C1q has been shown to recognize apoptotic cells, to enhance their uptake and to modulate cytokine release by phagocytes and thus promote immune tolerance. Surface-exposed calreticulin (CRT), known as a C1q receptor, is also considered to be an early eat-me signal that enhances the phagocytosis of apoptotic cells and is capable of eliciting an immunogenic response. However, the molecular mechanisms that trigger these functions are not clear. We hypothesized that CRT and C1q might act together in these processes. We first showed, by means of fluorescence resonance energy transfer (FRET), that CRT interacts with the C1q globular region at the surface of early apoptotic cells. Next, we pointed out that knockdown of CRT on early apoptotic HeLa cells impairs the enhancement effect of C1q on their uptake by THP-1 monocyte-derived macrophages. Furthermore, a deficiency of CRT induces contrasting effects on cytokine release by THP-1 macrophages, increasing interleukin (IL)-6 and monocyte chemotactic protein 1/CCL2 and decreasing IL-8. Remarkably, these effects were greatly reduced when apoptotic cells were opsonized by C1q, which counterbalanced the effect of the CRT deficiency. These results demonstrate that CRT-C1q interaction is involved in the C1q bridging function and they highlight the particular ability of C1q to control the phagocyte inflammatory status, i.e. by integrating the molecular changes that could occur at the surface of dying cells.

The cell-free synthesis is an efficient strategy to produce in large scale protein samples for structural investigations. In vitro synthesis allows significant reduction of production time, simplification of purification steps and enables production of both soluble and membrane proteins.The cell-free reaction is an open system and can be performed in presence of many additives such as cofactors, inhibitors, redox systems, chaperones, detergents, lipids, nanodiscs and surfactants to allow the expression of toxic, membrane or intrinsically disordered proteins. In this chapter we present protocols to prepare E. coli S30 cellular extracts, T7 RNA polymerase and their use for in vitro protein expression. Optimizations of the protocol are presented for preparation of protein samples enriched in deuterium, a prerequisite for the study of high molecular weight proteins by NMR spectroscopy. An efficient production of perdeuterated proteins is achieved together with a full protonation of all the amide NMR probes, without suffering from residual protonation on aliphatic carbons. Application to the production of the 468 kDa TET2 protein assembly for NMR investigations is presented.

Prefoldin is a heterohexameric protein assembly which acts as a co-chaperonin for the well conserved Hsp60 chaperonin, present in archaebacteria and the eukaryotic cell cytosol. Prefoldin is a holdase, capturing client proteins and subsequently transferring them to the Hsp60 chamber for refolding. The chaperonin family is implicated in the early stages of protein folding and plays an important role in proteostasis in the cytosol. Here, we report the assignment of 1 H N , 15 N, 13 C′, 13 C α , 13 C β , 1 H methyl , and 13 C methyl chemical shifts of the 87 kDa prefoldin from the hyperthermophilic archaeon Pyrococcus horikoshii, consisting of two α and four β subunits. 100% of the [ 13 C, 1 H]resonances of A b , I d1 , L d2 , T g2 , V g2 methyl groups were successfully assigned for both subunits.For the β-subunit, showing partial peak doubling, 80% of the backbone resonances were assigned.In the α subunit, large stretches of backbone resonances were not detectable due to slow (µs-ms) time scale dynamics. This conformational exchange limited the backbone sequential assignment of the α subunit to 57% of residues, which corresponds to 84% of visible NMR signals.