M Goethals

Identification of two regions in the N‐terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N‐terminus of ActA (residues 1–234) to the ω fragment of β‐galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N‐terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117–121) is critical for filament elongation, as shown by the absence of actin tail in a 117–121 deletion mutant or when motility assays are performed in the presence of anti‐region T antibodies. The second region (region C, amino acids 21–97), is more specifically involved in maintenance of the continuity of the process, probably by F‐actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21–97) leading to ‘discontinuous’ actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33–74 can interact with F‐actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N‐terminal domain of ActA.

The mammalian profilin isoforms display complementary affinities for PIP2 and proline‐rich sequences

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5‐bisphosphate (PIP2) and proline‐rich peptides derived from vasodilator‐stimulated phosphoprotein (VASP) and cyclase‐associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline‐rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline‐rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly‐L‐proline, since this isoform, but not profilin II, can be eluted from a poly‐L‐proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline‐rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross‐linking, complex formation in native gels and actin‐sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N‐terminal part needs to adopt an alpha‐helix for actin binding and interacts through a patch of hydrophobic residues (6M‐I‐F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin.

Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid beta peptides.

The Dutch (E22Q) and Flemish (A21G) mutations in the βAPP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three Aβ(12–42) variants [wild‐type (WT), Dutch and Flemish] were tested. At a concentration of 5 µm the aggregation of these peptides followed the order: Aβ(1–42) WT > Aβ(12–42) WT > Aβ(12–42) Flemish > Aβ(12–42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their α‐helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in β‐sheet and random coil content. Apoptosis was induced in neuronal cells by the Aβ(12–42) WT and Flemish peptides at concentrations as low as 1–5 µm, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase‐3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N‐truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length Aβ(1–42) peptide was weaker than that induced by its N‐truncated variant. These data suggest that N‐truncation enhanced the cytotoxic effects of Aβ WT and Flemish peptides, which may play a role in the accelerated progression of dementia.

Analogous F-actin Binding by Cofilin and Gelsolin Segment 2 Substantiates Their Structural Relationship

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047–1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366–369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369–373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin α-helix 112–128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2–3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long α‐helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197–226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F‐actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

Enhanced Efficiency Of A Targeted Fusogenic Peptide

Membrane targeting was investigated as a potential strategy to increase the fusogenic activity of an isolated fusion peptide. This was achieved by coupling the fusogenic carboxy-terminal part of the beta-amyloid peptide (Abeta, amino acids 29-40), involved in Alzheimers disease, to a positively charged peptide (PIP2-binding peptide, PBP) interacting specifically with a naturally occurring negatively charged phospholipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). Peptide-induced vesicle fusion was spectroscopically evidenced by: (i) mixing of membrane lipids, (ii) mixing of aqueous vesicular contents, and (iii) an irreversible increase in vesicle size, at concentrations five to six times lower than the Abeta(29-40) peptide. In contrast, at these concentrations the PBP-Abeta(29-40) peptide did not display any significant activity on neutral vesicles, indicating that negatively charged phospholipids included as targets in the membranes, are required to compensate for the lower hydrophobicity of this peptide. When the alpha-helical structure of the chimeric peptide was induced by dissolving it in trifluoroethanol, an increase of the fusogenic potential of the peptide was observed, supporting the hypothesis that the alpha-helical conformation of the peptide is crucial to trigger the lipid-peptide interaction. The specificity of the interaction between PIP2 and the PBP moiety, was shown by the less efficient targeting of the chimeric peptide to membranes charged with phosphatidylserine. These data thus demonstrate that the specific properties of both the Abeta(29-40) and the PBP peptide are conserved in the chimeric peptide, and that a synergetic effect is reached through chemical linkage of these two fragments.

Functional and profiling studies prove that prostate cancer upregulated neuroblastoma thymosin β is the true human homologue of rat thymosin β15

A peptide with a sequence identical to rat thymosin β(Tb)15 was reported to be upregulated in human prostate cancer. However, in this report we provide evidence that TbNB, initially identified in human neuroblastoma, is the only Tb isoform upregulated in human prostate cancer and that the Tb15 sequence is not present herein. In addition, we demonstrate that human TbNB has a higher affinity for actin in comparison to Tb4 and promotes cell migration. In combination, this experimentally validates TbNB as functional homologue of rat Tb15 in the human organism and clarifies the current composition of the human Tb family.