Protein modification by ubiquitin and ubiquitin-like modifiers (Ubls) is counteracted by ubiquitin- and Ubl-proteases collectively called DUBs. In contrast to other proteases of the ubiquitin-specific protease (USP) family, USP18 shows no reactivity towards ubiquitin but specifically deconjugates the interferon induced Ubl ISG15. To identify molecular determinants for this specificity, we solved the crystal structures of mouse USP18 and of mouse USP18 in complex with mouse ISG15. USP18 was crystallized in an open and a closed conformation revealing high flexibility of the enzyme. Structural data, biochemical and mutational analysis showed that only the C-terminal ubiquitin-like domain of ISG15 is recognized and essential for USP18 activity. A critical hydrophobic patch in USP18 interacts with a hydrophobic region unique to ISG15 providing evidence that ISG15 specificity of USP18 is mediated by a small interaction interface. Our results may provide the structural basis for the development of new drugs modulating ISGylation.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A–C2G, 13%–33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a ‘calcium-catching’ mechanism to respond to calcium influx during membrane repair.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phosphoubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.

1969, Factors affecting the yield and biological activity of lipid extracts of Listeria monocytoget~es. Can. J . Microbiol. 15: 421-428.The biological activity of lipid extracts of five strains of Listerin rnonocytogenes was studied, with special reference to their monocytosis-producing activity in mice. Two of the strains gave more active extracts than the others, and cells grown at 4 C produced more lipid with better activity than cells grown at room temperature or at 37 C. Extracts obtained from cells harvested at the various stages of log phase at 4 C all gave similar levels of monocytosis in mice. The level of monocyte response varied from animal to animal, but in general, older mice (28 to 48 weeks) responded better than young mice (9 to 15 weeks) and females gave a better response than males. These extracts also produced leucopenia in young mice, but this was less consistent and leucocytosis was more often observed in older mice. Granulocytosis and lymphopenia were commonly found in mice injected with lipid extracts of L . monocytogenes. These changes were affected by the strain of bacteria but not by the age and sex of the test animals. The changes in blood picture normally reached a maximum 1 to 2 days after injection and returned to normal after about 5 days.

Lipid extracts of Listeria monocytogenes, capable of producing monocytosis and lymphopenia in mice, were fractionated by column chromatography on acid-treated Florisil. The biological activity was associated with a phospholipid fraction and further separation of this fraction by thin-layer chromatography indicated that most of the activity was in the slower running components which gave a positive reaction with ninhydrin. Water-soluble ninhydrin-positive material was separated from either the crude lipids or the phospholipid fractions by techniques such as a Folch wash, chromatography on Sephadex, or dialysis of a chloroform solution of the lipids against water. These water-soluble materials were also able to produce monocytosis and lymphopenia in mice, but the remaining phospholipid was still ninhydrin-positive and biologically active. Most of the water-soluble material was dialyzable, but the biological activity appeared to be concentrated largely in the non-dialyzable fraction. This fraction contained protein, and digestion with Pronase appeared to enhance the biological activity and to make the active material more readily dialyzable. Extraction of the lipid-extracted bacterial residue with saline yielded additional non-dialyzable water-soluble material with activity comparable to that shown by the lipid extracts.