Several proteins with significant identity to ubiquitin have been characterized recently. In contrast to ubiquitin's main role in targeting proteins for degradation, a described function of one family of ubiquitin-related proteins, the Rub family, is to serve as a stable posttranslational modification of a complex involved in the G 1 -to-S cell cycle transition. Rub proteins have been found in animals, plants, and fungi and consist of 76 residues with 52-63% identity to ubiquitin. In this study three different RUB proteins within the plant Arabidopsis are identified; two differ by only 1 amino acid, while the third is only 77.6% identical to the other two. Genes encoding all three are expressed in multiple organs. In addition, we report the crystal structure of higher plant RUB1 at 1.7-Å resolution to help elucidate the functional differences between Rub and ubiquitin. RUB1 contains a single globular domain with a flexible COOH-terminal extension. The overall RUB1 structure is very similar to ubiquitin. The majority of the amino acid differences between RUB1 and ubiquitin map to the surface. These changes alter the electrostatic surface potential in two regions and likely confer specificity between ubiquitin and RUB1 and their ubiquitin-activating enzyme (E1) or E1-like activating enzymes.
A human anti-CD19 antibody was expressed in fucosyltransferase-deficient CHO cells to generate nonfucosylated MDX-1342. Binding of MDX-1342 to human CD19-expressing cells was similar to its fucosylated parental antibody. However, MDX-1342 exhibited increased affinity for FcγRIIIa-Phe158 and FcγRIIIa-Val158 receptors as well as enhanced effector cell function, as demonstrated by increased potency and efficacy in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. MDX-1342 showed dose-dependent improvement in survival using a murine B-cell lymphoma model in which Ramos cells were administered systemically. In addition, low nanomolar binding to cynomolgus monkey CD19 and increased affinity for cynomolgus monkey FcγRIIIa was observed. In vivo administration of MDX-1342 in cynomolgus monkeys revealed potent B-cell depletion, suggesting its potential utility as a B-lymphocyte depletive therapy for malignancies and autoimmune indications.
Aspartic proteinases are produced in the human body by a variety of cells. Some of these proteins, examples of which are pepsin, gastricsin, and renin, are secreted and exert their effects in the extracellular spaces. Cathepsin D and cathepsin E on the other hand are intracellular enzymes. The least characterized of the human aspartic proteinases is cathepsin E. Presented here are results of studies designed to characterize the binding specificities in the active site of human cathepsin E with comparison to other mechanistically similar enzymes. A peptide series based on Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu was generated to elucidate the specificity in the individual binding pockets with systematic substitutions in the P5-P2, and P2'-P3' based on charge, hydrophobicity, and hydrogen bonding. Also, to explore the S2 binding preferences, a second series of peptides based on Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu was generated with systematic replacements in the P2 position. Kinetic parameters were determined for both sets of peptides. The results were correlated to a rule-based structural model of human cathepsin E, constructed on the known three-dimensional structures of several highly homologous aspartic proteinases; porcine pepsin, bovine chymosin, yeast proteinase A, human cathepsin D, and mouse and human renin. Important specificity-determining interactions were found in the S3 (Glu-13) and S2 (Thr-222, Gln-287, Leu-289, Ile-300) subsites.
The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.
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