actin

Substoichiometric cytochalasin B (CB) inhibits both the rate of actin polymerization and the interaction of actin filaments in solution. The polymerization rate is reduced by inhibition of actin monomer addition to the "barbed" end of the filaments where monomers normally add more rapidly. 2 microM CB reduces the polymerization rate by up to 90%, but has little effect on the rate of monomer addition at the slow ("pointed") end of the filaments and no effect on the rate of filament annealing. Under most ionic conditions tested, 2 microM CB reduces the steady state high shear viscosity by 10-20% and increases the steady state monomer concentration by a factor of 2.5 or less. In addition to the effects on the polymerization process, 2 microM CB strongly reduces the low shear viscosity of actin filaments alone and actin filaments cross-linked by a variety of macromolecules. This may be due to inhibition of actin filament-filament interactions which normally contribute to network formation. Since the inhibition of monomer addition and of actin filament network formation have approximately the same CB concentration dependence, a common CB binding site, probably the barbed end of the filament, may be responsible for both effects.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.     

ADF/cofilin weakens lateral contacts in the actin filament.

Observed in vivo motility rates can only be accounted for if the rate of actin filament treadmilling in cells is considerably greater than has been quantified for purified actin in vitro. ADF/cofilin is uniquely suited to promote actin dynamics in cells, owing to its remarkable ability to change actin filament structure. In earlier work we showed that human cofilin chanRges filament twist by about 5 degrees per subunit and suggested that this contributes to increased filament turnover. Our initial structural modeling provided some insights into how the longitudinal actin-actin contacts might be disrupted following cofilin-induced twisting. Here we present direct evidence that cofilin also disrupts lateral actin-actin contacts in the filament and suggest a model showing how this could contribute to cofilin's novel effects on actin filament dynamics and assembly.     

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1–4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1–3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.     

Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1

BACKGROUND: Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo. RESULTS: Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo. CONCLUSIONS: We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.     

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.     

Profilin-mediated competition between capping protein and formin Cdc12p during cytokinesis in fission yeast

Fission yeast capping protein SpCP is a heterodimer of two subunits (Acp1p and Acp2p) that binds actin filament barbed ends. Neither acp1 nor acp2 is required for viability, but cells lacking either or both subunits have cytokinesis defects under stressful conditions, including elevated temperature, osmotic stress, or in combination with numerous mild mutations in genes important for cytokinesis. Defects arise as the contractile ring constricts and disassembles, resulting in delays in cell separation. Genetic and biochemical interactions show that the cytokinesis formin Cdc12p competes with capping protein for actin filament barbed ends in cells. Deletion of acp2 partly suppresses cytokinesis defects in temperature-sensitive cdc12-112 cells and mild overexpression of capping protein kills cdc12-112 cells. Biochemically, profilin has opposite effects on filaments capped with Cdc12p and capping protein. Profilin depolymerizes actin filaments capped by capping protein but allows filaments capped by Cdc12p to grow at their barbed ends. Once associated with a barbed end, either Cdc12p or capping protein prevents the other from influencing polymerization at that end. Given that capping protein arrives at the division site 20 min later than Cdc12p, capping protein may slowly replace Cdc12p on filament barbed ends in preparation for filament disassembly during ring constriction.     

Structural basis of actin sequestration by thymosin-beta4: implications for WH2 proteins

The WH2 (Wiscott-Aldridge syndrome protein homology domain 2) repeat is an actin interacting motif found in monomer sequestering and filament assembly proteins. We have stabilized the prototypical WH2 family member, thymosin-beta4 (Tbeta4), with respect to actin, by creating a hybrid between gelsolin domain 1 and the C-terminal half of Tbeta4 (G1-Tbeta4). This hybrid protein sequesters actin monomers, severs actin filaments and acts as a leaky barbed end cap. Here, we present the structure of the G1-Tbeta4:actin complex at 2 A resolution. The structure reveals that Tbeta4 sequesters by capping both ends of the actin monomer, and that exchange of actin between Tbeta4 and profilin is mediated by a minor overlap in binding sites. The structure implies that multiple WH2 motif-containing proteins will associate longitudinally with actin filaments. Finally, we discuss the role of the WH2 motif in arp2/3 activation.