Binding and cleavage of E. coli HUbeta by the E. coli Lon protease.

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUbeta, but not the related protein HUalpha. Here we show that the Lon protease binds to both HUbeta and HUalpha, but selectively degrades only HUbeta in the presence of ATP. Mass spectrometry of HUbeta peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUbeta-A20Q and HUbeta-A20D more slowly than HUbeta. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUalpha, HUbeta, and HUbeta-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUbeta, HUalpha, or HUbeta-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUbeta.

The interaction of lipopolysaccharide with membrane receptors on macrophages pretreated with extract of Reishi polysaccharides measured by optical tweezers.

Lipopolysaccharide (LPS), one of the cell wall components of Gram-negative bacteria, is recognized by and interacted with receptors on macrophages. In this paper, we report the trapping of LPS-coated polystyrene particles via optical tweezers and measured its interaction with murine macrophages (J774A.1 cells) for cells pre-treated with extract of Reishi polysaccharides (EORP) vs. those without EORP treatment. Our experimental results indicate that the cellular affinity for LPS increases when the macrophage is pretreated with EORP. We demonstrate for the first time by conventional biological methods and by tracking the dynamics of optically-trapped LPS-coated particles interacting with J774A.1 cells, that EORP not only enhances J774A.1 cells surface expression of TLR4 and CD14, two receptors on macrophages, as well as LPS binding and phagocytosis internalization, but also reduces the adhesion time constant and increases the force constant of the binding interaction. The application of optical tweezers allows us to study the effect on a single cell quantitatively in real-time with a spatial resolution ~ 1 mum within a single cell.