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abstracts
Toll-like receptors (TLRs) sense pathogen-associated molecular patterns originating from invading microorganism and evoke innate immune responses. Among TLRs, TLR3, TLR7, TLR8, and TLR9 are localized to endosomal membranes and are responsible for the recognition of nucleic acids. TLR7 and TLR8 recognize single stranded RNA. In addition, TLR7 and TLR8 are activated by small chemical compounds. TLR9 recognizes DNA containing Cytosine-phosphate-Guanine motif. Theses nucleic acid sensing TLRs are attractive therapeutic targets for the modulation of immune responses in the viral and bacterial infections and in the pathogenesis of autoimmune diseases. However, the structural basis for the nucleic acid recognition and signaling mechanisms remains to be elucidated. Therefore, we conducted crystallographic studies of these TLRs. Recently, we have determined the crystal structures of the extracellular domain of human TLR8 in the unliganded form and in the liganded forms with chemical ligands (Tanji et al., 2013). Both unliganded and liganded forms of TLR8 were dimer. Ligands were located at two equivalent positions in the dimerization interface. The ligand binding induced the reorganization of the preformed dimer to the activated dimer such that the C-terminal regions of the two protomers are in close proximity to enable the subsequent dimerization of the intracellular signaling domains and its interactions with adaptor proteins.
Keywords: Innate immunity.
abstracts
GM1-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-Galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation (ERAD) due to mutations in the β-Gal gene. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding, prevent ERAD and promote trafficking to the lysosome. Here, we present the enzymological properties of wild-type human β-Gal and two representative mutations in GM1 gangliosidosis Japanese patients (R201C and I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we determined the crystal structures of β-Gal in complex with these compouds and two structurally related analogues to elucidate the detailed atomic view of the recognition mechanism. All compounds bind to the active site of β-Gal with the sugar moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity and the PC potential are strongly affected by the mono or bicyclic structure of the core as well as the orientation, the nature and the length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds.
