Extracellular Histones Inhibit Complement Activation through Interacting with Complement Component 4.

Complement activation leads to membrane attack complex formation, which can lyse not only pathogens but also host cells. Histones can be released from the lysed or damaged cells and serve as a major type of damage-associated molecular pattern, but their effects on the complement system are not clear. In this study, we pulled down two major proteins from human serum using histone-conjugated beads: one was C-reactive protein and the other was C4, as identified by mass spectrometry. In surface plasmon resonance analysis, histone H3 and H4 showed stronger binding to C4 than other histones, with KD around 1 nM. The interaction did not affect C4 cleavage to C4a and C4b. Because histones bind to C4b, a component of C3 and C5 convertases, their activities were significantly inhibited in the presence of histones. Although it is not clear whether the inhibition was achieved through blocking C3 and C5 convertase assembly or just through reducing their activity, the outcome was that both classical and mannose-binding lectin pathways were dramatically inhibited. Using a high concentration of C4 protein, histone-suppressed complement activity could not be fully restored, indicating C4 is not the only target of histones in those pathways. In contrast, the alternative pathway was almost spared, but the overall complement activity activated by zymosan was inhibited by histones. Therefore, we believe that histones inhibiting complement activation is a natural feedback mechanism to prevent the excessive injury of host cells.

Structural Basis for Selective Interaction between the ESCRT Regulator HD-PTP and UBAP1.

Endosomal sorting complexes required for transport (ESCRTs) are essential for ubiquitin-dependent degradation of mitogenic receptors, a process often compromised in cancer pathologies. Sorting of ubiquinated receptors via ESCRTs is controlled by the tumor suppressor phosphatase HD-PTP. The specific interaction between HD-PTP and the ESCRT-I subunit UBAP1 is critical for degradation of growth factor receptors and integrins. Here, we present the structural characterization by X-ray crystallography and double electron-electron resonance spectroscopy of the coiled-coil domain of HD-PTP and its complex with UBAP1. The coiled-coil domain adopts an unexpected open and rigid conformation that contrasts with the closed and flexible coiled-coil domain of the related ESCRT regulator Alix. The HD-PTP:UBAP1 structure identifies the molecular determinants of the interaction and provides a molecular basis for the specific functional cooperation between HD-PTP and UBAP1. Our findings provide insights into the molecular mechanisms of regulation of ESCRT pathways that could be relevant to anticancer therapies.

Solid phase assays for studying ECM protein-protein interactions.

Solid-phase assays provide a simple, rapid and robust method for the analysis of protein-protein interactions; i.e., does protein A interacts with protein B? In this assay, protein A (here termed as 'receptor') is adsorbed to the wells of an enzyme-linked immunosorbent assay (ELISA) plate (solid phase). The plate is then blocked using bovine serum albumin (BSA), and biotin-labelled protein B (here termed as 'ligand') is added. After washing the wells to remove unbound ligand, bound ligand is detected by addition of an avidin-peroxidase conjugate followed by a colorimetric detection step. This type of assay is particularly well suited for studying the interaction of ECM proteins with integrins. The screening of antagonists of integrin-ligand interactions in the pharmaceutical industry is an important area in which this assay is finding use.