Edited by Ruma BanerjeeGlutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of ␥-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ϳ50% sequence identity. and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either ␥-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed.
Cellulase catalyzes the hydrolysis of β-1,4-linkages of cellulose to produce industrially relevant monomeric subunits. Cellulases find their applications in pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. These industries always have great demand for cellulases that can work efficiently even in harsh conditions such as high salt, heat, and acidic environments. While, cellulases with high thermal and acidic stability are already in use, existence of a high halotolerant cellulase is still elusive. Here, we report a novel cellulase Cel5R, obtained from soil metagenome that shows high halotolerance and thermal stability. The biochemical and functional characterization of Cel5R revealed its endoglucanase activity and high halostability. In addition, the crystal structure of Cel5R determined at 2.2 Å resolution reveals a large number of acidic residues on the surface of the protein that contribute to the halophilic nature of this enzyme. Moreover, we demonstrate that the four free and non-conserved cysteine residues (C65, C90, C231 and C273) contributes to the thermal stability of Cel5R by alanine scanning experiments. Thus, the newly identified endoglucanase Cel5R is a promising candidate for various industrial applications.
Transformed cells of macrophage lineage such as J774A.1, P388D1 and IC21 take up and degrade a conjugate of the antineoplastic drug, daunomycin, with maleylated BSA with high efficiency and saturation kinetics through the scavenger receptors expressed on the surface of these cells. By contrast, transformed cells of non-macrophage lineage, namely L929, EL4, Bowes melanoma and CHO (Chinese-hamster ovary), do not take up and degrade the conjugate, indicating that these cells are scavenger-receptor-deficient. In the conjugated form, about 0.1 microM-daunomycin cause 50% inhibition in the uptake of [3H]thymidine by the receptor-bearing J774A.1 cells, whereas the receptor-deficient Bowes-melanoma cells are not affected. Free daunomycin (0.1 microM) does not significantly affect the uptake of [3H]thymidine by either cell type. Treatment of cells derived from intraperitoneal tumours induced in BALB/C mice by J774A.1 cells with 0.4 microM-daunomycin in the conjugated form for 5 h abolished their ability to form tumours in BALB/C mice. By contrast, transplantation of untreated cells or cells treated with free daunomycin under identical conditions led to tumour formation and subsequent death of the BALB/C mice. These results indicate that this modality for selective elimination of scavenger-receptor-bearing neoplastic cells may be useful for the treatment of histiocytic malignancies in which cells of macrophage lineage turn malignant.
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