Tomonari Muramatsu

Physical and functional interactions between Pim-1 kinase and Cdc25A phosphatase. Implications for the Pim-1-mediated activation of the c-Myc signaling pathway.

The pim-1 oncogene encodes a serine/threonine kinase (Pim-1) involved in the transduction of cytokine-triggered mitogenic signals. Pim-1 is unique in that it closely cooperates with c-Myc not only in oncogenesis, but also in apoptosis induction. However, the molecular basis of Pim-1 function remains poorly understood, largely because the downstream effector molecule(s) for Pim-1 kinase has not been identified. Here we provide several lines of evidence that Cdc25A cell cycle phosphatase, a direct transcriptional target for c-Myc, is a substrate for Pim-1 kinase and functions as an effector for Pim-1. We found that Pim-1 physically interacts with Cdc25A both in vitro and in vivoand phosphorylates Cdc25A. We also observed that Pim-1-mediated phosphorylation of Cdc25A increases its phosphatase activity. In addition, wild-type Pim-1, but not kinase-inactive Pim-1, enhanced Cdc25A-mediated cellular transformation and apoptosis. Our results indicate that Cdc25A might be a key molecule that links Pim-1 and c-Myc and that also ties Pim-1-mediated mitogenic signals to cell cycle machinery.

Molecular mechanism of stop codon recognition by eRF1 : a wobble hypothesis for peptide anticodons

We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N‐terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.

Cleavage mechanism of ATP-dependent Lon protease toward ribosomal S2 protein

The Escherichia coli ATP‐dependent protease Lon degrades ribosomal S2 protein in the presence of inorganic polyphosphate (polyP). In this study, the process of the degradation was investigated in detail. During the degradation, 68 peptides with various sizes (4–29 residues) were produced in a processive fashion. Cleavage occurred at 45 sites, whose P1 and P3 positions were dominantly occupied by hydrophobic residues. These cleavage sites were located preferentially at the regions with rigid secondary structures and the P1 residues of the major cleavage sites appeared to be concealed from the surface of the substrate molecule. Furthermore, polyP changed not only the substrate preference but also the oligomeric structure of the enzyme.

The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1.

Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.

Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem (Reddy, M. N., Keim, P. S., Heinrikson, R. L., and Kézdy, F. J. (1975)J. Biol. Chem. 250, 1741–1750). It consists of eight isoinhibitors, and each isoinhibitor has a two-chain structure. In this study, the genomic DNA has been cloned and found to encode a precursor protein with 246 amino acids (M r = ∼27,500) containing three isoinhibitor domains (BI-III, -VI, and -VII) that are 93% identical to one another in amino acid sequences. The gene structure indicated that these isoinhibitors are produced by removal of the N-terminal pre-peptide (19 residues), 3 interchain peptides (each 5 residues), 2 interdomain peptides (each 19 residues), and the C-terminal pro-peptide (18 residues). Moreover, all the amino acid sequences of bromelain isoinhibitors could be explained by removal of one or two amino acids from BI-III, -VI, and -VII with exopeptidases. A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively. These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors.

The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli

Leucyl/phenylalanyl‐tRNA‐protein transferase (L/F‐transferase) is an N‐end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl‐tRNAs to exposed N‐terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 Å resolution crystal structure of L/F‐transferase (JW0868) from Escherichia coli, the first three‐dimensional structure of an L/F‐transferase. The L/F‐transferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N‐terminal domain forms a small lobe with a novel fold. The large C‐terminal domain has a highly conserved fold, which is observed in the GCN5‐related N‐acetyltransferase (GNAT) family. Most of the conserved residues of L/F‐transferase reside in the central cavity, which exists at the interface between the N‐terminal and C‐terminal domains. A comparison of the structures of L/F‐transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C‐terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F‐transferase suggest that this region is important for the enzyme catalysis.

Recognition of the Nucleoside in the First Position of the Anticodon of Isoleucine tRNA by Isoleucyl-tRNA Synthetase from Escherichia Coli

Abstract Escherichia coli tRNAIle 1 prepared in vitro by T7 RNA polymerase transcription was found to be charged with isoleucine by E. coli isoleucyl-tRNA synthetase (IleRS). Replacement of G in the first position of the anticodon with U, C, or A resulted in complete loss of isoleucine-accepting activity. This indicates that IleRS strictly recognizes the structural feature of guanosine in this position of tRNAIle 1 which is common to that of lysidine in E. coli tRNAIle 2.

Conformational Change of tRNA Upon Interaction of the Identity-Determinant Set with Aminoacyl-tRNA Synthetase

For correct interpretation of the genetic code, the twenty aminoacyl-tRNA synthetases are required to strictly recognize their cognate tRNA and amino acid. The amino acid specificity of tRNA depends on a set of a relatively small number of nucleotide residues (determinants for the identity of tRNA) such as the anticodon residues, the discriminator base at position 73, base pairs 1:72, 2:71, and 3:70 in the acceptor stem, base pair 10:25 in the D stem, and residue 20 (Shimura et al., 1972; Normanly et al., 1986; Hou and Schimmel, 1988; McClain and Foss, 1988ab; Muramatsu et al., 1988b; Schulman and Pelka, 1988; Normanly and Abelson, 1989; Schimmel, 1989; Himeno et al., 1990; Jahn et al., 1991; Putz et al., 1991; Franklyn et al., 1992; Muramatsu et al., 1992; Schulman, 1991; Tamura et al., 1992). The three-dimensional structures of the complexes of E. coli tRNAG1n and yeast tRNAAsp with their cognate aminoacyl-tRNA synthetases, GlnRS and AspRS, respectively, have been determined by X-ray crystallography (Rould et al., 1989; Rould et al., 1991; Ruff et al., 1991). These synthetases recognize the identity determinants mainly in the two ends of the L-shaped structure of tRNA (the anticodon loop and the 5’/3’ terminal region) (Jahn et al., 1991; Putz et al., 1991) and concomitantly change the conformations of these regions (Rould et al., 1989; Rould et al., 1991; Ruff et al., 1991).

Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

X-Ray Crystallographic Study of a Non-Pepsin-Type Acid Proteinase, Aspergillus Niger Proteinase A

Acid proteinase A, secreted by the fungus Aspergillus niger var. macrosporus, is a non-pepsin-type acid proteinase as judged from its distinct differences in various properties from the ordinary pepsin-type aspartic proteinase family [1,2]. It is insensitive to pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), and l,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) [3], and shows fairly different substrate specificity [4]. The enzyme is composed of two chains, and the relative molecular mass is 22,300, about half the size of the ordinary pepsin-type proteinases. No protein in the GenBank database is homologous in the primary structure with proteinase A except for Scytalidium lignicolum proteinase B [5] that shows about 50% identity [2]. Proteinase A has no consensus sequence, Asp-Thr/Ser-Gly, including the catalytic aspartic acid residue, present in the family of pepsin-type proteinases. It, thus, remains to be elucidated which residues participate in the catalysis and how the mechanism operates. Moreover, it is of interest to elucidate how the three-dimensional structure differs from pepsin-type enzymes. Comparison of the crystal structure of proteinase A with those of pepsin-type enzymes will enable us to explain the unusual properties of this enzyme. To approach these queries, we have started the crystallographic study of the enzyme, and have obtained an isomorphous heavy atom derivative of the crystals.