Genome-scale identification of cellular pathways required for cell surface recognition.

Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABAB receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABAB receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties. © 2018 Sharma et al.; Published by Cold Spring Harbor Laboratory Press

A PfRH5-Based Vaccine Is Efficacious against Heterologous Strain Blood-Stage Plasmodium falciparum Infection in Aotus Monkeys

SummaryAntigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans

The SHOCT domain: a widespread domain under-represented in model organisms.

We have identified a new protein domain, which we have named the SHOCT domain (Short C-terminal domain). This domain is widespread in bacteria with over a thousand examples. But we found it is missing from the most commonly studied model organisms, despite being present in closely related species. It's predominantly C-terminal location, co-occurrence with numerous other domains and short size is reminiscent of the Gram-positive anchor motif, however it is present in a much wider range of species. We suggest several hypotheses about the function of SHOCT, including oligomerisation and nucleic acid binding. Our initial experiments do not support its role as an oligomerisation domain

Domain architectures of selected proteins containing the SHOCT domain.

<p>Panel a shows proteins which are likely to be oligomeric, panel b shows enzymes and panel c shows binding proteins. Signal peptide and transmembrane domains are predicted using the Phobius web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057848#pone.0057848-Kall1" target="_blank">[46]</a>.</p

The SHOCT peptide does not multimerise the rat Cd4d3+4 protein.

<p>(<b>A</b>) Tissue culture supernatants containing biotinylated Cd4d3+4-F0QBY7.1 and Cd4d3+4-F0QBY7.1_shuffled were resolved by SDS-PAGE under reducing conditions, blotted and detected using streptavidin-HRP. (<b>B</b>) Purified Cd4d3+4-F0QBY7.1 and Cd4d3+4-F0QBY7.1_shuffled were resolved on a Superdex 2000 Tricorn 10/600 column. The elution volumes of protein standards are marked in red. The expected monomer size for Cd4d3+4-peptide is 33 kDa. (<b>C</b>) Purified Cd4d3+4-F0QBY7.1 and Cd4d3+4-F0QBY7.1_shuffled were resolved by SDS-PAGE under non-reducing conditions before (Ni) and after size exclusion chromatography (SE) and detected Coomassie Brilliant Blue R-250 staining.</p

Three-dimensional structure prediction of the SHOCT domain of UniProtKB B0PET9.1 (residues 20–50) generated with QUARK using the default parameters[<b>12</b>] and viewed using MarkUs [<b>44</b>].

<p>Surface electrostatic potential for the model is calculated using the program GRASP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057848#pone.0057848-Petrey2" target="_blank">[45]</a> accessed through MarkUs. The positively charged areas of the protein surface are shown in blue, and negatively charged areas in red, the two alpha helices are overlaid in grey.</p

Histogram showing the bit scores distribution of the SHOCT domain HMM compared to a reversed version of the SHOCT domain HMM, searched against the UniProtKB database.

<p>The UniProtKB database was searched using an HMM constructed from the SHOCT seed alignment (unfilled bars) and an HMM from the reversed version of this alignment (green bars). The vertical line represents the sequence inclusion threshold.</p

A Library of <i>Plasmodium vivax</i> Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

<div><p>Background</p><p>A vaccine targeting <i>Plasmodium vivax</i> will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of <i>P</i>. <i>vivax</i> biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between <i>P</i>. <i>vivax</i> Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential <i>P</i>. <i>vivax</i> merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of <i>Plasmodium</i> proteins in a functionally-active form in mammalian cells and initiated a large-scale study of <i>P</i>. <i>vivax</i> merozoite proteins that are potentially involved in reticulocyte binding and invasion.</p><p>Methodology/Principal Findings</p><p>We selected 39 <i>P</i>. <i>vivax</i> proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to <i>P</i>. <i>falciparum</i> vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express <i>P</i>. <i>falciparum</i> invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between <i>P</i>. <i>vivax</i> 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.</p><p>Conclusions/Significance</p><p>We produced a new library of recombinant full-length <i>P</i>. <i>vivax</i> ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying <i>P</i>. <i>vivax</i> proteins with vaccine potential, and studying <i>P</i>. <i>vivax</i> malaria pathogenesis and immunity.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT00663546" target="_blank">NCT00663546</a></p></div