INTRODUCTION Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. Gluconeogenesis (de novo synthesis of glucose) is, in effect, a reversal of glycolysis, in which glucose is converted to pyruvate. Some enzymatic steps are shared between gluconeogenesis and glycolysis, but other steps are confined to one of the two pathways. In the yeast Saccharomyces cerevisiae, the gluconeogenesis-specific enzymes are fructose-1,6-bisphosphatase (Fbp1), isocitrate lyase (Icl1), malate dehydrogenase (Mdh2), and phosphoenolpyruvate carboxykinase (Pck1). We found that Gid4, a subunit of the oligomeric GID ubiquitin ligase, is the recognition component of a proteolytic pathway termed the Pro/N-end rule pathway, which conditionally destroys gluconeogenic enzymes. The N-end rule pathway is a set of proteolytic systems whose unifying feature is their ability to recognize and polyubiquitylate proteins containing N-terminal degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome. In eukaryotes, the previously known branches of this system are the Arg/N-end rule pathway and the Ac/N-end rule pathway. The Arg/N-end rule pathway targets specific unacetylated N-terminal residues of cellular proteins, including Asn, Gln, Glu, Asp, Arg, Lys,His, Leu, Phe, Tyr, Trp, Ile, and Met (if Met is followed by a bulky hydrophobic residue). The pathway’s other branch, called the Ac/N-end rule pathway, targets proteins for degradation by recognizing their Nα-terminally acetylated (Ntacetylated) residues. About 90% of human proteins are cotranslationally and irreversibly Nt-acetylated. Many, possiblymost, Nt-acetylated proteins bear N-degrons of the Ac/N-end rule pathway. RATIONALE We wished to identify the recognition component of themultisubunit GID ubiquitin ligase and also to determine whether GID, which was known to mediate the conditional degradation of gluconeogenic enzymes, might recognize them through their N-terminal Pro residues, and also through Pro at position 2. RESULTS The successful strategy involved a version of two-hybrid assay for in vivo protein interactions. Themain discovery identified Gid4, a subunit of the GID ubiquitin ligase, as the recognition component (termed Pro/N-recognin) of the Pro/N-end rule pathway. Gid4 was shown to target the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 (and possibly other yeast proteins as well) through the binding to their N-terminal Pro residues in the presence of cognate adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2. Gid4 was also required for the degradation of Pck1 through the ability ofGid4 to target the Pro residue of Pck1 at position 2. The properties of Gid4 discovered so far indicate that its substrate binding groove can recognize either the N-terminal Pro residue or Pro at position 2 in the presence of cognate adjacent sequence motifs. The recognition flexibility of Gid4 suggests that the true diversity of Gid4 substrates is o...

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This paper intends to discuss the potential tactile need of human and resort to design--especially industrial design--to fulfill it, in other words, to rediscover the basic need and encourage people to notice it. Touch feeling may be not only a subordinative consideration in industrial design and we expect the possible popularity of future touch-contented products. The paper consists of four parts: the importance and potential need of touch feeling, the current design research and products connecting touch feeling and the future design trend to satisfy touch feeling. It will be based on observations, interviews of typical people and a questionnaire survey, which intertwine together thus to make the analysis more convincing, to achieve the final result.

For the efficient biotransformation of cucurbitacin B 2-o-β-d-glucoside (CuBg) to cucurbitacin B (CuB) in Cucumis melo pedicel extracts, the β-glucosidase gene bglS—consisting of 1344 bp (447 amino acids) from Streptomyces sp. RW-2—was cloned and expressed in Escherichia coli BL21(DE3). The activity of recombinant β-glucosidase with p-nitrophenyl-β-d-glucoside (pNPG) as a substrate was 3.48 U/mL in a culture. Using the recombinant β-glucosidase for the biotransformation of C. melo pedicel extracts, CuBg was converted into CuB with a conversion rate of 87.6% when the concentration of CuBg was 0.973 g/L in a reaction mixtures. The concentration of CuB in C. melo pedicel extracts was improved from 13.6 to 20.2 g/L after biotransformation. The present study provides high-efficiency technology for the production of CuB from its glycoside by biotransformation.

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