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Capillary morphogenesis gene 2 (CMG2/ANTXR2) is a cell surface receptor for both collagen VI and anthrax toxin. Biallelic loss-of-function mutations in CMG2 lead to a severe condition, hyaline fibromatosis syndrome (HFS). We have here dissected a network of dynamic interactions between CMG2 and various actin interactors and regulators, describing a different behavior from other extracellular matrix receptors. CMG2 binds talin, and thereby the actin cytoskeleton, only in its ligand-free state. Extracellular ligand binding leads to src-dependent talin release and recruitment of the actin cytoskeleton regulator RhoA and its effectors. These sequential interactions of CMG2 are necessary for the control of oriented cell division during fish development. Finally, we demonstrate that effective switching between talin and RhoA binding is required for the intracellular degradation of collagen VI in human fibroblasts, which explains why HFS mutations in the cytoskeleton-binding domain lead to dysregulation of extracellular matrix homeostasis.

A third of the human genome encodes N-glycosylated proteins. These are co-translationally translocated into the lumen/membrane of the endoplasmic reticulum (ER) where they fold and assemble before they are transported to their final destination. Here, we show that calnexin, a major ER chaperone involved in glycoprotein folding is palmitoylated and that this modification is mediated by the ER palmitoyltransferase DHHC6. This modification leads to the preferential localization of calnexin to the perinuclear rough ER, at the expense of ER tubules. Moreover, palmitoylation mediates the association of calnexin with the ribosome-translocon complex (RTC) leading to the formation of a supercomplex that recruits the actin cytoskeleton, leading to further stabilization of the assembly. When formation of the calnexin-RTC supercomplex was affected by DHHC6 silencing, mutation of calnexin palmitoylation sites or actin depolymerization, folding of glycoproteins was impaired. Our findings thus show that calnexin is a stable component of the RTC in a manner that is exquisitely dependent on its palmitoylation status. This association is essential for the chaperone to capture its client proteins as they emerge from the translocon, acquire their N-linked glycans and initiate folding.

Hyaline Fibromatosis Syndrome (HIS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized lg-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the lg-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.

Some cell-surface receptors activate downstream signal transduction pathways not only from the cell surface but also from endosomes, suggesting that signalling pathways can be regulated by compartmentalization. A further twist is that different internalization routes seem to predetermine whether transforming growth factor β (TGF-β) receptors will trigger a signalling response or be degraded.

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ß-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis., Author Summary Insects are potential reservoirs for microbes and ideal vectors for their transmission due to their motility and capacity to live in bacteria-rich environments. This is exemplified by fruit flies that live in rotting fruits and are capable of transmitting phytopathogenic bacteria. Insects are notably resistant to microbial infection allowing them to colonize these microbe-rich environments. To study how pathogenic bacteria disrupt gut homeostasis, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Ingestion of P. entomophila inflicts severe damage to the Drosophila intestine. How damages are inflicted, however, remains unknown. In this study, we identified a secreted protein that plays an important role in the damage inflicted by P. entomophila to the Drosophila gut. We showed that this protein is a pore-forming toxin (PFT) that we named Monalysin. Our study reveals that Monalysin oligomerizes into ring-like structures that form pores into the plasma membrane of target cells leading to the disruption of membrane permeability and cell death. Our work together with studies on the insecticidal Cry toxins produced by Bacillus thuringiensis suggests that production of PFTs is a common strategy of entomopathogenic bacteria to interfere with insect gut homeostasis.