| (I) Disturbance of the balance between oxidant and reducer lead to the appearance of many free radicals. The process that the cells respond and eliminate these free radicals is called oxidative stress response. Oxidative stress plays a crucial role during the occurrence of many diseases, such as heart diseases, idiopathic pulmonary fibrosis, falling sickness, and neurodegenerative diseases. Constructing the net of oxidative stress response would help us understand the mechanisms of these diseases and reveal informations for drug design.Besides the thioredoxins (Trxs), a group of glutathione (GSH)-dependent oxidoreductases called glutaredoxins (Grxs), also function in responsing to oxidative stress and sustaining the redox homeostasis. Primary sequence alignment easily distinguishes most Grxs from Trxs. However, from the viewpoint of3-D structure, Grxs share a highly similar fold and topology with the Trx family, which is characterized by a central p-pleated sheet surrounded by five helices, and a CXX(C/S) active site motif. Grxs reduce disulfide bonds in substrate proteins using electrons from reduced glutathione. As previously reported, Grxs carry out a number of biological functions, including reduction of ascorbate, activation of ribonucleotide reductase and3’-phosphoadenylylsulfate reductase, and regulation of the DNA-binding capacity of nuclear factors. Recently, a variety of Grxs or Grx-like proteins were found that execute distinct functions, like signal transduction, and iron-sulfur (Fe-S) cluster assembly.The yeast Saccharomyces cerevisiae encodes multiple Grx isoforms, making it an ideal model for exploring the diversity of Grx subcellular localizations and molecular functions. To date, eight Grxs have been found in yeast, named Grx1to Grx8in chronological order of identification. The yeast Grx6is a monothiol Grx and localize in the Golgi compartments and endoplasmic reticulum. Grx6consists of three parts, a predicted signal peptide (Met1-Ile36), an N-terminal domain (Lys37-Thr110) and a C-terminal Grx domain (Lyslll-Asn231, designated Grx6C). Grx6has a lower glutathione-disulfide reductase (EC1.8.1.7) activity but a higher glutathione S-transferase (EC2.5.1.18) activity, in compare with the classic dithiol glutaredoxin Grxl. Moreover, Grx6binds GSH via an iron-sulfur cluster in vitro, like human Grx2. The N-terminal domain is necessary for noncovalent dimerization, but not contributes to either of the above activities. The crystal structure of Grx6C at1.5A resolution revealed a novel two-strand antiparallel β-sheet at the opposite of the GSH binding groove. The extra β-sheet probably also exist in some lower organisms Grxs and yeast Grx7. These informations enable us to define a new subfamily of Grxs. (II) Many kinds of approaches have been used for obtaining proteins that bind to cancer-specific (biomedically relevant) carbohydrates with high selectivity and affinity. Most often, monoclonal antibodies are isolated from mice immunized with the glycan of interest, but this process is laborious and most human glycans are not particularly immunogenic in mice, probably due to their conserved structures in these species, which gives rise to self-tolerance. Other strategies include directed evolution of antibodies or lectins, microarrays of carbohydrate-binding peptides, and systematic evolution of glycan-specific aptamers by exponential enrichment, but none have proven generally useful.We recently described a rapid and cost-effective strategy using yeast surface display for isolating monoclonal variable lymphocyte receptors (VLRs) from lamprey that selectively bind various human glycans, including the pancarcinoma antigens T-nouvelle (GalNaca) and Thomsen-Friedenreich (TFa; Galβ1-3GalNAca). VLRs are leucine-rich repeat (LRR) proteins that mediate adaptive immunity in jawless vertebrates (hagfish and lamprey). Although VLRs are structurally unrelated to the immunoglobulin-(Ig) based antibodies of jawed vertebrates, they are able to recognize as broad a spectrum of antigens as antibodies, with altogether comparable affinity and specificity. VLR genes are assembled from multiple LRR-encoding cassettes by DNA recombination in a process that generates a vast repertoire of more than101unique receptors, which is sufficiently diverse to recognize most, if not all, antigens.VLRs were shown to recognize, glycans, such as the tumor-associated Thomsen-Friedenreich antigen, with a selectivity rivaling equal to or beyond that of lectins and antibodies. To understand the basis for TFa recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters of binding interaction. And we determined the VLRB.aGPA.23-TFα complex structure to2.2A resolution. In the structure, a tight hydrophobic cage formed by4Trp-residues covers the TFa disaccharide. This cage, together with hydrogen bonds, elucidates the delicate glycan selectivity of VLRB.aGPA.23. Comapared to the topology of glycan-binding site of lectins and antibodies, which typically consist of long, flat grooves for the oligosaccharide, that of VLRB.aGPA.23is very different. Instead, the TFa disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the p-strands of the LRR modules. VLRs can utilize the LRR module to recognize glycans with an affinity equal to that of lectins and antibodies, and will be a very promising class of natural glycan-binding proteins for basis research and clinical applications. |
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