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Histones are located in the cell nucleus. They are positively charged small proteins which became extracellular upon apoptosis, necrosis, and infection – induced cell death. The mixture of extracellular Histones was shown to bundle Actin filaments and digested by bacterial proteases, which was inhibited by DNA and Actin. Here we studied the interaction of five major family of Histones, H2A, H2B, H3.1, H1 and H4, with DNA and Actin filaments. We found that all the Histones studied bound to DNA, increased the viscosity of Actin containing solutions and bundled Actin filaments in various degrees. The bundling of Actin filaments by Histones was inhibited by DNA, NaCl and DNase1. DNA and Actin filaments also inhibited the proteolysis of the five Histones by Subtilisin, Fusolisin and Pseudomonas Aeruginosa bacterial proteases. Both the degree of the proteolysis and its inhibition was different with various Histones. The results indicate that all the Histones studied bind strongly to the negatively charged DNA and to the Actin filaments.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.

There are two A-type cyclins in higher vertebrates, cyclin A1 and A2. Targeted mutagenesis has shown that cyclin A2 is essential for early embryonic development while cyclin A1 is required only for male meiosis. The embryonic lethality of cyclin A2 knockout mice has obviated understanding its role in other aspects of mammalian development, including the germ line. We reported previously that cyclin A2 expression in the male germ line is consistent with a role in both mitotic and meiotic cell cycles. Usingin situhybridization and immunohistochemistry, we now observe high levels of cyclin A2 in granulosa cells and less-abundant but readily detectable expression in ovarian and ovulated oocytes. A decrease in cyclin A2 protein was observed in oocytes from embryonic stages to post-natal and adult ovaries. Interestingly, cyclin A2 protein was nuclear in oocytes from embryonic day 13.5 to 15.5, changing to largely cytoplasmic in oocytes from embryonic day 16.5 to post-natal and adults. Readily detectable expression of the cyclin-dependent kinases Cdk1 and Cdk2, two common partners for the A-type cyclins, was observed in granulosa cells and oocytes at all stages of folliculogenesis. Cdk1 was predominantly cytoplasmic, whereas Cdk2 was both cytoplasmic and nuclear in oocytes. No cyclin A1 expression, at either the mRNA level or the protein level was detected in either embryonic or adult ovaries, consistent with the full fertility observed in female cyclin A1-deficient mice. These results suggest that in the female germ line, cyclin A2 but not cyclin A1 has distinct roles in both mitosis and meiosis.

Aberrant expression of several key regulators controlling the G1/S phase of the cell cycle has been implicated in human male germ cell tumorigenesis. Given the critical role of cyclin A2 at both the G1/S and G2/M transitions and the essential role for cyclin A1 in male germ cell development, our present study focused on the involvement of the A-type cyclins in the transformation and progression of male germ cell tumors (GCTs). The expression of the A-type cyclins and their catalytic partners Cdk1 and Cdk2 was examined in all types and stages of human male GCTs, including carcinoma in situ(CIS), seminoma and non-seminoma GCTs, along with normal testis samples. Elevated levels of cyclin A2, Cdk1 and Cdk2 were detected in the majority of GCTs and were correlated with the invasiveness of the tumors (p < 0.05). Cyclin A1 expression was virtually undetectable in CIS and seminoma, but was aberrantly expressed in all non-seminomatous GCTs. Cyclin A2 expression was strongly correlated with that of its catalytic partners Cdk1 and Cdk2 in all types of testicular tumors examined (p < 0.05), whereas a strong correlation between cyclin A1 and Cdk1 or Cdk2 was only seen in non-seminomatous GCTs (p < 0.05). Histone kinase activities of cyclin A1/Cdks and cyclin A2/Cdks were found to be elevated in tumors. Our data suggest that aberrant expression of A-type cyclins and their Cdks is a significant factor in male germ cell tumorigenesis. The abundant ectopic expression of cyclin A1 in non-seminomatous GCTs and its absence in CIS and seminomas is likely linked to the tumor transformation and progression and may be relevant to clinical prognosis.

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

The atomic structures for several myosin head isoforms in different nucleotide states have been determined in recent years. The comparison of these structures is complicated by the use of myosin subfragment 1 (S1) constructs of different length in different studies. Several atomic structures of the S1 nucleotide complex were obtained using Dictyostelium discoideum S1dC, a genetically truncated form of S1 lacking the light chain binding domain (LCBD) and both light chains. The goal of the present study has been to assess the effects of such a truncation on the solution properties of S1 and in particular, on its active site, actin binding site and the converter region. The nucleotide and actin binding properties, CD spectra and the reactivities of Lys-84 (corresponds to the 'reactive lysine', Lys-83 in rabbit skeletal S1) and Cys-678 (corresponds to the 'SH2-group', Cys-697 in rabbit S1) were compared for the full length (flS1) and the truncated (S1dC) forms of Dictyostelium S1. The two forms showed similar nucleotide binding properties. However, SldC had a lower structural stability and a significantly higher Km value for actin-activated ATPase as compared to flS1. Differences were found also in the near-UV CD spectrum between flS1 and S1dC. SH2 reactivity in SldC appeared to be greatly inhibited compared with that in flS1. The modification of Lys-84 caused a greater increase in the MgATPase activity in S1dC than in flS1. ADP inhibited this activation for both SldC and flS1. Taken together our results identify both truncation-caused differences between S1dC and flS1, as well as isoform-related differences between skeletal and Dictyostelium S1.

Cys 10 is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys 10 was modified with fluorescence probes N -(iodoacetyl) N′ -(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of Drewes and Faulstich (1991, J. Biol. Chem. 266:5508–5513). The specificity of Cys 10 modification was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48–375), which contains all of the actin thiols but Cys 10 , is not fluorescent. Cys 10 modification exposed a new site on actin to subtilisin cleavage. Edman degradation revealed this site to be between Ala 19 and Gly 20 . The modification slightly increased the rate of ϵATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys 10 -modified F-actin showed small probe-dependent changes in the values of V max and K M . The sliding speed of actin filaments in the in vitro motility assay remained unchanged upon modification of Cys 10 . These results indicate that although the labeling of Cys 10 perturbs the structure of subdomain 1, the modified actin remains fully functional. The binding of S1 to actin filaments decreases the accessibility of Cys 10 probes to acrylamide and nitromethane quenchers. Because Cys 10 does not participate directly in either actin polymerization or S1 binding, our results indicate that actin-actin and actin-myosin interactions induce dynamic, allosteric changes in actin structure.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801–17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41–Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38–52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.

Bertrand et al. [Bertrand, R., Derancourt, J.Kassab, R. (1995) Biochemistry 34, 9500-9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the epsilon-amino group of Lys553 reacts with FHS. Addition of 0.4 M KCl reduced the rate of reaction significantly, which indicates ionic strength-dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP.BeF(x), ADP.AlF(4), ADP.V(i) and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M(**)ADP.P(i) state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1.ADP.AlF(4)(-) and S1.ADP.V(i) complexes, which are believed to mimic the M(**)ADP.P(i) state. This indicates that the conformation of the S1-ADP.AlF(4)(-) and S1.ADP.V(i) complexes in the vicinity of Lys553 does not resemble the structure of the M(**)ADP.P(i) state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40 degreesC and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeFx), aluminum fluoride (AlF4-), or vanadate (Vi) phosphate analogues, while the S1.PhAEDP complex is stable in the presence BeFx but much less stable with AlF4- and Vi. These results indicate that the S1.PhAEDP.Pi state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths≥240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.

G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The cross-linking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636–642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038–14046). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2mM MgCl2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATPase activity of the cross-linked S1 was not activated by actin, whereas the K+(EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Polyclonal site-directed peptide antibodies were raised against the 567-574 and 238-246 sequences of the rabbit skeletal muscle myosin heavy chain. These sequences, which are located in the subfragment 1 (S1) segment of myosin, have been implicated by former studies in actin and nucleotide binding of the molecule and in the communication between the two binding sites. The antibodies obtained from rabbit sera were found to be conformation-sensitive since they specifically reacted with S1 in solid-phase binding assay but not in Western blot. The binding of both antibodies to S1 was strongly inhibited by actin. The antibody against the 567-574 sequence, Ab567-574, moderately decreased the binding of S1 to actin filaments in rigor but not in the weakly-attached state, while Ab238-246 did not influence the binding of S1 to actin under either conditions. Both antibodies inhibited the actin activation of the MgATPase of S1 but did not affect MgATPase without actin or the Ca- and K(EDTA)-activated ATPase activities of S1. The sliding velocity of actin filaments in the in vitro motility assays were also reduced in the presence of the antibodies. Ab567-574 had especially strong inhibitory effect on the movement of actin filaments. The results indicate that the binding of antibodies may induce conformational changes, which propagate in the S1 structure, perturb the coupling between the binding sites and impair the motor function of myosin.

In this study, we use fluorescent probes and proteolytic digestions to demonstrate structural coupling between distant regions of actin. We show that modifications of Cys-374 in the C-terminus of actin slow the rate of nucleotide exchange in the nucleotide cleft. Conformational coupling between the C-terminus and the DNasal loop in subdomain II is observed in proteolytic digestion experiments in which a new C-terminal cleavage site is exposed upon DNasel binding. The functional consequences of C-terminal modification are evident from S-1 ATPase activity and the in vitro motility experiments with modified actins. Pyrene actin, labeled at Cys-374, activates S-1 ATPase activity only half as well as control actin. This reduction is attributed to a lower Vmax value because the affinity of pyrene actin to S-1 is not significantly altered. The in vitro sliding velocity of pyrene actin is also decreased. However, IAEDANS labeling of actin (also at Cys-374) enhances the Vmax of acto-S-1 ATPase activity and the in vitro sliding velocity by approximately 25%. These results are discussed in terms of conformational coupling between distant regions in actin and the functional implications of the interactions of actin-binding proteins with the C-terminus of actin.

THE degradation of messenger RNA in eukaryotic cells is initiated by endonucleolytic cleavage1,2 or by shortening of the poly(A) tail3–6, which for some mRNAs activates a deadenylation-depen-dent decapping reaction7. One type of rapid mRNA degradation in eukaryotes is caused by premature termination of translation8,9. This turnover process prevents the translation of aberrant mRNAs10,11, may affect the abundance and splicing pattern of nuclear transcripts12,13, and may be involved in the aetiology of human genetic disease14. Here we show that premature translational termination in yeast triggers decapping, independent of deadenylation, thereby exposing the transcript to 5′-to-3′ degradation. Inactivation of the 5′-to-3′ exonuclease reveals an additional 3′-to-5′ pathway of mRNA turnover. These observations provide in vivo evidence for two new mechanisms of mRNA decay.

The antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools for examination of the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to the routinely used p-phenylene maleimide (pPDM) for APD detection, allowing for fast and efficient cross-linking under conditions of actin polymerization at neutral pH. We report also that pyrene-labeled yeast actin mutant A167C/C374A (C167PM) forms significant amounts of stable APD in solution, without chemical cross-linking or polymerization-affecting compounds, and that the kinetics of APD transformation and decay upon actin polymerization can be easily monitored. The dimerization of C167PM has been characterized in sedimentation equilibrium experiments (K(d) approximately 0.3 microM). This new system offers the advantage of assessing the effects of the APD under physiological conditions (pH, ionic strength, and Mg(2+) concentration) and testing for conformational transitions in the APD during nucleation-polymerization reactions or/and in the presence of actin-interacting factors. The results obtained using two different systems (C167PM actin and polylysine-induced polymerization of alpha-actin) show that the APD decays at a rate slower than that at which the filaments elongate, revealing its transient incorporation into filaments, and confirm that it inhibits the nucleation and elongation of actin filaments.

Bax inhibitor 1 (BI-1), a transmembrane protein with Ca2+ channel-like activity, has antiapoptotic and anticancer activities. Cells overexpressing BI-1 demonstrated increased cell adhesion. Using a proteomics tool, we found that BI-1 interacted with gamma-actin via leucines 221 and 225 and could control actin polymerization and cell adhesion. Among BI-1-/- cells and cells transfected with BI-1 small interfering RNA (siRNA), levels of actin polymerization and cell adhesion were lower than those among BI-1+/+ cells and cells transfected with nonspecific siRNA. BI-1 acts as a leaky Ca2+ channel, but mutations of the actin binding sites (L221A, L225A, and L221A/L225A) did not change intra-endoplasmic reticulum Ca2+, although deleting the C-terminal motif (EKDKKKEKK) did. However, store-operated Ca2+ entry (SOCE) is activated in cells expressing BI-1 but not in cells expressing actin binding site mutants, even those with the intact C-terminal motif. Consistently, actin polymerization and cell adhesion were inhibited among all the mutant cells. Compared to BI-1+/+ cells, BI-1-/- cells inhibited SOCE, actin polymerization, and cell adhesion. Endogenous BI-1 knockdown cells showed a similar pattern. The C-terminal peptide of BI-1 (LMMLILAMNRKDKKKEKK) polymerized actin even after the deletion of four or six charged C-terminal residues. This indicates that the actin binding site containing L221 to D231 of BI-1 is responsible for actin interaction and that the C-terminal motif has only a supporting role. The intact C-terminal peptide also bundled actin and increased cell adhesion. The results of experiments with whole recombinant BI-1 reconstituted in membranes also coincide well with the results obtained with peptides. In summary, BI-1 increased actin polymerization and cell adhesion through Ca2+ regulation and actin interaction.

Beryllium and aluminum fluorides are good phosphate analogues. These compounds, like orthovanadate, form stable complexes with myosin subfragment 1 (S1) in the presence of MgADP. The formation of the stable S1-nucleotide complexes is characterized by the loss of ATPase activity. For the complete loss of ATPase activity there was necessary a higher concentration of aluminum than of beryllium or vanadate. In the presence of MgATP the onset of the inhibition is delayed, which indicates that stable complexes cannot form when a specific site is occupied by the gamma-phosphate of ATP or by P(i) derived from the gamma-phosphate. The half-lives of the S1-MgADP-(BeF3-), S1-MgADP-(AlF4-), and S1-MgADP-Vi complexes at 0 degrees C are 7, 2, and 4 days, respectively. In the presence of actin the rate of decomposition of all of the complexes is significantly enhanced; however, the order of decomposition is reversed, the fastest rate being observed with beryllium and the slowest with aluminum. The formation of the S1-MgADP-(BeF3-) and S1-MgADP-(AlF4-) complexes is accompanied by an increase in tryptophan fluorescence similar to that observed upon addition of MgATP to S1. The fluorescence increase develops rather slowly, by suggesting that the rate-limiting step in the formation of the stable complex is an isomerization. The rate of the fluorescence change accompanying the formation of the Be complex is faster than that for the Al complex. Addition of vanadate to S1 causes a static quenching of the tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Cofilin is essential for cell viability and for actin-based motility. Cofilin severs actin filaments, which enhances the dynamics of filament assembly. We investigated the mechanism of filament severing by cofilin with direct fluorescence microscopy observation of single actin filaments in real time. In cells, actin filaments are likely to be attached at multiple points along their length, and we found that attaching filaments in such a manner greatly increased the efficiency of filament severing by cofilin. Cofilin severing increased and then decreased with increasing concentration of cofilin. Together, these results indicate that cofilin severs the actin filament by a mechanism of allosteric and cooperative destabilization. Severing is more efficient when relaxation of this cofilin-induced instability of the actin filament is inhibited by restricting the flexibility of the filament. These conclusions have particular relevance to cofilin function during actin-based motility in cells and in synthetic systems.

Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway that recognizes and degrades aberrant mRNAs containing premature stop codons. A critical protein in NMD is Upf1p, which belongs to the helicase super family 1 (SF1), and is thought to utilize the energy of ATP hydrolysis to promote transitions in the structure of RNA or RNA–protein complexes. The crystal structure of the catalytic core of human Upf1p determined in three states (phosphate-, AMPPNP- and ADP-bound forms) reveals an overall structure containing two RecA-like domains with two additional domains protruding from the N-terminal RecA-like domain. Structural comparison combined with mutational analysis identifies a likely single-stranded RNA (ssRNA)-binding channel, and a cycle of conformational change coupled to ATP binding and hydrolysis. These conformational changes alter the likely ssRNA-binding channel in a manner that can explain how ATP binding destabilizes ssRNA binding to Upf1p.

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.

High concentration of the cosolvent poly(ethylene glycol) (PEG) induces reversible aggregation of skeletal myosin subfragment 1 (S1) and inhibition of its Mg-ATPase activity [Highsmith et al. (1998) Biophys. J. 74, 1465-1472]. In the present work the effect of aggregation on the various steps of the ATPase cycle was studied. The isomerization and hydrolysis steps of the cycle were not affected by S1 aggregation since the formation of the "trapped" S1.MgADP.phosphate analogue complexes, which mimic the prehydrolysis M*ATP and posthydrolysis M**ADP.P(i) transition states, proceeded without any hindrance. Similar conclusions could be reached from the chemical modification of Lys-83 and Cys-707 in the presence of MgATP and MgATPgammaS, which indicated that the most populated intermediate of the cycle in solubilized and aggregated S1 is M**ADP.P(i). The dissociation of the trapped S1.MgADP.phosphate analogue complexes resembling the M**ADP.P(i) state was strongly inhibited by PEG-6000, showing that the transition from this intermediate is prevented by the aggregation. This step is presumably inhibited because the coupled swinging of the lever arm from the closed to the open position is constrained by the close packing of aggregated S1.

The major pathways of mRNA turnover in eukaryotic cells are initiated by shortening of the poly(A) tail. Recent work has identified Ccr4p and Pop2p as components of the major cytoplasmic deadenylase in yeast. We now demonstrate that CCR4 encodes the catalytic subunit of the deadenylase and that Pop2p is dispensable for catalysis. In addition, we demonstrate that at least some of the Ccr4p/Pop2p-associated Not proteins are cytoplasmic, and lesions in some of the NOT genes can lead to defects in mRNA deadenylation rates. The Ccr4p deadenylase is inhibited in vitro by addition of the poly(A) binding protein (Pab1p), suggesting that dissociation of Pab1p from the poly(A) tail may be rate limiting for deadenylation in vivo. In addition, the rapid deadenylation of the COX17 mRNA, which is controlled by a member of the Pumilio family of deadenylation activators Puf3p, requires an active Ccr4p/Pop2p/Not deadenylase. These results define the Ccr4p/Pop2p/Not complex as the cytoplasmic deadenylase in yeast and identify positive and negative regulators of this enzyme complex.

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.

The mRNA surveillance system is known to rapidly degrade aberrant mRNAs that contain premature termination codons in a process referred to as nonsense-mediated decay. A second class of aberrant mRNAs are those wherein the 3' UTR is abnormally extended due to a mutation in the polyadenylation site. We provide several observations that these abnormally 3'-extended mRNAs are degraded by the same machinery that degrades mRNAs with premature nonsense codons. First, the decay of the 3'-extended mRNAs is dependent on the same decapping enzyme and 5'-to-3' exonuclease. Second, the decay is also dependent on the proteins encoded by the UPF1, UPF2, and UPF3 genes, which are known to be specifically required for the rapid decay of mRNAs containing nonsense codons. Third, the ability of an extended 3' UTR to trigger decay is prevented by stabilizing sequences within the PGK1 coding region that are known to protect mRNAs from the rapid decay induced by premature nonsense codons. These results indicate that the mRNA surveillance system plays a role in degrading abnormally extended 3' UTRs. Based on these results, we propose a model in which the mRNA surveillance machinery degrades aberrant mRNAs due to the absence of the proper spatial arrangement of the translation-termination codon with respect to the 3' UTR element as defined by the utilization of a polyadenylation site.

Trinitrophenylation of the reactive lysine (Lys84) in skeletal myosin subfragment 1 (S1) introduces a chiral probe (TNP) into an interface of the catalytic and lever arm domains of S1 [Muhlrad (1977) Biochim. Biophys. Acta 493, 154-166]. Characteristics of the TNP absorption and circular dichroism (CD) spectra in TNP-modified S1 (TNP-Lys84-S1), and the Lys84 trinitrophenylation rate in native S1, indicate a one-to-one correspondence between ATPase transients and trapped phosphate analogues. Phosphate analogue-induced structures of TNP-Lys84-S1 were modeled using the crystallographic coordinates of S1 [Rayment et al. (1993) Science 261, 50-58] with swivels at Gly699 and Gly710 to approximate conformational changes during ATPase. The CD and absorption spectral characteristics of the model structures were compared to those observed for analogue-induced structures. The model calculations, first tested on a trinitrophenylated hexapeptide with known structure, were applied to TNP-Lys84-S1. They showed that ATP binding initiates swiveling at Gly699 and that swiveling at both Gly710 and Gly699 accompanied ATP splitting just prior to product release. The computed lever arm trajectory during ATPase suggests (i) a plausible mechanism for the nucleotide-induced inhibition of Lys84 trinitrophenylation, and (ii) trinitrophenylation-induced changes in S1 Mg2+- and K+-EDTA ATPase are from collision of the lever arm with TNP at Lys84. TNP is a site-specific structural perturbant of S1 and a chiral reporter group for the effect of Lys84 modification on dynamic S1 structure. As such, TNP-Lys84-S1 is equivalent to a genetically engineered mutant with intrinsic sensitivity to structure local to the modified residue.

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

Rabbit skeletal muscle myosin subfragment-1 (S-1) was reacted with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S-1. In order to assign the most reactive tryptophan of the 5 S-1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS-treated S-1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS-treated S-1 and was further cleaved at a single cysteine residue by 2-nitro-5-thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122–204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S-1.

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals it increases osteogenesis and hemopoiesis. In stromal cell cultures OGP stimulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP in high abundance is present in normal human and animal serum mainly complexed to OGP binding protein (OGPBP) or proteins. Here we show the presence of two OGPBPs, OGPBP-1, and OGPBP-2, in cultures of osteoblastic MC3T3 E1 cells. Immunoreactive OGP (irOGP) also accumulates in the medium of these cultures and in cultures of NIH 3T3 fibroblasts. A large amount of irOGP was released by heat inactivation of OGPBP-2 and purified by ultrafiltration and hydrophobic HPLC. The purified irOGP was identical to OGP obtained previously from rat regenerating bone marrow and human serum in terms of its amino acid sequence, immunoreactivity, and mitogenicity. Osteoblastic and fibroblastic cell proliferation can be arrested by anti-OGP antibodies and rescued by exogenous OGP, indicating that in the absence of serum or other exogenous growth stimulators the endogenously produced OGP is both necessary and sufficient for baseline proliferation. The OGP production is up- and down-regulated, respectively, by low and high doses and exogenous OGP in a manner consistent with an autoregulated feedback mechanism. The most effective OGP dose in MC3T3 E1 cells is at least two orders of magnitude lower than that in non-osteoblastic cell systems. This differential sensitivity of the osteoblastic cells could result in a preferential anabolic effect of OGP in bone. J. Cell. Biochem. 65:359–367. © 1997 Wiley-Liss, Inc.

Recent crystallographic studies have suggested structural differences between the complexes of S1 ˙ Mg ˙ ADP with the phosphate analogs aluminium fluoride (AlF−4), vanadate (VO3-4) and beryllium fluoride (BeFx) [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960–8972; Smith, R. & Rayment, I. (1996) Biochemistry 35, 5404–54171. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707 – Cys697 (SH1-SH2) cross-linking, were used to compare the S1 ˙ ADP ˙ BeFx, S1 ˙ ADP ˙ AlF−4 and S1 ˙ ADP ˙ VO3-4 complexes with specific states of the myosin-ATPase pathway. Modification of Cys707 with the fluorescent monofunctional reagents 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin and N-iodoacetyl-N′-(5-sulfo-1-naphtyl)ethylenediamine has shown that the reactivity of the SH1 group depends on the nucleotide bound to S1. The observed rates of Cys707 modification at 20°C lead to the conclusion that S1˙ ADP ˙ BeFx is similar to S1*˙ ATP, while S1 ˙ ADP ˙ AIF−4 and S1 ˙ ADP ˙ VO43- are more similar to S1**˙ ADP ˙ Pi. The conformations of the analog states were also compared by monitoring the dissociation of the fluorescent nucleotide analog 1-N6-ethenoadenosine diphosphate (ADP[C2H2]) from the active site of Cys707-modified (by N-ethylmaleimide) and Cys707-Cys697-cross-linked (by N,N′-p-phenylene dimaleimide) S1 ˙ ADP[C2H2] ˙ AlF−4 and S1 ˙ ADP[C2H2] ˙ BeFx. Our results suggest that the conformations of the S1 ˙ ADP ˙ AlF−4, S1 ˙ ADP ˙ VO3-4 and S1 ˙ ADP ˙ BeFx, complexes in the Cys707–Cys697 region are distinct from each other, with the former two at least partially resembling the S1**˙ ADP ˙ Pi state, while the latter is similar to the prehydrolyzed S1*˙ ATP state.

Myosin belongs to the family of motor proteins. Its interaction with actin coupled with hydrolysis of ATP is the molecular basis of muscle contraction. The head segment of myosin, called subfragment 1 (S1), contains the distinct binding sites for ATP and actin and responsible for the ATPase activity. The rate-limiting step of the ATP hydrolysis is the dissociation of the S1-MgADP-Pi complex which is accelerated by actin. The substitution of Pi with phosphate analogs (PA), such as vanadate (Vi) or beryllium fluoride (BeF(x)), highly stabilizes the complex. We studied the role of the divalent cations in the ATPase activity and in the formation and decomposition of PA-containing stable complexes by substituting Mg2+ with Fe2+, Mn2+, Ni2+, Co2+, and Ca2+. These metal ions supported the actin activation of S1 ATPase and affected the obtained kinetic parameters, Km and V(max). The ATPase activity of S1 in the absence of actin increased with the increasing ionic radius of the metal (Me) ions. These ions also substituted for Mg2+ in the formation of the stable ternary S1-MeADP-PA complexes, which cannot be generated in the absence of divalent cations. Upon formation of stable ternary complexes, S1 reversibly loses its ability to catalyze the hydrolysis of ATP. The formation of the complexes can be followed by monitoring the disappearance of the ATPase activity. The rate of the complex formation depends on the divalent cation present and decreases in the order Mn > Fe > Ni > Co > Mg and Ca > Mn > Fe > Mg > Co in the Vi- and BeF(x)-containing complexes, respectively. The ATPase activity of S1 is recovered upon addition of actin, which causes the decomposition of the complex. The spontaneous decomposition of the complexes was studied in the presence of ethylenediaminetetraacetic acid (EDTA), which chelates the metal divalent cations released from the complex and prevents its reformation. The rate of decomposition was assessed by monitoring the recovery of the ATPase activity of S1 in the presence of EDTA. The rate of decomposition of the Vi- and BeF(x)-containing complexes follows the order Mn > Fe > Co > Mg > Ni and Ca >> Mn > Fe > Co > Mg, respectively. The rate of decomposition increases with the increasing ionic radius of the metal ions, similarly as observed in the case of ionic radius dependence of the ATPase activity. On the basis of this similarity, it is assumed that the decomposition of the complexes consists of two steps, the first step being the very slow release of PA followed by a rapid dissociation of MeADP from S1. The stability of the complexes has been calculated from the formation and decomposition rates. Except in the case of Mg, the stabilities of the BeF(x) complexes are higher than those containing Vi. The results indicate that the metal cations have a significant role in maintaining the proper structure of the transient state complex in the myosin-catalyzed ATP hydrolysis.

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl(2) or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.

The first step in the decay of several yeast mRNAs is the shortening of the poly(A) tail, which for the MFA2 transcript triggers decapping and 5'-to-3' degradation. To understand the basis for differences in mRNA decay rates, it is important to determine if deadenylation-dependent decapping is specific to the unstable MFA2 transcript or is a general mechanism of mRNA degradation. To this end, we analyzed the turnover of the stable PGK1 mRNA by monitoring the decay of a pulse of newly synthesized transcripts while using two strategies to trap decay intermediates. First, we used strains deleted for the XRN1 gene, which encodes a major 5'-to-3' exonuclease in Saccharomyces cerevisiae. In xrn1 delta cells, PGK1 transcripts lacking the 5' cap structure and a few nucleotides at the 5' end were detected after deadenylation. Second, we inserted into the PGK1 5' untranslated region strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates. These secondary structures led to the accumulation of PGK1 mRNA fragments, following deadenylation, trimmed from the 5' end to the site of the secondary structure. The insertion of strong secondary structures into the 5' untranslated region also inhibited translation of the mRNA and greatly stimulated the decay of the PGK1 transcripts, suggesting that translation of the PGK1 mRNA is required for its normally slow rate of decay. These results suggest that one mechanism of degradation of the PGK1 transcript is deadenylation followed by decapping and subsequent 5'-to-3' exonucleolytic degradation. In addition, by blocking the 5'-to-3' degradation process, we observed PGK1 mRNA fragments that are consistent with a 3'-to-5' pathway of mRNA turnover that is slightly slower than the decapping/5'-to-3' decay pathway. These observations indicate that there are multiple mechanisms by which an individual transcript can be degraded following deadenylation.

Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- but not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263, 17429-17436) and to affect the three-dimensional structure of filaments by stabilizing the subdomain 2 region of the actin promoter (Orlova, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In this work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase, and in vitro motility assays. The results of proteolysis studies show that BeFx binds also to MgADP-G-actin and renders its subdomain 2 region more similar to that in MgATP-G-actin. This is manifested in enhanced subtilisin and decreased tryptic digestions in subdomain 2 of G-actin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: both the tryptic and subtilisin digestions in subdomain 2 were completely inhibited. Significant protection against proteolysis in this region was observed even at 1:14 molar ratios of BeFx to actin indicating cooperative effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces changes in the subdomain 1 region of F-actin. This is revealed via reduced rates of Cys-374 alkylation with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and an increased subtilisin cleavage near the C terminus of actin in the presence of BeFx. The BeFx-induced structural changes in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by decreasing Vmax without affecting KM. Additionally, the binding of BeFx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structure and the local differences between monomeric and polymeric forms of actin.

The first step in the decay of some eukaryotic mRNAs is the shortening of the poly(A) tail. To examine how the transcript body was degraded after deadenylation, we followed the decay of a pulse of newly synthesized MFA2 transcripts while utilizing two strategies to trap intermediates in the degradation pathway. First, we inserted strong RNA secondary structures, which can slow exonucleolytic digestion and thereby trap decay intermediates, into the MFA2 5' UTR. Following deadenylation, fragments of the MFA2 mRNA trimmed from the 5' end to the site of secondary structure accumulated as full-length mRNA levels decreased. In addition, in cells deleted for the XRN1 gene, which encodes a major 5' to 3' exonuclease in yeast, the MFA2 transcript is deadenylated normally but persists as a full-length mRNA lacking the 5' cap structure. These results define a mRNA decay pathway in which deadenylation leads to decapping of the mRNA followed by 5'-->3' exonucleolytic degradation of the transcript body. Because the poly(A) tail and the cap structure are found on essentially all mRNAs, this pathway could be a general mechanism for the decay of many eukaryotic transcripts.

Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K + (EDTA)-, Ca 2+ -, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of acting-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the «consensus» ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the bindings and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the «consensus» site through a loop. The 74-kDa cleavage site a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction

It has been reported recently that the isolated and renatured 23-kDa N-terminal fragment of rabbit skeletal muscle myosin binds tightly to F-actin in an ATP-dependent manner [Muhlrad, A. (1989) Biochemistry 28, 4002-4010]. The binding to actin is of electrostatic nature and may involve a positively charged cluster of residues on the 23-kDa fragment stretching from Arg-143 to Arg-147. An octapeptide containing this positive cluster was synthesized and coupled to BSA through a cysteine residue added to the N-terminus of the peptide. Polyclonal antibody was raised against the BSA-coupled peptide in rabbits which recognized the N-terminal 23-kDa fragment of rabbit skeletal myosin subfragment 1, and a peptide comprised of residues 122-204 of the 23K fragment in Western blots. The purified antibody [IgG and F(ab)] inhibited the actin-activated ATPase activity of S1 without affecting its Mg2(+)- and K+(EDTA)-modulated ATPase activity. Both IgG and F(ab) decreased the binding of S1 to F-actin in a sedimentation assay, and actin inhibited the binding of both IgG and F(ab) to S1 in a competitive binding assay. The cysteine thiol of the synthetic octapeptide was labeled by the fluorescent thiol reagent monobromobimane, and the labeled peptide was found to bind to actin in a sedimentation assay. The results support the possibility that the positively charged Arg-143 to Arg-147 stretch of residues on the 23-kDa fragment participates in actin binding of myosin and may represent an essential constituent of the actin-S1 interface.

Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.

Myosin contains reactive lysine residues which are trinitrophenylated by 2,4,6-trinitrobenzene sulfonate much faster than the rest of the lysines. Here we find the location of these residues in the primary and spatial structure of myosin with the help of an anti-trinitrophenyl antibody. This antibody was raised against trinitrophenyl hemocyanin in rabbits. It reacted with trinitrophenylated myosin, and with some of the tryptic fragments of trinitrophenylated myosin. By analyzing the reaction with Western blots, it was found that the antibody preferentially reacts with the 27 kDa N-terminal fragment of the myosin head, and more weakly with the light meromyosin region of the myosin rod. The 27 kDa fragment contains the most reactive lysine residue, while the intermediate lysine residue is located in the light meromyosin region. The locations of the epitopes of the antibody were visualized on electron microscope images of rotary-shadowed trinitrophenylated myosin-antibody complexes. The distances of the epitopes to the head-rod junction of myosin were measured as 13 and 113 nm for the epitope on the head (reactive lysine residue) and for that on the rod (intermediary reactive lysine residue), respectively.

β-Naphthoquinone-4-sulfonate was used for chemical modification of amino groups of myosin. The reagent was found to affect also the sulfhydryl groups if the reaction was not prevented by previous disulfide exchange with cystamine. When cystamine protection was employed the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was enhanced in the presence of Mg2+ and decreased in the presence of K+ or Ca2+, a pattern typical of myosin with blocked essential amino groups. On addition of ATP or ADP a blueshift was observed in the fluorescent emission spectrum of β-naphthoquinone-4-sulfonate bound by myosin, presumably owing to conformational changes in the environment of essential amino groups induced by the binding of nucleotides.

Myosin was reacted with 2,4,6-trinitrobenzene sulphonate (TNBS) in the presence or absence of Mg-pyrophosphate. The reaction led to trinitrophenylation of lysyl residues which could be divided on the basis of the reaction into three classes: (i) two rapidly reacting lysyl residues (RLR), one residing on each head of myosin, whose rate of reaction depends on the presence of Mg-pyrophosphate; (ii) two lysyl residues which react with intermediate rate (ILR) and reside on the rod segment of myosin; and (iii) the remaining lysyl residues of myosin which react slowly with TNBS. The rate of the trinitrophenylation of RLR was followed spectrophotometrically and enzymatically, measuring an absorbance change at 345 nm, and also changes in K+ (EDTA)-, Mg2+- and Ca2+-activated ATPase activities, respectively. According to analysis of the kinetics of the reaction, Mg-pyrophosphate inhibited the rate of trinitrophenylation in both heads of myosin, not in one head only as was suggested by Miyanishi et al. (J. Biochem Tokyo 85; 1979). Myosin heads (myosin subfragment-1, S-1) were prepared by digesting myosin trinitrophenylated in the absence and presence of Mg-pyrophosphate with chymotrypsin. S-1, with trinitrophenylated RLR, was separated from non-trinitrophenylated S-1 by DEAE cellulose column chromatography. The trinitrophenylated S-1 had a high Mg2+- and a low K+(EDTA)-activated ATPase while the non-trinitrophenylated species had the usual high K+(EDTA)- and low Mg2+-ATPase activity. This results excluded the possibility suggested by Miyanishi et al., that the myosin head, which is resistant to trinitrophenylation in the presence of Mg-pyrophosphate, did not possess K+(EDTA)-activated ATPase activity. The presence of Mg-pyrophosphate during trinitrophenylation substantially affected the enzymic characteristics of the modified myosin. The myosin trinitrophenylated in the presence of Mg-pyrophosphate had a higher K+(EDTA)- and a lower Mg2+-ATPase activity. SH1 (Cys-707) also probably becomes a target of the reaction if myosin is trinitrophenylated in the presence of Mg-pyrophosphate. This is deduced from the following findings: (i) the addition of dithiothreitol after trinitrophenylation partially reversed the loss in the K+(EDTA)-ATPase activity; and (ii) the specific alkylation of the SH1 thiol by 1,5-IAEDANS prior to trinitrophenylation prevented the effect of dithiothreitol on the ATPase activity of myosin. The results indicated that Mg-pyrophosphate induced structural changes in the myosin molecule which influenced the course and possibly the target(s) of trinitrophenylation.

Human platelets were induced to aggregate by either ADP or collagen. Both aggregated and non-aggregated (resting) platelets were then washed twice, lyzed by osmotic shock in the presence of several protease inhibitors and fractionated. The cytoplasmic fraction (Fraction I) was separated from the rest of the cell by centrifugation, the pellet was suspended in 0.8 M KC1 and the extract (Fraction II) was separated from the particulate fraction (Fraction III). The relative amounts of three contractile proteins, myosin, actin and actin-binding protein were evaluated and the activities of myosin, K + -(EDTA)-activated ATPase, and of actomyosin, Mg 2+ -mediated ATPase, were determined in all fractions. Following ADP- or collagen-induced aggregation the specific activities of both myosin and actomyosin ATPases increased significantly in the non-soluble Fraction III.

Identification and isolation of extracellular matrix vesicles were performed from an osteosarcona. They were characterized as trilaminated membrane-bound organelles with diameters ranging between 0.05 – 0.45 μm. The vesicle fraction showed remarkably high alkaline phosphatase, pyrophosphatase and K + (EDTA) mediated ATPase activities when compared to the membrane and cellular fractions. Occurrence of K + (EDTA) mediated ATPase indicated the presence of myosin. Electrophoretic bands, corresponding to the contractile proteins, actin and myosin, supported the findings of enzymatic assays.