Biomedical subjects

C Roustan

Publications and source records attributed to C Roustan.

At least 19 recordsLinked to original sources

PubMed2004

Biochemical characterization of the L-plastin-actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin-binding domains of the molecule.

Actin interaction with L-plastin, a plastin/fimbrins isoform of the alpha-actinin family of molecules, is poorly characterized, from the biochemical point of view. Besides, molecular modeling of the T-isoform has recently provided a complete model of interaction with filamentous actin [Volkmann, N., DeRosier, D., Matsudaira, P., and Hanein, D. (2001) J. Cell Biol. 153, 947-956]. In this study, we report that recombinant L-plastin binds actin in a manner that strongly resembles that of the alpha-actinin-actin interface. The similitudes concern the absence of specificity toward the actin isoform and the inhibition of the binding by phosphoinositides. Furthermore, the participation of actin peptides 112-125 and 360-372 in the interface together with an inhibition of the rate of pyrenyl F-actin depolymerization is in favor of a lateral binding of the plastin isoform along the filament axis and strenghtens the similitudes in the way L-plastin and alpha-actinin bind to actin. We have also investigated the functional aspect and the putative equivalence of the two actin-binding domains of L-plastin toward actin binding. We demonstrate for the first time that the two recombinant fragments, expressed as single domains, have different affinities for actin. We further analyzed the difference using chemical cross-linking and F-actin depolymerization experiments assayed by fluorescence and high-speed centrifugation. The results clearly demonstrate that the two actin-binding domains of plastin display different modes of interaction with the actin filament. We discuss these results in light of the model of actin interaction proposed for T-plastin.

Actinin↗

PubMed2001

Postmortem degradation of white fish skeletal muscle (sea bass, Dicentrarchus labrax): fat diet effects on in situ dystrophin proteolysis during the prerigor stage.

All during fish postmortem evolution, structural muscle proteins are targets for various proteases. During the prerigor period (24 hours at 4 degrees C for sea bass), cytoskeletal proteins are affected by the first proteolytic events. These cleavages disrupt connections between myofibrils and the extracellular matrix, induce segmentation of myofibril cores, and modify the rheological properties of tissue. Dystrophin, a cytoskeletal actin-binding protein, is a relevant in situ marker for muscular proteolysis in the prerigor period. The immunodetection of dystrophin allowed the monitoring of early proteolysis during fish storage. Using antidystrophin antibodies directed toward the carboxy-terminal region, a highly sensitive domain exposed to calpain activity, we showed that proteolysis kinetics are strongly influenced by the muscular lipid content. In particular, comparison between low-fat diets (11.3% lipid) and high-fat diets (30% lipid), used during sea bass farming (90 days), revealed a faster proteolysis rate during the first 8 hours of storage at 0 degrees C with the high-fat diet. The origin of this faster proteolysis is discussed on the basis of a possible activation or translocation of calpains related to lipid accumulation in muscle fibers and cytoskeleton alterations.

Journal Article↗

PubMed2001

Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.

Actin Depolymerizing Factors↗

PubMed2001

The second ADF/cofilin actin-binding site exists in F-actin, the cofilin-G-actin complex, but not in G-actin.

ADF/cofilins are actin binding proteins that bind actin close to both the N- and C-termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112-125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893-28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol. 138, 771-781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G-actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G-actin by FRET induced by cofilin, to reveal the second cofilin-binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation.

Actin Depolymerizing Factors↗

PubMed2000

Interaction of actin with the capping protein, CapZ from sea bass (Dicentrarchus labrax) white skeletal muscle.

We have compared the functional properties of CapZ from fish white skeletal muscle with those of CapZ from chicken muscle. CapZ is a heterodimer, which enhances actin nucleation and inhibits the depolymerization process by binding to the barbed ends of microfilaments. Here, we report the interaction of CapZ not only with F-actin, but also with monomeric actin. The affinity of sea bass CapZ for G-actin estimated by enzyme-linked immunosorbent assay (ELISA) was in the microM range. This association was PIP2 dependent. Binding contacts with the barbed end of actin were delimited by both ELISA and fluorescence approaches. One site (actin sequence 338-348) was located in a helical region of the subdomain 1, region already implicated in the interaction with other actin binding proteins such as gelsolin. Another site implicates the C-terminal region (sequence 360-372) of actin. Finally, the partial competition of antibodies directed against CapZ alpha or beta-subunits towards CapZ interaction with actin filaments suggests both subunits participate in the complex with actin.

Actins↗

PubMed1999

The identification of a second cofilin binding site on actin suggests a novel, intercalated arrangement of F-actin binding.

The cofilins are members of a protein family that binds monomeric and filamentous actin, severs actin filaments, and increases monomer off-rate from the pointed end. Here, we characterize the cofilin-actin interface. We confirm earlier work suggesting the importance of the lower region of subdomain 1 encompassing the N and C termini (site 1) in cofilin binding. In addition, we report the discovery of a new cofilin binding site (site 2) from residues 112-125 that form a helix toward the upper, rear surface of subdomain 1 in the standard actin orientation (Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347, 37-44). We propose that cofilin binds "behind" one monomer and "in front" of the other longitudinally associated monomer, accounting for the fact that cofilin alters the twist in the actin (McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997) J. Cell Biol. 138, 771-781). The characterization of the cofilin-actin interface will facilitate an understanding of how cofilin severs and depolymerizes filaments and may shed light on the mechanism of the gelsolin family because they share a similar fold with the cofilins (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagiki, F. (1996) Cell 85, 1047-1055).

Actin Depolymerizing Factors↗

PubMed1999

Use of a chaotropic anion iodide in the purification of Z-line proteins: isolation of CapZ from fish white muscle.

In the present study, we have described an improved method allowing the isolation of proteins which form tightly associated complexes in organized structures such as Z line in skeletal muscle. This procedure is based on both extraction and chromatography in the presence of a chaotropic agent. KI at medium concentration (0.6 M) was selected, taking into account its dissociating activity and mild effect on the native state of proteins. This procedure was applied to purify and to characterize for the first time a CapZ from fish white muscle, a protein involved in the stabilization of the filaments in Z line. The alpha and beta CapZ subunits were identified using anti-synthetic peptide antibodies directed against conserved sequences derived from chicken CapZ. The protocol can be also used for the isolation of other muscular proteins such as alpha-actinin and actin. Finally this technique may be utilized to obtain a good amount of capping protein which could be employed in experiments of microfilament dynamics.

Acetone↗

PubMed1999

Alpha actinin-CapZ, an anchoring complex for thin filaments in Z-line.

CapZ is a widely distributed and highly conserved, heterodimeric protein, that nucleates actin polymerization and binds to the barbed ends of actin filaments, preventing the addition or loss of actin monomers. CapZ interaction with actin filaments was shown to be of high affinity and decreased in the presence of PIP2. CapZ was located in nascent Z-lines during skeletal muscle myofibrillogenesis before the striated appearance of thin filaments in sarcomers. In this study, the stabilization and the anchorage of thin filaments were explored through identification of CapZ partners in the Z-line. Fish (sea bass) striated white muscle and its related Z-line proteins were selected since they correspond to the simplest Z-line organization. We report here the interaction between purified CapZ and alpha-actinin, a major component of Z filaments and polar links in Z-discs. Affinity of CapZ for alpha-actinin, estimated by fluorescence and immunochemical assays, is in the microM range. This association was found to be independent of actin and shown to be weakened in the presence of phosphoinositides. Binding contacts on the alpha-actinin molecule lie in the 55 kDa repetitive domain. A model including CapZ/alpha-actinin/titin/actin interactions is proposed considering Luther's 3D Z-line reconstruction.

Actin Cytoskeleton↗

PubMed1999

Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts.

The fluorescence parameters of the environment-sensitive acrylodan, selectively attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrations. The formation of the F-actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21-nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three-dimensional reconstruction and chemical crosslinking of the F-actin-calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F-actin-dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145-182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 A was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin-calponin complex suggesting that the F-actin effect was allosteric reflecting a global conformational change in the C-terminal domain of calponin.

2-Naphthylamine↗

PubMed1998

Interaction of caldesmon with actin subdomain-2.

The polymerization-resistant maleimidobenzoyl-G-actin (MBS-G-actin), which behaves as a functional analogue of native G-actin [Bettache, N., Bertrand, R. & Kassab, R. (1989) Proc. Natl Acad. Sci. USA 86, 6028-6032; Bettache, N., Bertrand, R. & Kassab, R. (1990) Biochemistry 29, 9085-9091) has been employed to probe the solution interaction between monomeric actin and smooth muscle caldesmon, using fluorescence measurements, limited proteolysis and covalent cross-linking reactions. MBS-G-actin associates, without polymerization, to turkey gizzard caldesmon, at about 50 mM ionic strength and 25 degrees C, with a high affinity (Kd approximately 0.04 microM) and with a 1:1 stoichiometry. However, the binding strength of the complex including caldesmon and MBS-G-actin cleaved at the subdomain-2 loop with subtilisin decreased fivefold (Kd approximately 0.20 microM). Conversely, caldesmon strongly protected subdomain-2 of MBS-G-actin from tryptic digestion at the susceptible peptide bond at positions 68-69. Furthermore, caldesmon induced the dissociation of native G-actin from its complex with DNase I, as assessed by cosedimentation assays, and increasing concentrations of the latter protein inhibited the MBS-G-actin-caldesmon interaction, suggesting mutual exclusion binding of caldesmon and DNase I to monomeric actin. MBS-G-actin was specifically coupled, via a maleimidobenzoyl group incorporated into its subdomain-2, to caldesmon, producing in high yield a 205-kDa covalent complex consisting of one actin monomer joined to Cys 580 of caldesmon. A similar conjugation process was observed with the complex of caldesmon and polymerized MBS-F-actin. MBS-G-actin could be also cross-linked to caldesmon by 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide, producing a three-band pattern identical to that of F-actin and caldesmon and previously shown to reflect the covalent union between the NH2-terminal segment of actin and the COOH-terminal actin-binding domain of caldesmon. The overall data point to a direct interaction of the latter region with actin subdomain-2 and suggest that during its binding to monomeric or filamentous actin, the caldesmon functional domain spans the entire length of a single actin and closely contacts the bottom of its subdomain-1 as well as the top portion of its subdomain-2.

Actins↗

PubMed1998

Binding of a native titin fragment to actin is regulated by PIP2.

Titin is a giant protein which extends from Z-line to M-line in striated muscles. We report here the purification of a 150-kDa titin fragment, obtained after V8 protease treatment of myofibrils. This polypeptide was located at the N1-line level, in a titin part known to exhibit stiff properties correlated to an association with actin. By solid or liquid phase binding assays and cosedimentation, we have clearly demonstrated a direct, saturable and relative high affinity binding of the native titin fragment to F-actin. The 150-kDa titin fragment was also shown to accelerate actin polymerization. Furthermore, the actin-titin interaction was found to be inhibited by phosphoinositides.

Actins↗

PubMed1998

Aggregates of an amphiphilic synthetic peptide bind and deliver all-trans retinol and all-trans retinoic acid into fibroblast cells.

The structure and conformational behaviour of a vector peptide, designed by association of a fusion peptide and a nuclear localization sequence, are described. A beta-sheet domain is observed in which fluorescence measurements show that ten peptide molecules bind one all-trans retinol or all-trans retinoic acid molecule with a strong affinity (K'd = 40 nM). Stoichiometry and affinity of the binding can be compared with those of cellular retinoid binding proteins, the structure of which is an anti-parallel beta barrel. Analogy between the system under study and cellular retinoid-binding proteins is discussed. Peptide-helped internalization and subsequent perinuclear localization of retinol in human fibroblast cells confirm this analogy. Also, this last result shows that the peptide is an efficient carrier for insoluble substances like retinoids.

Amino Acid Sequence↗

PubMed1998

Capping and dynamic relation between domains 1 and 2 of gelsolin.

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2-3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments.

Actins↗

PubMed1998

Coevolution of actin and associated proteins: an alpha-actinin-like protein in a cyanobacterium (Spirulina platensis).

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that alpha-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an alpha-actinin-like protein using specific antibodies.

Actinin↗

PubMed1997

Interaction of F-actin with synthetic peptides spanning the loop region of human cardiac beta-myosin heavy chain containing Arg403.

The atomic model of the F-actin-myosin subfragment 1 complex (acto-S-1) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50-kDa subdomain of myosin subfragment 1 (S-1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the beta-heavy chain sequence, a residue whose mutation into Gln is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F-actin with this particular S-1 loop region, we have synthesized four 17-residue peptides corresponding to the sequence Gly398-Gly414 of the human beta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross-linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, with almost identical affinities (Kd approximately 40 microM). The binding strength of the F-actin-GG17 peptide complex was increased fivefold (Kd = 8 microM) in the presence of subsaturating concentrations of added skeletal S-1 relative to actin, without apparent competition between the peptide and S-1. Each of the three actin-binding peptides inhibited the steady-state actin-activated MgATPase of skeletal S-1 by specifically decreasing about twofold the Vmax of the reaction without changing the actin affinity for the S-1-ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F-actin that uncouples its binding to S-1 from ATP hydrolysis, probably by interfering with the proper attachment of the S-1 loop segment to the interdomain connection of actin.

Actins↗

PubMed1997

Localization of a calcium sensitive binding site for gelsolin on actin subdomain I: implication for severing process.

The binding of the N-terminal domain (S1) of gelsolin to monomeric actin has been extensively documented. In contrast, the location of the C-terminal calcium dependent domains (S4-6) interacting with the actin filament during the severing process remains uncertain. In this study, we have identified a new interface that supports calcium dependent gelsolin binding to actin. This site is located in a critical position towards actin-actin contact in the filament and in the vicinity of the phalloidin site. Using specific antibody and synthetic peptides derived from actin sequence within 105-132 residues, this interface was finally ascribed to the segment 112-120 on the actin subdomain-1.

Actins↗

PubMed1997

Conformational and functional studies of three gelsolin subdomain-1 synthetic peptides and their implication in actin polymerization.

Gelsolin, a calcium and inositol phospholipid-sensitive protein, regulates actin filament length. Its activity is complex (capping, severing, etc.) and is supported by several functional domains. The N-terminal domain alone (S1), in particular, is able to impede actin polymerization. Our investigations were attempted to precise this inhibitory process by using synthetic peptides as models mimicking gelsolin S1 activity. Three peptides issued from S1 and located in gelsolin-actin interfaces were synthesized. The peptides (15-28, 42-55, and 96-114 sequences) were tested for their conformational and actin binding properties. Although the three peptides interact well with actin, only peptide 42-55 affects actin polymerization. A detailed kinetic study shows that the latter peptide essentially inhibits the nucleation step during actin polymerization. In conclusion, the present work shows that the binding of a synthetic peptide to a small sequence located outside the actin-actin interface is essential in the actin polymerization process.

Actins↗

PubMed1997

Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ.

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.

Actins↗