Identifying viruses with zoonotic potential on the basis of their ability to enter human cells is a critical component of pandemic prediction, prevention and preparedness. Here using a computational approach that retains maximum phylogenetic diversity, we selected an optimal subset of alphacoronavirus spike proteins to screen against broad coronavirus receptor libraries. Most of the selected spike proteins did not use any of the established coronavirus receptors. However, the pseudotyped spike protein of Cardioderma cor (heart-nosed bat) coronavirus KY43 (CcCoV-KY43) could enter human cells. Using a recombinant CcCoV receptor-binding domain (RBD) and a human receptor screening platform, we identified direct interactions with the human CEACAM proteins CEACAM3, CEACAM5 and CEACAM6. Overexpression of human CEACAM6—a protein widely expressed in the human lung—conferred permissivity to otherwise refractory human cells. A crystal structure showed that the RBD binds the amino-terminal IgV-like domain of human CEACAM6. Immune surveillance studies using sera of individuals from the Taveta region of Kenya, where CcCoV-KY43 was identified, did not show significant evidence of recent spillover. Wider characterization of alphacoronaviruses related to CcCoV-KY43 showed that human CEACAM6 is used by two other CcCoVs collected in Kenya. Moreover, there was more restricted nonhuman CEACAM6 tropism for viruses isolated from Rhinolophus bats from Russia and China. Thus, alphacoronaviruses that use CEACAM6 are probably geographically widespread, and viruses from East Africa show potential for transmission to humans.

Resistance to BRAF/MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms allowing tumor cells to evade therapy. We explored early signaling responses to BRAF and MAPK inhibition in a BRAFV600E-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is linked to resistance and immune evasion. Through an innovative systems biology approach integrating multi-omics datasets, we identified critical resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, suppressing PRKD1 activation, increasing JUN activity, and disrupting PKC dynamics via elevated RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity. ARID1A-KO also reduced HLA-related protein expression and enhanced extracellular matrix components, potentially limiting immune infiltration and immunotherapy efficacy. Our multi-omics analysis revealed PRKD1, JUN, and NCK1 as key resistance nodes, offering potential targets for therapeutic strategies to counter resistance in melanoma.

Cell surface protein display is widely used in biotechnology as a platform for interrogating and engineering protein function in mammalian systems. Current display systems, including pDisplay™, are based on type-I membrane topology and enforce an extracellular N-terminus and cytosolic C-terminus. While suitable for secreted proteins and antibody fragments, they are incompatible with type-II membrane proteins, which instead possess an inverted topology. Despite encompassing an important class of proteins, type-II proteins lack robust and generalisable surface display tools. Here, we report a transferrin receptor 1 (TfR1)-based vector for type-II membrane protein display in mammalian cells. By leveraging the native cytoplasmic and transmembrane architecture of TfR1, the system enforces a correct type-II topology and enables efficient trafficking to the plasma membrane. Using TMEM106b as a representative type-II protein, we show that TfR1 fusion drives robust cell-surface localisation, confirmed by fluorescence-activated cell sorting and confocal microscopy.

Animal African trypanosomiasis (AAT) is an infectious wasting disease of economically important livestock caused by Trypanosoma spp. parasites. The disease is primarily caused by two species: Trypanosoma congolense and Trypanosoma vivax, which are endemic in many African countries. AAT is managed by therapeutic and prophylactic drugs; however, resistance is now widely reported, and the development of new drugs has been impeded due to a chronic lack of investment. Recently, we identified an invariant flagellar-associated cell surface protein (IFX) that could elicit protective immune responses when used as a vaccine against T. vivax. We showed that a complement-recruiting anti-IFX monoclonal antibody can prevent infection when used prophylactically. Here, we show that this same unmodified antibody can be used to cure T. vivax infections in a murine experimental model. Importantly, we show that infections can be treated with a single dose and demonstrate full cure by the lack of detectable parasites in peripheral tissues even after immunosuppression. Using structural modeling and site-directed mutagenesis, we localize the protective antibody epitope, thereby identifying targetable regions on IFX to improve vaccine design. Together, these findings validate IFX as both a prophylactic and curative drug target that could be useful in the management of AAT.IMPORTANCETrypanosoma vivax is a parasite that causes animal African trypanosomiasis (AAT), a chronic wasting disease that infects economically important livestock animals, which is a particular problem in African countries south of the Sahara. The impact of this disease is significant: it is responsible for over 3 million cattle deaths and an estimated $4.5 billion of annual lost productivity. There is a desperate need to develop new control measures because resistance is now widely reported to the drugs commonly used to treat this infection. We show here that a single dose of an unmodified monoclonal antibody that recognizes IFX—a parasite cell surface protein localized to the flagellum—is sufficient to cure an established T. vivax infection with no parasite reservoirs detectable in peripheral tissues. Our finding validates IFX as a new drug target and provides a rationale route to the development of new drugs to target AAT.

Helminth infections persist by influencing host immunity through the release of immunomodulatory proteins which prevent immune ejection. The intestinal nematode Heligmosomoides polygyrus bakeri (Hpb) secretes multiple families of immunomodulatory proteins, many of which are composed of consecutive Complement Control Protein (CCP) domains. We hypothesised that further CCP domain proteins are secreted by the parasite to interact with the host. We identified an unusually large number of CCP domain-containing proteins in the genome of Hpb, and cloned a range of these for screening in an Avidity-based Extracellular Interaction Screening (AVEXIS) assay, focussing on interactions with host immune proteins. This screen confirmed the binding of known immunomodulators (HpBARI, TGM1) for their targets (ST2, TGFBR2) and identified a new interaction between a 2 CCP domain Hpb protein and mouse resistin-like molecule beta (RELMβ), a host protein demonstrated to have anti-helminth properties. This protein was named Binder of RELMβ (HpBoRB). This interaction was specific and heat-labile, and was confirmed in ELISA, competition assays, size exclusion chromatography and surface plasmon resonance experiments, identifying a subnanomolar affinity interaction between HpBoRB and RELMβ. These data may indicate that Hpb interferes with the potent anti-helminth host protein RELMβ and adds to our knowledge of the host-parasite interactions mediated by Hpb secreted proteins.

The safety, quality and supply of donor-derived platelet units intended for transfusion have improved over the past decades but significant problems still remain. In vitro-derived platelets offer a possible alternative but up-scaling production is hindered by our limited understanding of thrombopoiesis (the release of platelets by their mother cell, the megakaryocyte [MK]). Here, we have developed an integrated strategy aiming to mimic ex vivo the bone marrow physiological niche that promotes thrombopoiesis by mature MK. The screening of a panel of 259 recombinant transmembrane proteins derived from cells known to promote platelet production through direct contact with MK enabled us to show that ACVR1B, CRTAM, MUCEN and BTN1A1 improve platelet production from either cord blood- (ACVR1B) or pluripotent stem cells-derived (CRTAM, MUCEN and BTN1A1) MK. Using two different methodologies, we functionalize either collagen- or silk-based 3-dimensional scaffolds and confirm increased functional platelet production by up to 2-fold. This unbiased approach has allowed us to identify novel proteins whose role in platelet formation was previously unknown and highlights the potential gain of recreating the MK niche to allow in vitro platelets to become a viable alternative for transfusion.

Malaria transmission from humans to mosquitoes is essential to the parasite life cycle. In the human malaria parasite, Plasmodium falciparum, the rate of commitment to produce the sexual transmission stages, or gametocytes varies and is governed by genetic, epigenetic and environmental factors. The sexually committed parasite has so far remained elusive due to the lack of markers to efficiently isolate these parasites for subsequent functional studies including proteomic analysis of the isolated population. Here, we demonstrate that MSRP1 is a highly specific sexual commitment marker. Using this marker, we generated and validated reporter parasite lines for subsequent FACS-based isolation of sexually and asexually committed parasites. Proteomics of isolated parasites defined distinct protein signatures, including several merozoite surface proteins, indicating functional differences between the two parasite populations. This study provides a blueprint for systematic characterization of the parasite stage at this crucial juncture in the life cycle.

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Resistance to BRAF and MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms that allow tumour cells to evade therapy. Here, we examined early signalling responses to single and combined BRAF and MAPK inhibition in a BRAFV600E, drug-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is associated with resistance and immune evasion. Using an innovative systems biology approach that integrates transcriptomics, proteomics, phosphoproteomics, and functional kinomics through matrix factorization and network analysis, we identified key signalling alterations and resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, bypassing feedback sensitivity observed in parental cells. This rewiring suppressed PRKD1 activation, increased JUN activity - a central resistance network node - and disrupted PKC dynamics through elevated basal RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity post-treatment. ARID1A mutations also reduced HLA-related protein expression and enriched extracellular matrix components, potentially limiting immune infiltration and reducing immunotherapy efficacy. Our graph-theoretical multi-omics approach uncovered novel resistance-associated signalling pathways, identifying PRKD1, JUN, and NCK1 as critical nodes. While receptor activation redundancies complicate single-target therapies, they also present opportunities for combination strategies. This study highlights ARID1A's role in reshaping signalling and immune interactions, offering new insights into melanoma resistance mechanisms. By identifying actionable targets, including JUN and immune pathways, we provide a foundation for developing integrated therapeutic strategies to overcome resistance in BRAF/MAPK inhibitor-treated melanoma. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Egg-sperm binding and fusion are the central events of fertilization and are essential for the formation of a new individual. While decades of research have provided a thorough understanding of the cellular processes, the identity of molecules orchestrating these events, along with their mechanism of action, remain elusive. Often the interactions established by cell surface proteins are weak and transient, making them challenging to deal with using standard biochemical methods, like purifications that involve stringent washing and denaturing conditions. Therefore, our knowledge of membrane proteins required for the sperm-egg interaction remains scarce and the research is further complicated by the limited amount of biological material, especially the eggs. Thanks to experimental approaches specifically developed to investigate cell surface interactions, we identified the first binding pair essential for fertilization in mammals 1. The pair is made of the egg protein JUNO and the sperm protein IZUMO1. Recently another membrane protein, the Fc receptor-like 3 (MAIA), has been indicated as a binding partner of IZUMO1 and suggested to be required for human fertilization2, but the data needs to be further investigated. Indeed, the same biochemical methods used to identify the JUNO-IZUMO1 binding pair, found no direct evidence of binding between MAIA and IZUMO13. In the last decade more sperm proteins have been shown to be essential for sperm-egg interaction other than IZUMO1, and all findings have been validated using transgenic mouse models. Among these sperm proteins, Tmem95 was firstly discovered in bulls, it was later confirmed to be required for fertilization in mice, and new evidence suggests that its role is conserved in humans4,5. Notably, the role played by each of the novel sperm candidates remains elusive and little is known on whether and how these cell surface molecules interact with one another. Insights into the architecture of the mammalian egg-sperm fusion synapse are now emerging with the use of AlphaFold2, an artificial intelligence-based program capable of predicting proteins’ 3D structure. Remarkably, AlphaFold2 not only predicts the structure of extracellular proteins essential for fertilization, also suggests the formation of a pentameric complex composed of three sperm (IZUMO1, SPACA6 and TMEM81) and two egg (JUNO and CD9) proteins which is likely to be important for gamete recognition and fusion6,7. In a high throughput screening we have recently identified a novel binding partner of IZUMO1, expressed in human leukocytes, potentially linking the reproductive and immune system (unpublished). References 1. Bianchi, E., Doe, B., Goulding, D. & Wright, G. J. Juno is the egg Izumo receptor and is essential for mammalian fertilization. Nature 508, 483–487 (2014). 2. Vondrakova, J. et al. MAIA, Fc receptor-like 3, supersedes JUNO as IZUMO1 receptor during human fertilization. Sci Adv 8, eabn0047 (2022). 3. Bianchi, E. et al. No evidence for a direct extracellular interaction between human Fc receptor–like 3 (MAIA) and the sperm ligand IZUMO1. Science Advances 10, eadk6352 (2024). 4. Lamas-Toranzo, I. et al. TMEM95 is a sperm membrane protein essential for mammalian fertilization. eLife 9, e53913 (2020). 5. Tang, S. et al. Human sperm TMEM95 binds eggs and facilitates membrane fusion. Proceedings of the National Academy of Sciences 119, e2207805119 (2022). 6. Elofsson, A., Han, L., Bianchi, E., Wright, G. J. & Jovine, L. Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse. eLife 13, (2024). 7. Deneke, V. E. et al. A conserved fertilization complex of Izumo1, Spaca6, and Tmem81 mediates sperm-egg interaction in vertebrates. 2023.07.27.550750 Preprint at https://doi.org/10.1101/2023.07.27.550750 (2023).

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Fertilization involves the recognition and fusion of sperm and egg to form a previously unidentified organism. In mammals, surface molecules on the sperm and egg have central roles, and while adhesion is mediated by the IZUMO1-JUNO sperm-egg ligand-receptor pair, the molecule/s responsible for membrane fusion remain mysterious. Recently, MAIA/FCRL3 was identified as a mammalian egg receptor, which bound IZUMO1 and JUNO and might therefore have a bridging role in gamete recognition and fusion. Here, we use sensitive assays designed to detect extracellular protein binding to investigate the interactions between MAIA and both IZUMO1 and JUNO. Despite using reagents with demonstrable biochemical activity, we did not identify any direct binding between MAIA/FCRL3 and either IZUMO1 or JUNO. We also observed no fusogenic activity of MAIA/FCRL3 in a cell-based membrane fusion assay. Our findings encourage caution in further investigations on the role played by MAIA/FCRL3 in fertilization.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor–interacting protein that regulates lateralized neuronal identity in the zebrafish brain.

The parasitic nematode Heligmosomoides polygyrus bakeri secretes the HpARI family, which bind to IL-33, either suppressing (HpARI1 and HpARI2) or enhancing (HpARI3) responses to the cytokine. We previously showed that HpARI2 also bound to DNA via its first complement control protein (CCP1) domain. Here, we find that HpARI1 can also bind DNA, while HpARI3 cannot. Through the production of HpARI2/HpARI3 CCP1 domain-swapped chimeras, DNA-binding ability can be transferred, and correlates with in vivo half-life of administered proteins. We found that HpARI1 and HpARI2 (but not HpARI3) also binds to the extracellular matrix component heparan sulphate (HS), and structural modelling showed a basic charged patch in the CCP1 domain of HpARI1 and HpARI2 (but not HpARI3) which could facilitate these interactions. Finally, a mutant of HpARI2 was produced which lacked DNA and HS binding, and was also shown to have a short half-life in vivo. Therefore, we propose that during infection the suppressive HpARI1 and HpARI2 proteins have long-lasting effects at the site of deposition due to DNA and/or extracellular matrix interactions, while HpARI3 has a shorter half-life due to a lack of these interactions.

Proteins on the surfaces of cells serve as physical connection points to bridge one cell with another, enabling direct communication between cells and cohesive structure. As biomedical research makes the leap from characterizing individual cells toward understanding the multicellular organization of the human body, the binding interactions between molecules on the surfaces of cells are foundational both for computational models and for clinical efforts to exploit these influential receptor pathways. To achieve this grander vision, we must assemble the full interactome of ways surface proteins can link together. This review investigates how close we are to knowing the human cell surface protein interactome. We summarize the current state of databases and systematic technologies to assemble surface protein interactomes, while highlighting substantial gaps that remain. We aim for this to serve as a road map for eventually building a more robust picture of the human cell surface protein interactome.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE:Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Sporozoite invasion of hepatocytes is a necessary step prior to development of malaria, with similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the malaria blood-stage, efforts to identify host-pathogen protein-protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host-pathogen protein-protein interactions involved are poorly understood. Here, we performed a systematic screen to identify such interactions. We substantially extended previous Plasmodium falciparum and human surface protein ectodomain libraries, creating new libraries containing 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, we used a plate-based assay capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. We were able to test 7540 sporozoite-hepatocyte protein pairs under conditions likely to be sensitive for interaction. We report and characterise an interaction between human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34, and describe an additional interaction between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15. Strategies to inhibit these interactions may have value in malaria prevention, and the modified interaction screening assay and protein expression libraries we report may be of wider value to the community.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8-151 and MAD8-502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.