Distinct roles of four gelsolin-like domains of Caenorhabditis elegans gelsolin-like protein-1 in actin filament severing, barbed end capping, and phosphoinositide binding.

Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) is a new member of the gelsolin family of actin regulatory proteins [Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008) J. Biol. Chem. 283, 26071-26080]. It is an unconventional gelsolin-related protein with four gelsolin-like (G) domains (G1-G4), unlike typical gelsolin-related proteins with three or six G domains. GSNL-1 severs actin filaments and caps the barbed end in a calcium-dependent manner similar to that of gelsolin. In contrast, GSNL-1 has properties different from those of gelsolin in that it remains bound to F-actin and does not nucleate actin polymerization. To understand the mechanism by which GSNL-1 regulates actin dynamics, we investigated the domain-function relationship of GSNL-1 by analyzing activities of truncated forms of GSNL-1. G1 and the linker between G1 and G2 were sufficient for actin filament severing, whereas G1 and G2 were required for barbed end capping. The actin severing activity of GSNL-1 was inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and a PIP2-sensitive domain was mapped to G1 and G2. At least two actin-binding sites were detected: a calcium-dependent G-actin-binding site in G1 and a calcium-independent G- and F-actin-binding site in G3 and G4. These results reveal both conserved and different utilization of G domains between C. elegans GSNL-1 and mammalian gelsolin for actin regulatory functions.

A novel human Cl(-) channel family related to Drosophila flightless locus.

Large conductance chloride (maxi-Cl(-)) currents have been recorded in some cells, but there is still little information on the molecular nature of the channel underlying this conductance. We report here that tweety, a gene located in Drosophila flightless, has a structure similar to those of known channels and that human homologues of tweety (hTTYH1-3) are novel maxi-Cl(-) channels. hTTYH3 mRNA was found to be distributed in excitable tissues. The whole cell current of hTTYH3 was large enough to be discriminated from the control but emerged only after treatment with ionomycin. Analysis of pore mutants suggested that positively charged amino acids contributed to anion selectivity. Like a maxi-Cl(-) channel in situ, the hTTYH3 single channel showed 26-picosiemen linear current voltage, complex kinetics, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid sensitivity, subconductance, and the permeability order of I(-) > Br(-) > Cl(-). Similarly, hTTYH2 encoded an ionomycin-induced maxi-Cl(-) channel, but TTYH1 encoded a Ca(2+)-independent and swelling-activated maxi-Cl(-) channel. Therefore, the hTTYH family encoded maxi-Cl(-) channels of mammals. Further studies on the hTTYH family should lead to the elucidation of physiological and pathophysiological roles of novel Cl(-) channel molecules.

Correlations between biological activity and structural properties for two short homologous sequences in thymosin beta4 and gelsolin.

Gelsolin and thymosin beta4 appear to be two important actin-associated proteins involved in the regulation of actin polymerization. It has been widely demonstrated that thymosin is the major cellular actin-sequestering factor shifting the polymerization equilibrium of actin towards a monomeric state. At the same time gelsolin, a Ca2+ and inositol phosphate sensitive protein, regulates actin filament length. The interactions of these two proteins with actin are rather complex and require the participation of several complementary peptide sequences. We have identified a common motif, (I, V)EKFD, in the two proteins in the functional sequences so far examined. Gelsolin- and thymosin beta4-related peptides including the common motif were synthesized and their structural and functional properties studied. These two sequences exert a major inhibitory effect on salt-induced actin polymerization. We used circular dichroism and Fourier-transform infrared spectroscopy to show that the two synthetic peptides present some secondary structure in solution. As far as the peptide derived from the thymosin sequence was concerned, alpha-helical structure was induced by trifluoroethanol as observed with the full-length molecule. These experiments underscore the importance of the conformational state of peptide fragments in their biological activities. ELISA and fluorescence measurements have been used to identify the binding regions of these fragments to a C-terminal region (subdomain 1) of the actin sequence. Our results also emphasize the relationship between the propensity of small sequences to form secondary structures and their propensity for biological activity as related to actin interaction and inhibition of actin polymerization.