The complement system is an efficient anti-microbial effector mechanism. On the other hand abnormal complement activation is involved in the pathogenesis of multiple inflammatory and hemolytic diseases. As general inhibition of the complement system may jeopardize patient health due to increased susceptibility to infections, the development of pathway-specific complement therapeutics has been a long-lasting goal over the last decades. In particular, pathogen mimicry has been considered as a promising approach for the design of selective anti-complement drugs. The C-terminal domain of staphylococcal superantigen-like protein 7 (SSL7), a protein secreted by Staphylococcus aureus, was recently found to be a specific inhibitor of the terminal pathway of the complement system, providing selective inhibition of cell lysis mediated by the membrane attack complex (MAC). We describe here the selection by phage display of a humanized single-domain antibody (sdAb) mimicking the C-terminal domain of SSL7. The antibody, called sdAb_E4, binds complement C5 with an affinity in the low micromolar range. Furthermore, sdAb_E4 induces selective inhibition of MAC-mediated lysis, allowing inhibition of red blood cell hemolysis and inhibition of complement deposition on apopto-necrotic cells, while maintaining efficient bactericidal activity of the complement terminal pathway. Finally, we present preliminary results indicating that sdAb_E4 may also be efficient in inhibiting hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. Our data provide a proof of concept for the design of a selective MAC inhibitor capable of retaining complement bacteriolytic activity and this study opens up promising perspectives for the development of an sdAb_E4-derived therapeutics with application in the treatment of complement-mediated hemolytic disorders.

Complement is an ancient part of the innate immune system that plays a pivotal role in protection against invading pathogens and helps to clear apoptotic and necrotic cells. Upon complement activation, a cascade of proteolytic events generates the complement effectors, including the anaphylatoxins C3a and C5a. Signalling through their cognate G-protein coupled receptors, C3aR and C5aR, leads to a wide range of biological events promoting inflammation at the site of complement activation. The function of anaphylatoxins is regulated by circulating carboxypeptidases that remove their C-terminal arginine residue, yielding C3a-desArg and C5a-desArg. Whereas human C3a and C3a-desArg adopt a canonical four-helix bundle fold, the conformation of human C5a-desArg has recently been described as a three-helix bundle. Here, the crystal structures of an antagonist version of human C5a, A8(Δ71-73), and of murine C5a and C5a-desArg are reported. Whereas A8(Δ71-73) adopts a three-helix bundle conformation similar to human C5a-desArg, the two murine proteins form a four-helix bundle. A cell-based functional assay reveals that murine C5a-desArg, in contrast to its human counterpart, exerts the same level of activition as murine C5a on its cognate receptor. The role of the different C5a conformations is discussed in relation to the differential activation of C5a receptors across species.

L-Oligonucleotide aptamers (Spiegelmers) consist of non-natural L-configured nucleotides and are of particular therapeutic interest due to their high resistance to plasma nucleases. The anaphylatoxin C5a, a potent inflammatory mediator generated during complement activation that has been implicated with organ damage, can be efficiently targeted by Spiegelmers. Here, we present the first crystallographic structures of an active Spiegelmer, NOX-D20, bound to its physiological targets, mouse C5a and C5a-desArg. The structures reveal a complex 3D architecture for the L-aptamer that wraps around C5a, including an intramolecular G-quadruplex stabilized by a central Ca(2+) ion. Functional validation of the observed L-aptamer:C5a binding mode through mutational studies also rationalizes the specificity of NOX-D20 for mouse and human C5a against macaque and rat C5a. Finally, our structural model provides the molecular basis for the Spiegelmer affinity improvement through positional L-ribonucleotide to L-deoxyribonucleotide exchanges and for its inhibition of the C5a:C5aR interaction.

In humans, several members of the CEACAM receptor family have been shown to interact with intestinal pathogens in an inflammatory context. While CEACAMs have long been thought to be only present in mammals, recent studies have identified ceacam genes in other vertebrates, including teleosts. The function of these related genes remains however largely unknown. To gain insight into the function of CEACAM proteins in fish, we undertook the study of a putative member of the family, CEACAMz1, identified in Danio rerio. Sequence analysis of the ceacamz1 gene product predicted a GPI-anchored extracellular protein containing eleven immunoglobulin domains but revealed no evident orthology with human CEACAMs. Using a combination of RT-PCR analyses and in situ hybridization experiments, as well as a fluorescent reporter line, we showed that CEACAMz1 is first expressed in discrete cells on the ventral skin of zebrafish larvae and later on in the developing gills. This distribution remains constant until juvenile stage is reached, at which point CEACAMz1 is almost exclusively expressed in gills. We further observed that at late larval stages, CEACAMz1-expressing cells mostly localize on the afferent side of the branchial filaments and possibly in the inter-lamellar space. Using immunolabelling and 3D-reconstructions, we showed that CEACAMz1 is expressed in cells from the uppermost layer of skin epidermis. These cells are embedded within the keratinocytes pavement and we unambiguously identified them as proton-pump rich ionocytes (HR cells). As the expression of ceacamz1 is turned on concomitantly to that of other known markers of HR cells, we propose that ceacamz1 may serve as a novel marker of mature HR cells from the zebrafish epidermis.

S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.

Receptor for activated C-kinase 1 (RACK1) serves as a scaffolding protein in numerous signaling pathways involving kinases and membrane-bound receptors from different cellular compartments. It exists simultaneously as a cytosolic free form and as a ribosome-bound protein. As part of the 40S ribosomal subunit, it triggers translational regulation by establishing a direct link between protein kinase C and the protein synthesis machinery. It has been suggested that RACK1 could recruit other signaling molecules onto the ribosome, providing a signal-specific modulation of the translational process. RACK1 is able to dimerize both in vitro and in vivo. This homodimer formation has been observed in several processes including the regulation of the N-methyl-d-aspartate receptor by the Fyn kinase in the brain and the oxygen-independent degradation of hypoxia-inducible factor 1. The functional relevance of this dimerization is, however, still unclear and the question of a possible dimerization of the ribosome-bound protein is still pending. Here, we report the first structure of a RACK1 homodimer, as determined from two independent crystal forms of the Saccharomyces cerevisiae RACK1 protein (also known as Asc1p) at 2.9 and 3.9 Å resolution. The structure reveals an atypical mode of dimerization where monomers intertwine on blade 4, thus exposing a novel surface of the protein to potential interacting partners. We discuss the significance of the dimer structure for RACK1 function.