Actin Degradation Research Articles

Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.

Glucocorticoids in intracellular catabolism of myosin and actin

The catabolic effect of glucocorticoids on the structural proteins of contractile apparatus seems to be realized through the increased alkaline proteinase activity and accelerated synthesis of light enzyme subunits. The administration of large amounts of glucocorticoids increased the excretion of 3-methylhistidine in rats by 60%. Digestion of isolated myofibrils with alkaline proteinase resulted in the degradation of myosin heavy chain and actin. The turnover rate of actin and myosin heavy chain was decreased.

Increased actin content in cytochalasin D-treated cells is not caused by a selective decrease in actin degradation

Increased actin content in cytochalasin D-treated cells is not caused by a selective decrease in actin degradation

Actin synthesis in tumorigenic and non-tumorigenic human hybrid cells.

We have previously shown that the total actin content of tumorigenic HeLa/fibroblast somatic cell hybrids is significantly lower than that of the non-tumorigenic hybrid cells. A measure of actin synthesis, relative to total protein synthesis, was obtained for these cells to determine whether the reduced actin content of the tumorigenic cells is due to specific suppression of actin synthesis. Actin synthesis was measured in cells labelled with [35S]methionine using DNase I affinity chromatography to isolate the actin quantitatively. The results show that actin synthesis is not specifically suppressed, since the relative amount of actin synthesized is constant for the tumorigenic and non-tumorigenic cell lines. The reduced actin content of the tumorigenic cells is therefore most likely to be the result of increased degradation of actin.

Degradation of actin and vimentin by calpain II, a Ca 2+-dependent cysteine proteinase, in bovine lens

Calpain II, a high Ca 2+-requiring form of Ca 2+-dependent cysteine proteinase (EC 3.4.22.17), isolated from bovine lens was found to cleave actin and vimentin, two major cytoskeletal elements of the lens. Polyacrylamide gel electrophoresis revealed that actin ( M r 43000) was broken down through intermediary products of approximate M r 42000 and 40000, while vimentin ( M r 57000) was rapidly cleaved into several fragments ranging from M r 44000 to 20000. The cleavage was dependent on Ca 2+ and could be blocked by calpastatin, a calpain-specific inhibitor. These findings suggest that calpain might play a role in age-related degradation of the lens cytoskeleton.

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca2+-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.

Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmin.

A calcium (Ca2+)-activated, neutral proteinase has been purified from Ehrlich ascites tumor cells. The protocol used has resulted in a 3,600-fold purification of the enzyme in a yield of 21% from the Ehrlich ascites tumor cell postnuclear supernatant. The purified proteinase has a high substrate specificity for the intermediate filament subunit proteins, vimentin and desmin, and showed no activity towards other intermediate filament proteins except a 60,000-dalton protein of the cytokeratins. Also, there was no degradation of actin, tubulin, the major constituent proteins of myofibrils and several standard proteins. Characterization of the purified proteinase has shown that it is activated by Ca2+ (10 to 100 microM), is probably calmodulin-independent and irreversibly loses activity when incubated in the presence of Ca2+ without substrate. The enzyme has a Km of 1.7 x 10(-8) M for vimentin and 5.2 x 10(-7) M for desmin. The proteinase has a major subunit of 72,000 daltons which has the catalytic center and a minor component of 29,000 daltons; by gel permeation chromatography it has an apparent molecular weight of 100,000. It requires a reduced sulfhydryl group for activity and can be inhibited by sulfhydryl-blocking reagents. The high substrate specificity of the proteinase indicates that it is involved in the regulation of the distribution and turnover of vimentin- and desmin-containing intermediate filaments.

Effect of phalloidin on liver actin distribution, content, and turnover.

Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Planimetric analyses of SDS-polyacrylamide gels showed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 micrograms/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dualisotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.

Spectrin extractability from erythrocyte in duchenne muscular dystrophies and the effect of proteases on erythrocyte ghosts

We studied the erythrocyte membrane proteins from patients with Duchenne muscular dystrophy (DMD) using SDS-polyacrylamide gel electrophoresis. Our observations were the following: (1) The electrophoretic densitogram of freshly prepared DMD-ghosts was similar to that of controls. After the extraction of spectrin from ghosts with 1 mmol/l EDTA, pH 8.0, the unextractable spectrin remained more firmly bound in the DMD ghost residues than in controls. Extractability of spectrin from DMD ghosts was decreased about 20%. In addition, several minor bands were detected between spectrin and Band 3 in the DMD ghost residues (treated ghosts). (2) Ca 2+-activated, neutral protease reacted more effectively with spectrin of DMD ghosts and ATP-depleted ghosts than with that of controls. (3) Erythrocyte actin (Band 5) of DMD ghosts was more fragile than that of controls and of ATP-depleted actin in the EDTA extracts. Gradually, partial degradation of actin was observed for three weeks at 4°C. (4) The intracellular ATP level and the activities of membrane-bound (Mg 2+/uCa 2+) ATPases in DMD erythrocytes were unchanged. We suggest that spectrin from DMD ghosts as well as actin may be subject to increased degradation.

Actin content and synthesis in differentiating Drosophila cells in culture

In response to the addition of 20-hydroxyecdysone, Drosophila line Kc cells extend filopodia, become motile and aggregate. An investigation was carried out to determine whether the appearance of motility was correlated with an increase in intracellular actin content or actin synthesis, or a decrease in actin degradation. With the exception of actin content, measured by DNAse I inactivation, treated and untreated cells were indistinguishable for all parameters. DNAse I inactivation studies indicated a three- to four-fold increase in actin content during the two days following hormone exposure. These data are interpreted by a model in which an inactive pool of actin becomes available for microfilament assembly.

Degradation of canine cardiac myosin and actin by cathepsin D isolated from homologous tissue.

The ability of canine cardiac cathepsin D to catalyse hydrolysis of myosin and actin isolated from homologous tissue was monitored using both SDS polyacrylamide gel electrophoresis and the release of trichloroacetic acid soluble radioactivity from the radioactively (14C) labelled proteins. Cathepsin D readily catalysed the hydrolysis of myosin and actin. The pH-activity profile for myosin hydrolysis exhibited two optima with a major peak at pH 2.6 together with a smaller peak at pH 3.4. This was distinct from the pH-activity profile for actin hydrolysis where a single optimum at pH 4.2 was observed. Incubation of cathepsin D with myosin (pH 2.6, 37°C) resulted in complete degradation of myosin light chains (MW =28000±700 and 18600±350, n=4) within 10 min while heavy chains (MW= 190400±2500, n=4) were degraded into three proteins of molecular weight 32000 ±750; 37000 ±1000 and 49000 ±750 (n=4) within 4 h. Further incubation (24 h) of cathepsin D with myosin resulted in two proteins of MW 29000±2000 and 48800±3100 (n=4). In the case of actin (MW 44800± 1600, n=6) polypeptides produced as a result of hydrolytic cleavage by cathepsin D (pH 4.2, 37°C) were not observed on SDS gels, although the overall size of the actin band was markedly decreased at 24 h. Cathepsin D catalysed the hydrolysis of myosin at near neutral pH (6.5) although the overall rate of degradation was considerably reduced compared to that observed at pH 2.6. Pepstatin A was a potent inhibitor of the hydrolysis of myosin and actin at all pH values investigated indicating that cathepsin D and not a contaminant was responsible for the observed degradation.

Degradation of myofibrillar proteins by cathepsins B and D.

1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.

Actin turnover during encystation in Acanthamoeba

A thin-slab, SDS polyacrylamide gel electrophoresis system is described in which actin within whole cell homogenates can be quantitated within a wide range of protein values (0.05–1.4 μg/band). After demonstrating the absence of appreciable contaminants in the actin band, and the lack of appreciable reincorporation of label during pulse-chase experiments, the turnover of actin was examined in pre-labeled cells during normal log growth and during induced encystation in Acanthamoeba. During log growth, no actin degradation was detected. However, as the cells approached the end of log phase growth and entered stationary phase, a dramatic increase in the amount of actin/cell and the percentage of total protein represented by actin was recorded. The encystation process per se was accompanied by a rapid reduction in these values to preencystment levels.

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.