Different functional role of domain boundaries of Toll-like receptor 4.

Toll-like receptors (TLRs) recognize molecules representing danger signals via their ectodomain, while signal transduction is provided by the cytosolic TIR domain that recruits adapter proteins upon dimerization. Since in crystal structures both domains dimerize as rigid bodies, any structural adjustment must be provided by the intermediate segments between the domains. We investigated domain coupling by inserting flexible linkers between the structural domains of TLR4. Insertion of linkers between the transmembrane and cytosolic TIR domain did not affect activation, indicating that TIR domain dimerization is triggered by proximity. In contrast, insertion of a linker between the transmembrane and ectodomain or within the ectodomain decreased activation proportionally with the length of the linker. This suggests the requirement for tight coupling of the ectodomain to the membrane, which may facilitate its interaction with ligand, promote dimerization and prevent interaction with the cell-membrane surface. Native linker sizes of TLR4 orthologs support these conclusions.

Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases.

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.