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The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks., RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/M011194/1 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 721613 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 659029 EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 695669

Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH), Funder: U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID), Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

Funder: Cambridge Commonwealth, European and International Trust (Cambridge Commonwealth, European & International Trust); doi: https://doi.org/10.13039/501100003343, Funder: The Nehru Trust for Cambridge University, Funder: H.F. and K.S. were supported in part by the Innovative Drug Discovery Infrastructure through the Functional Control of Biomolecular Systems Priority Issue1 in Post-K Supercomputer Development from MEXT (Project ID: hp190171, hp170255, hp180191, and hp150269. K.S. was also supported in part by JSPS KAKENHI Grant Number JP20H05453., Funder: H.F. and K.S. were supported in part by the Innovative Drug Discovery Infrastructure through the Functional Control of Biomolecular Systems Priority Issue1 in Post-K Supercomputer Development from MEXT (Project ID: hp190171, hp170255, hp180191, and hp150269)., Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.

Tumors consist of cancer cells and a network of non-cancerous stroma. Cancer-associated fibroblasts (CAF) are known to support tumorigenesis, and are emerging as immune modulators. Neutrophils release histone-bound nuclear DNA and cytotoxic granules as extracellular traps (NET). Here we show that CAFs induce NET formation within the tumor and systemically in the blood and bone marrow. These tumor-induced NETs (t-NETs) are driven by a ROS-mediated pathway dependent on CAF-derived Amyloid β, a peptide implicated in both neurodegenerative and inflammatory disorders. Inhibition of NETosis in murine tumors skews neutrophils to an anti-tumor phenotype, preventing tumor growth; reciprocally, t-NETs enhance CAF activation. Mirroring observations in mice, CAFs are detected juxtaposed to NETs in human melanoma and pancreatic adenocarcinoma, and show elevated amyloid and β-Secretase expression which correlates with poor prognosis. In summary, we report that CAFs drive NETosis to support cancer progression, identifying Amyloid β as the protagonist and potential therapeutic target., The tumor microenvironment is composed of many cell types that crosstalk to modulate local immunity. Here the authors show that Amyloid β proteins from cancer-associated fibroblasts (CAF) induce neutrophil extracellular trap (NET) production by neutrophils, while NET feeds back to activate CAF, thereby implicating Amyloid β as a potential therapy target.

Funder: Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation); doi: https://doi.org/10.13039/501100004063, Funder: Alzheimerfonden; doi: https://doi.org/10.13039/501100008599, Alzheimer’s disease is linked to amyloid β (Aβ) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aβ aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aβ42 in human CSF through kinetic experiments at several Aβ42 monomer concentrations (0.8–10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.