Endolysins, the cell wall lytic enzymes encoded by bacteriophages to release the phage progeny, are among the top alternatives to fight against multiresistant pathogenic bacteria; one of the current biggest challenges to global health. Their narrow range of susceptible bacteria relies, primarily, on targeting specific cell-wall receptors through specialized modules. The cell wall-binding domain of Cpl-7 endolysin, made of three CW_7 repeats, accounts for its extended-range of substrates. Using as model system the cell wall-binding domain of Cpl-7, here we describe the molecular basis for the bacterial cell wall recognition by the CW_7 motif, which is widely represented in sequences of cell wall hydrolases. We report the crystal and solution structure of the full-length domain, identify N-acetyl-D-glucosaminyl-(β1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW_7 motifs, and characterize feasible GMDP-CW_7 contacts. Our data suggest that Cpl-7 cell wall-binding domain might simultaneously bind to three PG chains, and also highlight the potential use of CW_7-containing lysins as novel anti-infectives.

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.

Background: The fundamental process of protein secretion from eukaryotic cells has been well described for many years, yet gaps in our understanding of how this process is regulated remain. Methods: With the aim of identifying novel genes involved in the secretion of glycoproteins, we used a screening pipeline consisting of a pooled genome-wide CRISPR screen, followed by secondary siRNA screening of the hits to identify and validate several novel regulators of protein secretion. Results: We present approximately 50 novel genes not previously associated with protein secretion, many of which also had an effect on the structure of the Golgi apparatus. We further studied a small selection of hits to investigate their subcellular localisation. One of these, GPR161, is a novel Golgi-resident protein that we propose maintains Golgi structure via an interaction with golgin A5. Conclusions: This study has identified new factors for protein secretion involved in Golgi homeostasis.