Background: Ubiquitin (E3) ligases interact with specific ubiquitin conjugating (E2) enzymes to ubiquitinate particular substrate proteins. As the combination of E2 and E3 dictates the type and biological consequence of ubiquitination, it is important to understand the basis of specificity in E2:E3 interactions. The E3 ligase CHIP interacts with Hsp70 and Hsp90 and ubiquitinates client proteins that are chaperoned by these heat shock proteins. CHIP interacts with two types of E2 enzymes, UbcH5 and Ubc13-Uev1a. It is unclear, however, why CHIP binds these E2 enzymes rather than others, and whether CHIP interacts preferentially with UbcH5 or Ubc13-Uev1a, which form different types of polyubiquitin chains.

The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.

The Crystal Diagnostics MultiPath System™ provides rapid detection of Escherichia coli O157 in fresh raw ground beef, raw beeftrim, and spinach. The Crystal Diagnostics system combines patented Liquid Crystal technology with antibody-coated paramagnetic microspheres to selectively capture and detect E. coli O157 in food matrixes. This is the only liquid crystal-based biosensor commercially available for the detection of pathogens. The Crystal Diagnostics system expeditiously provides the sensitivity and accuracy of the U.S. Department of Agriculture Food Safety Inspection Service (USDA-FSIS) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) methods for detecting as low as one CFU of E. coli O157 per 375 g of raw ground beef and raw beef trim, or 200 g of raw spinach. An internal inclusivity validation demonstrated detection of all 50 tested strains of . coli O157. The internal and independent laboratory tests demonstrate that the method is rapid and sensitive for detecting of E. coli O157 in fresh raw ground beef, beef trim, and spinach.