Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function.

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis.

Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptor endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. Here, we show that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. We demonstrate further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. Based on these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.

The nonaspanins TM9SF2 and TM9SF4 regulate the plasma membrane localization and signalling activity of the peptidoglycan recognition protein PGRP-LC in Drosophila.

Transmembrane 9 (TM9) proteins, or nonaspanins, are a family of proteins conserved throughout evolution and characterized by 9 transmembrane domains. In Drosophila, TM9 superfamily protein member 4 (TM9SF4) and its closest paralogue, TM9SF2, contribute to phagocytosis of various types of particles, while TM9SF4 displays non-redundant requirement in Gram-negative bacteria engulfment. In addition, the two TM9 proteins control the actin cytoskeleton in larval haemocytes and in Drosophila S2 cells. Here, we show that TM9SF4 and TM9SF2 co-immunoprecipitate with the peptidoglycan recognition protein (PGRP)-LC, which triggers the Drosophila immune response to bacterial infection. Furthermore, both TM9 proteins co-localize with this receptor in intracellular vesicles and at the plasma membrane in Drosophila S2 cells in culture and in the fly fat body. Silencing TM9SF4 prevents plasma membrane localization of PGRP-LC, whereas silencing TM9SF2 does not, which may account for the non-redundant role of TM9SF4 in phagocytosis of Gram-negative bacteria. Finally, we provide a set of data suggesting that TM9 proteins can prevent inappropriate signalling from the unstimulated receptor.

Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome.

BACKGROUND: Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created and screened a focused RNA interference library on Drosophila NF-κB-like pathways Toll and Imd in cultured S2 cells, and further analysed the function of selected genes in vivo. RESULTS: We report here that USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NF-κB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. We further show that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. CONCLUSION: Our work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis.