LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA.

The DExD/H box RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (mda-5) sense viral RNA in the cytoplasm of infected cells and activate signal transduction pathways that trigger the production of type I interferons (IFNs). Laboratory of genetics and physiology 2 (LGP2) is thought to influence IFN production by regulating the activity of RIG-I and mda-5, although its mechanism of action is not known and its function is controversial. Here we show that expression of LGP2 potentiates IFN induction by polyinosinic-polycytidylic acid [poly(I:C)], commonly used as a synthetic mimic of viral dsRNA, and that this is particularly significant at limited levels of the inducer. The observed enhancement is mediated through co-operation with mda-5, which depends upon LGP2 for maximal activation in response to poly(I:C). This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with mda-5. In contrast, although RIG-I can also be activated by poly(I:C), LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signaling. Thus the level of LGP2 expression is a critical factor in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low

High-Speed Force Spectroscopy for Single Protein Unfolding

International audienceSingle-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain

SAP97 Controls the Trafficking and Resensitization of the Beta-1-Adrenergic Receptor through Its PDZ2 and I3 Domains

Previous studies have determined that the type-1 PDZ sequence at the extreme carboxy-terminus of the ß(1)-adrenergic receptor (ß(1)-AR) binds SAP97 and AKAP79 to organize a scaffold involved in trafficking of the ß(1)-AR. In this study we focused on characterizing the domains in SAP97 that were involved in recycling and resensitization of the ß(1)-AR in HEK-293 cells. Using a SAP97 knockdown and rescue strategy, we determined that PDZ-deletion mutants of SAP97 containing PDZ2 rescued the recycling and resensitization of the ß(1)-AR. Among the three PDZs of SAP97, PDZ2 displayed the highest affinity in binding to the ß(1)-AR. Expression of isolated PDZ2, but not the other PDZs, inhibited the recycling of the ß(1)-AR by destabilizing the macromolecular complex involved in trafficking and functional resensitization of the ß(1)-AR. In addition to its PDZs, SAP97 contains other protein interacting domains, such as the I3 sequence in the SRC homology-3 (SH3) domain, which binds to AKAP79. Deletion of I3 from SAP97 (ΔI3-SAP97) did not affect the binding of SAP97 to the ß(1)-AR. However, ΔI3-SAP97 could not rescue the recycling of the ß(1)-AR because it failed to incorporate AKAP79/PKA into the SAP97-ß(1)-AR complex. Therefore, bipartite binding of SAP97 to the ß(1)-AR and to AKAP79 is necessary for SAP97-mediated effects on recycling, externalization and functional resensitization of the ß(1)-AR. These data establish a prominent role for PDZ2 and I3 domains of SAP97 in organizing the ß(1)-adrenergic receptosome involved in connecting the ß(1)-AR to trafficking and signaling networks