Biochemical and immunological detection of physical interactions between penta-EF-hand protein ALG-2 and its binding partners.

Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or -macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca(2+)-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using biotinylated ALG-2.

Identification of the P-body component PATL1 as a novel ALG-2-interacting protein by in silico and far-Western screening of proline-rich proteins.

ALG-2 (also named PDCD6) is a 22-kDa Ca(2+)-binding protein that belongs to the penta-EF-hand family including calpain small subunit and interacts with various proteins such as ALIX and Sec31A at their specific sites containing an ALG-2-binding motif (ABM) present in their respective Pro-rich region (PRR). In this study, to search for novel ALG-2-interacting proteins, we first performed in silico screening of ABM-containing PRRs in a human protein database. After selecting 17 sequences, we expressed the PRR or full-length proteins fused with green fluorescent protein (GFP) in HEK293T cells and analysed their abilities to bind to ALG-2 by Far-Western blotting using biotinylated ALG-2 as a probe.

Hepatitis C virus hijacks P-body and stress granule components around lipid droplets.

The microRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of the microRNA effectors DDX6, Lsm1, Xrn1, PATL1, and Ago2, but not the decapping enzyme DCP2, and dynamically redistributed these microRNA effectors to the HCV production factory around lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed the stress granule components GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), ataxin-2 (ATX2), and poly(A)-binding protein 1 (PABP1) to the HCV production factory.