Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.

The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.     

Role for Geminin in sustaining the activity of hematopoietic stem cells

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.     

MicroRNA-dependent regulation of the microenvironment and the epithelial stromal cell interactions in the mouse mammary gland

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.     

A long way to stemness

Comment on: Tanaka Y, et al. Proc Natl Acad Sci USA 2012; 109:4515-20.     

Sensing senescence in preterm birth

Comment on: Hirota Y, et al. Proc Natl Acad Sci USA 2011; 108:18073-8     

A long way to stemness

Comment on: Tanaka Y, et al. Proc Natl Acad Sci USA 2012; 109:4515-20.     

DEC1 and MIC-1

Comment on: Qian Y, et al. Proc Natl Acad Sci USA 2012; 109:11300-5.     

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell- free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.     

The yeast DNA repair proteins RAD1 and RAD7 share similar putative functional domains

Sequence information on eukaryotic DNA repair proteins provided so far only few clues concerning possible functional domains. Since the DNA repair process involves a strict sequential complex formation of several proteins [1988) FASEB J. 2, 2696-2701], we searched for special protein-protein interacting domains, which consist of tandemly repeated leucine rich motifs (LRM). Search algorithms, capable of detecting even largely divergent repeats by assessing their significance due to the tandem repetitivity, revealed that the yeast DNA repair proteins RAD1 and RAD7 contain 9 and 12 tandem LRM repeats, respectively. These results represent the first clues concerning specific domains in these proteins and assign them to the LRM superfamily, which includes such members as yeast adenylate cyclase, cell surface protein receptors and ribonuclease/angiogenin inhibitor, all exerting their function by specific protein-protein interactions involving LRM domains [( 1988) EMBO J. 7, 4151-4156; (1990) Proc. Natl. Acad. Sci. USA 87, 8711-8715; (1989) Science 245, 494-499; (1990) Mol. Cell. Biol. 10, 6436-6444; (1989) Proc. Natl. Acad. Sci. USA 86, 6773-6777].     

Proposal for sequencing DNA using ligation of hexamers to generate sequential elongation primers (SPEL-6)

The efficiency of the directed-priming proposal of Studier [Proc. Natl. Acad. Sci. USA 86 (1989) 6917-1921] can be improved upon by employing a library of less than 4094 hexamers, allowing for the easy construction of all required primers using template-directed automated ligation. This 'top-down' sequencing procedure should be several times more efficient than the present 'bottom-up' procedures [see Watson, Science 248 (1990) 44-49].     

Consistencies of individual DNA base-amino acid interactions in structures and sequences.

Amino acid-amino acid interaction energies have been derived from crystal structure data for a number of years. Here is reported the first derivation of normalized relative interaction from binding data for each of the four bases interacting with a specific amino acid, utilizing data from combinatorial multiplex DNA binding of zinc finger domains [Desjarlais, J. R. and Berg, J. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 11099-11103]. The five strongest interactions are observed for lysine-guanine, lysine-thymine, arginine-guanine, aspartic acid-cytosine and asparagine-adenine. These rankings for interactions with the four bases appear to be related to base-amino acid partial charges. Also, similar normalized relative interaction energies are derived by using DNA binding data for Cro and lambda repressors and the R2R3 c-Myb protein domain [Takeda, Y., Sarai, A. and Rivera, V. M. (1989) Proc. Natl. Acad. Sci. USA, 86, 439-443; Sarai, A. and Takeda, Y. (1989) Proc. Natl. Acad. Sci. USA, 86, 6513-6517; Ogata, K. et al. (1995) submitted]. These energies correlate well with the combinatorial multiplex energies, and the strongest cases are similar between the two sets. They also correlate well with similar relative interaction energies derived directly from frequencies of bases in the bacteriophage lambda operator sequences. These results suggest that such potentials are general and that extensive combinatorial binding studies can be used to derive potential energies for DNA-protein interactions.     

Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry.

Protein complexes assembled on mRNA precursors can be separated by gel filtration chromatography to yield spliceosomal and H complex fractions (Reed R, Griffith J, Maniatis T, 1988, Cell 53:949-961; Reed R, 1990, Proc Natl Acad Sci USA 87:8031-8035.). Here we use Nano electrospray mass spectrometry (Wilm M, Mann M, 1994, Int J Mass Spectrometry Ion Processes 136:167-180) to identify proteins complexed with Adeno-pre-mRNA in the H complex peak. Four of the major hnRNP proteins, A1, B1, C1, and G, were identified by database analysis based on peptide mass and sequence information. A fifth protein in the H complex peak, corresponding to hnRNP P2, is shown to be the product of the TLS/FUS gene. This was originally identified as a chimeric oncogene formed by the chromosome translocation t(12;16) that is responsible for myxoid liposarcoma. The involvement of hnRNP P2 in oncogenesis provides a clear example of the importance of hnRNP proteins in molecular disease.     

Molecular structure of the Japanese hepatitis C viral genome

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et. al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524–9528)indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase. NTPase and RNA-dependent RNA polymerase     

c[D-pro-Pro-D-pro-N-methyl-Ala] adopts a rigid conformation that serves as a scaffold to mimic reverse-turns

Naturally occurring cyclic tetrapeptides (CTPs) such as tentoxin (Halloin et al., Plant Physiol 1970, 45, 310-314; Saad, Phytopathology 1970, 60, 415-418), ampicidin (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147), HC-toxin (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447), and trapoxin (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328; Itazaki et al., J Antibiot (Tokyo) 1990, 43, 1524-1532) have a wide range of biological activity and potential use ranging from herbicides (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447; Judson, J Agric Food Chem 1987, 35, 451-456) to therapeutics (Loiseau, Biopolymers 2003, 69, 363-385) for malaria (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147) and cancer (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328). To elucidate scaffolds that have few low-energy conformations and could serve as semirigid reverse-turn mimetics, the flexibility of CTPs was determined computationally. Four analogs of cyclic tetraproline c[Pro-pro-Pro-pro] with alternating L- and D-prolines, namely c[pro-Pro-pro-NMe-Ala], c[pip-Pro-pip-Pro], c[pro-Pip-pro-Pro], and c[Ala-Pro-pip-Pro] were synthesized and characterized by NOESY NMR. Both molecular mechanics and Density Functional Theory quantum calculations found these head-to-tail CTPs to be constrained to one or two relatively stable conformations. NMR structures, while not always yielding the same lowest energy conformation as expected by in silico predictions, confirmed only one or two highly populated solution conformations for all four peptides examined. c[pro-Pro-pro-NMe-Ala] was shown to have a single all trans-amide bond conformation from both in silico predictions and NMR characterization, and to be a reverse-turn mimetic by overlapping four Calpha-Cbeta bonds with those for approximately 6.5% (Tran, J Comput Aided Mol Des 2005, 19, 551-566) of reverse-turns in the Protein Data Bank PDB with a RMSD of 0.57 A.     

Oncolytic herpes virus retargeted to HER-2

Comment on: Menotti L, et al. Inhibition of human tumor growth in mice by an oncolytic herpes simplex virus designed to target solely HER-2-positive cells. Proc Natl Acad Sci USA 2009;106:9039-44.     

Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis

We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.     

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.     

The complete amino acid sequence of the shorter form of human basic fibroblast growth factor receptor deduced from its cDNA

We have isolated a full-length cDNA for human basic fibroblast growth factor (bFGF) receptor-like protein from a human placenta cDNA library. Determination of the nucleotide sequence of the cDNA allows elucidation of the complete amino acid sequence of the receptor (731 amino acids) which has two extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The receptor has remarkable amino acid similarity (98% identity) to the shorter form of murine bFGF receptor reported recently (H.H.Reid et al. (1990) Proc.Natl.Acad.Sci. USA 87, 1596-1600). The receptor described here is expected to be the shorter form of human bFGF receptor.