The Interaction of Bovine Pancreatic Deoxyribonuclease I and Skeletal Muscle Actin

Mannherz, Hans Georg; Goody, Roger S.; Konrad, Manfred; Nowak, Ewa

doi:10.1111/j.1432-1033.1980.tb04437.x

Cited by 204 publications

(161 citation statements)

References 28 publications

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“…Full-length actin binds to and inhibits DNase I with an association constant of 109 M-1 (35), and actin-DNase complexes have been described in vivo (35,36), yet a physiological rationale for these observations has not been reported. If in healthy nonapoptosing cells one of the functions of actin is to inhibit DNase I, then it is conceivable that, at the onset of apoptosis, ICE may reverse this inhibition by cleaving actin into fragments that have a reduced capacity to inhibit the endonucleolytic activity of DNase I.…”

Section: Resultsmentioning

confidence: 99%

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

Kayalar

Örd

Testa

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

269

164

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Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction ofcell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1.8-converting enzyme. Actin cleaved by interleukin lp-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin. These findings suggest a mechanism for the coordination of the proteolytic, endonucleolytic, and morphogenetic aspects of apoptosis.

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Section: Resultsmentioning

confidence: 99%

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

Kayalar

Örd

Testa

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

269

164

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“…While in the latter, due to the extremely high affinity of DNase I for actin (KD ca. 1 nM [24]), no free actin was visible, the crosslinking product shows an additional faint band, which from the mobility may represent a small amount of TiM-actin (lane 4) or free actin (lane 5). The dominant band of lane 2 corresponds to the presence of by far the largest part of actin as a complex of the actin derivative, DNase I and Cys6TIM .…”

Section: Resultsmentioning

confidence: 99%

The ternary complex of DNase I, actin and thymosin β4

et al. 1996

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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

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“…As the nuclear staining with anti-thymosin β 4 varied, we also tested the possibility that there might have been an unequal distribution of monomeric actin in these cells. Therefore, we counterstained the cells with Oregon Green-labelled deoxyribonuclease I (DNase I), which is known to bind with high affinity and specificity to monomeric actin (Mannherz et al, 1980). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Nuclear localisation of the G-actin sequestering peptide thymosin β4

Huff

Rosorius

Otto

et al. 2004

Journal of Cell Science

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Thymosin β4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin β4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin β4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin β4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin β4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin β4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded.

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The Interaction of Bovine Pancreatic Deoxyribonuclease I and Skeletal Muscle Actin

Cited by 204 publications

References 28 publications

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

Cleavage of actin by interleukin 1 beta-converting enzyme to reverse DNase I inhibition.

The ternary complex of DNase I, actin and thymosin β4

Nuclear localisation of the G-actin sequestering peptide thymosin β4

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