The ternary complex of DNase I, actin and thymosin β4

Abstract

Key words: Actin; Thymosin 134; DNase I; Ternary complex; Thiol-specific crosslinking the differently positioned thiol groups in TIM come close enough to distinct thiol groups in actin to allow the formation of crosslinks. In this way two contact sites between the two proteins could be identified, one between the C-terminus of actin (Cys 374) and position 6 of TIM and a second between the )'-phosphate of the actin-bound nucleotide in actin and the sequence 17 28 in TIM.X-ray analysis of monomeric actin took advantage of a facilitated crystallization of actin when complexed with DNase I. Provided DNase I exerted a similar effect on TIMactin as well, co-crystallization with DNase I would allow the interface of actin and TIM in the ternary complex to be studied. Corresponding complexes have been reported for DNase I, actin and profilin, or ADF/cofilin, respectively [14,15]. However, the ternary complex can be expected to form only if the binding of DNase I and that of TIM to actin do not strongly interfere. We therefore examined whether DNase I affects the affinity of TIM for actin by studying one of the crosslinking reactions mentioned above in the presence of DNase I. Materials and methods In~oductionThymosin 134 (TIM) is one of the small proteins in nonmuscle cells that bind to monomeric actin and thus prevent unregulated polymerization [14]. From the TiM-complexed monomers, polymerization can be started in vitro either by decapping barbed ends of filaments [5,6], or by adding nucleus stabilizers such as myosin S1 or phalloidin [7][8][9][10][11]. The relatively low affinity of T~4 for actin (ca. 1 ~tM [4]) may be functionally significant in allowing the instant release of polymerizable actin into cytoplasm, if required.Since an X-ray analysis of the actin-TiM complex has so far not been achieved we have tried to identify contact sites between the two proteins by a chemical approach [12]. For this, five TIM analogs were synthesized, each of them with one cysteine residue substituted for a hydrophobic amino acid in the TIM chain. Using a set of thiol-specific crosslinkers of varying length, we assayed whether in the complex with actin

Gelsolin activity controls efficient early HIV-1 infection

“…These data were confirmed by quantifying total levels of F-actin expression by flow cytometry (Figure 1B, right histogram ), which revealed that gelsolin-EGFP overexpression promoted actin filament severing and decreased the levels of cortical F-actin in CEM.NKR-CCR5 permissive lymphocytes. Our data demonstrate that the functional gelsolin construct and the endogenous gelsolin protein both co-localize with F-actin, in agreement with previous reports describing the binding of gelsolin to actin filaments, even during actin filament processing [20,21]. …”

“…Gelsolin binds to actin filaments with high affinity (Kd of 50 nM) and it is thought to sever actin filaments (reviewed in [14]). After severing, gelsolin remains attached to the barbed ends of the filaments as a cap, thereby preventing the reannealing of actin fragments, and it constitutively co-localizes with the modified actin-filaments [21]. We found that overexpression of functional gelsolin diminished the amount of F-actin in permissive lymphocytes, which had a thinner F-actin cortex than the corresponding controls.…”

Models of the Actin‐Bound Forms of the β‐Thymosins

“…Cross‐linking studies between thymosin‐β4, actin and DNase I have shown that DNase I competes with thymosin‐β4 in binding to actin 19 . In contrast, an earlier report had described the ternary complex between these three components 20 6. depicts the superposition of the DNase I:actin complex onto the β‐thymosin:actin model.…”

“…19 In contrast, an earlier report had described the ternary complex between these three components. 20 FIG URE 6 depicts the superposition of the DNase I:actin complex onto the ␤-thymosin:actin model. There is a clear clash between the ␤-thymosin C-terminal helix, and DNase I, which rules out their simultaneous binding to the pointed end of actin.…”

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

“…On the other hand, stabilization of antiparallel dimers by certain toxins or proteins of pathogenic origin 51 is likely to underlie their destructive effects on the actin cytoskeleton of the host cell. It remains to be established whether LD form spontaneously under in vivo conditions where the monomer-sequestering proteins thymosin-β 4 and profilin interacting with subdomain 1 of actin [54][55][56] might prevent this type of monomer association by competing for the antiparallel dimer interface.…”