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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5'to 3'degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3'UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3'UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery. [ABSTRACT FROM AUTHOR]

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. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.© 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10–14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10–14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr[sup 10] or its replacement by Ala or d-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr[sup 10] amino group. No further loss occurred consequent to the monoiodination of desaminoTyr[sup 10] on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr[sup 10] phenolic or the Phe[sup 12] aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly[sup 11] with l- or d-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly[sup 14] or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and l- or d-Ala substitution of Gly[sup 13] resulted in 70–97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10–14). [ABSTRACT FROM AUTHOR]

Studies the effect of actin, ATP, ADP and ADP.PA complexes on the conformational changes in the vicinity of Lys533 in myosin subfragment 1. Modification of Lys533 S1 by 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS); Fluorescence intensity of FHS; Decrease in the rate of FHS modification by ATP.

Abstract: The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10–14). The OGP(10–14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10–14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP–OGP binding protein (OGPBP) complex formation, sOGP(10–14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10–14) is a naturally occuring human and murine mitogen. In addition, the data suggests that the OGP(10–14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP–OGPBP complexes. [ABSTRACT FROM AUTHOR]

The structure of myosin subfragment 1 (S1) in the weakly attached complex with actin was studied at three specific sites, at the 50-kDa/20-kDa and 27-kDa/50-kDa junctions, and at the N-terminal region, using tryptic digestion as a structure-exploring tool. The structure of S1 at the vicinity of the 50-kDa/20-kDa junction is pH dependent in the weakly attached state because the tryptic cleavage at this site was fully protected by actin at pH 6.2, but the protection was only partial at pH 8.0. Since the actin protection is complete in rigor at both pH values, the results indicate that the structure of S1 at the 50-kDa/20-kDa junction differs in the two states at pH 8.0, but not at pH 6.2. Actin restores the ADP-suppressed tryptic cleavage after Lys213 at the 27-kDa/50-kDa junction in the strongly attached state, but not in the weakly attached state, which indicates structural difference between the two states at this site. ATP and ADP open a new site for tryptic cleavage in the N-terminal region of the S1 heavy chain between Arg23 and Ile24. Actin was found to suppress this cleavage in both weakly and strongly attached states, which shows that, in the vicinity of this site, the structure of S1 is similar in both states. The results indicate that the binding of S1 to actin induces localized changes in the S1 structure, and the extent of these changes is different in the various actin-S1 complexes. [ABSTRACT FROM AUTHOR]

We have incorporated Fe2+ into the high-affinity metal-ion-binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced freeradical generation, with the intent of causing peptide cleavage at the metal-ion-binding site. By analysis of the resulting fragments from actin in the F-form, we have deduced that cuts occurred at positions 159-160 and 301-302 (at the latter location we could not be sure if more than one cut occurred). We considered that these two cuts occurred in the chain strand coursing from the outer to the inner domain and vice-versa. Our results harmonize very well with the recently reported atomic structure of actin [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37-44] and remove ambiguities that had remained in the structure. The results partly bear out the homology-based prediction of Strzelecka-Golaszewska et al. [Strzelecka-Golaszewska, H., Boguta, G., Zmorzynshi, S. & Moraczcwska, J. (1989) Eur. J. Biochem. 182, 299-305]. [ABSTRACT FROM AUTHOR]

Myosin subfragment 1 (S1) is cleaved by near-ultraviolet irradiation in the presence of vanadate at three sites located at 23, 31 and 74 kDa from the N-terminus. Since vanadate is considered to be a good structural analogue of phosphate, it is assumed that the cleavage sites participate in forming the phosphate-binding site(s) of S1. In this work, the effect of various ions on the vanadate-induced photocleavage of S1 was studied. Monovalent anions were found to inhibit photocleavage in the 50-200 mM range. The inhibition is more expressed at a site 74 kDa from the N-terminus than at the 23-kDa and 31-kDa sites. The inhibitory effect of the monovalent anions increases in the order acetate F- < Cl- < Br- < I- = SCN-. The order of the inhibitory effect is identical to the protein-structure-damaging effect of monovalent anions in the von Hippel series [von Hipel, P. H. & Wong, K. Y. (1964) Science 145,577-581]. Therefore, it is assumed that decreased photocleavage is due to local perturbations of structure, especially at the 74-kDa site, in addition to increased ionic strength. Divalent anions, sulfate and thiosulfate, strongly inhibit photocleavage at 2 mM. The inhibition is very pronounced at the 23-kDa and 31-kDa sites, while the 74-kDa site is hardly affected. Since photocleavage at the 23-kDa and 31-kDa sites is regulated jointly and independently from cleavage at the 74-kDa site, it is assumed that SI has two distinct phosphate- binding sites: the regions of the 23-kDa and 31-kDa cleavage sites, which are proximal to each other in the spatial structure, participate in forming the first phosphate-binding site, while the 74-kDa site is part of the second binding site. Sulfate was also found to inhibit the trapping of vanadate and to facilitate its release from the SI- MgADP-Vi (Vi, inorganic vanadate) complex. Photocleavage of S1 takes place at all three sites, both in the presence or absence of divalent cations, indicating that these, including Mg2+, are not essential for cleavage. [ABSTRACT FROM AUTHOR]

Myosin subfragment-1 from rabbit skeletal muscle was digested by thermolysin at 25°, 12° and 0°C. Thermolysin cleaves subfragment-1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N-terminus [Applegate, D. & Reisler, E. (1983) Proc. Natl Acad. Sci. USA 80, 7109-7112]. The rate of digestion at 25°C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12°C and completely eliminated at 0 °C. The results can be explained by assuming that there are two subfragment-1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004- 2012], and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment-1 at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment-1. Actin inhibition of thermolysin digestion also prevailed when subfragment-1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment-1 modified by 1 ,4-phenylenediamine, which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin-susceptible site of subfragment-1 at the 28 -- 70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states. [ABSTRACT FROM AUTHOR]

Recent crystallographic studies have suggested structural differences between the complexes of S1 · Mg · ADP with the phosphate analogs aluminium fluoride (AlF4-), vanadate (VO43-) and beryllium fluon,de (BeF4) [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, 5404-5417]. In this work, chemical modifications, namely labeling of Cys707 (the reactive SH1 thiol) and Cys707Cys697 (SH1-SH2) cross-linking, were used to compare the S1 · ADP · BeF4, St1· ADP · AlF4- and S1 · ADP · VO43- 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 · AI.F4- and S1 · ADP · VO43- are more similar to S1** · ADP · P1. 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-Cys6'97cross-linked (by N,N'-p-phenylene dimaleimide) S1 · ADP[C2H2] · AlF4- and S1 · ADP[C2H2] · BeFx. Our results suggest that the conformations of the S1 · ADP AlF4- S1 · ADP · VO43- 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 S 1* · ATP state. [ABSTRACT FROM AUTHOR]

Discusses the immunochemical probing of the functional role of the 238-246 and 567-574 sequences of myosin heavy chain. Antibodies obtained from rabbit sera; Binding of antibodies; Sliding velocity of actin filaments.