Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry.

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

The Immunoglobulin Superfamily Receptome Defines Cancer-Relevant Networks Associated with Clinical Outcome.

Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome. Using a high-throughput technology to interrogate most single transmembrane receptors for binding to 445 IgSF proteins, we identify over 500 interactions, 82% previously undocumented, and confirm more than 60 receptor-ligand pairs using orthogonal assays. Our study reveals a map of cell-type-specific interactions and the landscape of dysregulated receptor-ligand crosstalk in cancer, including selective loss of function for tumor-associated mutations. Furthermore, investigation of the IgSF interactome in a large cohort of cancer patients identifies interacting protein signatures associated with clinical outcome. The IgSF interactome represents an important resource to fuel biological discoveries and a framework for understanding the functional organization of the surfaceome during homeostasis and disease, ultimately informing therapeutic development.

An Unbiased Screen for Human Cytomegalovirus Identifies Neuropilin-2 as a Central Viral Receptor.

Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.

Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1.

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

A membrane protein display platform for receptor interactome discovery.

Cell surface receptors are critical for cell signaling and constitute a quarter of all human genes. Despite their importance and abundance, receptor interaction networks remain understudied because of difficulties associated with maintaining membrane proteins in their native conformation and their typically weak interactions. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor-ligand interactions in membranes. We demonstrate that this platform is broadly applicable to a variety of membrane proteins, enabling enhanced detection of extracellular interactions over a wide range of binding affinities. We were able to recapitulate and expand the interactome for prominent members of the B7 family of immunoregulatory proteins such as PD-L1/CD274 and B7-H3/CD276. Moreover, when applied to the orphan cancer-associated fibroblast protein, LRRC15, we identified a membrane-dependent interaction with the tumor stroma marker TEM1/CD248. Furthermore, this platform enabled profiling of cellular receptors for target-expressing as well as endogenous extracellular vesicles. Overall, this study presents a sensitive and easy to use screening platform that bypasses membrane protein purification and enables characterization of interactomes for any cell surface-expressed target of interest in its native state.

Cutting Edge: IL-17B Uses IL-17RA and IL-17RB to Induce Type 2 Inflammation from Human Lymphocytes.

IL-17 family cytokines are critical to host defense responses at cutaneous and mucosal surfaces. Whereas IL-17A, IL-17F, and IL-17C induce overlapping inflammatory cascades to promote neutrophil-mediated immunity, IL-17E/IL-25 drives type 2 immune pathways and eosinophil activity. Genetic and pharmacological studies reveal the significant contribution these cytokines play in antimicrobial and autoimmune mechanisms. However, little is known about the related family member, IL-17B, with contrasting reports of both pro- and anti-inflammatory function in rodents. We demonstrate that in the human immune system, IL-17B is functionally similar to IL-25 and elicits type 2 cytokine secretion from innate type 2 lymphocytes, NKT, and CD4+ CRTH2+ Th2 cells. Like IL-25, this activity is dependent on the IL-17RA and IL-17RB receptor subunits. Furthermore, IL-17B can augment IL-33-driven type 2 responses. These data position IL-17B as a novel component in the regulation of human type 2 immunity.

A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies.

ß-Arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism.

T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified ß-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that ß-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the ß-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that ß-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of ß-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.

Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection.

Herpes simplex virus type 1 (HSV-1) and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG) from HSV-2, known to modulate immune mediators (chemokines), also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2) increases nerve growth factor (NGF)-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (Trk)A-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems.

Herpes simplex virus particles interact with chemokines and enhance cell migration.

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively) are among the most prevalent human pathogens, causing a variety of diseases. HSV modulation of the chemokine network remains poorly understood. We have previously identified secreted glycoprotein G (SgG) as the first viral chemokine-binding protein that enhances chemokine function as a novel viral immunomodulatory mechanism. However, gG is also present at the viral envelope and its role in the virus particle remains unknown. Here we have addressed the chemokine-binding capacity of HSV particles and the functionality of such interaction in vitro. We adapted surface plasmon resonance assays and demonstrated the ability of HSV particles to bind a specific set of human chemokines with high affinity. Moreover, we identified gG as the envelope glycoprotein mediating such interaction, as shown by the lack of binding to a HSV-1 gG mutant. In contrast to HSV-1, HSV-2 gG is cleaved and the chemokine-binding domain is secreted (SgG2). However, we found that HSV-2 particles retain the ability to bind chemokines, potentially through SgG2 associated to the viral envelope or non-processed precursor protein. Moreover, we found that HSV particles increase cell migration independently of chemokine binding to envelope gG. This work provides insights into HSV manipulation of the host immune system.

Enhancement of chemokine function as an immunomodulatory strategy employed by human herpesviruses.

Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses.

Cell-based receptor discovery identifies host factors specifically targeted by the SARS CoV-2 spike.

Receptor-ligand interactions on the plasma membrane regulate cellular communication and play a key role in viral infection. Despite representing main targets for drug development, the characterization of these interactions remains challenging in part due to the dearth of optimal technologies. Here, we build a comprehensive library of human proteins engineered for controlled cell surface expression. Coupled to tetramer-based screening for increased binding avidity, we develop a high throughput cell-based platform that enables systematic interrogation of receptor-ligand interactomes. Using this technology, we characterize the cell surface proteins targeted by the receptor binding domain (RBD) of the SARS-CoV spike protein. Host factors that specifically bind to SARS CoV-2 but not SARS CoV RBD are identified, including proteins that are expressed in the nervous system or olfactory epithelium. Remarkably, our results show that Contactin-1, a previously unknown SARS CoV-2 spike-specific receptor that is upregulated in COVID-19 patients, significantly enhances ACE2-dependent pseudotyped virus infection. Starting from a versatile platform to characterize cell surface interactomes, this study uncovers host factors specifically targeted by SARS CoV-2, information that may help design improved therapeutic strategies against COVID-19.

Receptor Interactome Discovery with RDIMIS, a Membrane Protein Interaction Screen Using Recombinant Extracellular Vesicles.

Membrane protein interactions are challenging to identify because of the unique biophysical characteristics of both transmembrane proteins and membrane environments. The Receptor Display in Membranes Interaction Screen (RDIMIS) platform overcomes these challenges by screening transmembrane and membrane-proximal proteins in a membrane environment using recombinant extracellular vesicles (rEVs). The screen has been used to successfully identify interactions for difficult-to-study receptors in an unbiased manner. In this report, we detail how we generate rEVs, characterize the rEVs to ensure screen-readiness, and perform the full interaction screening, with emphasis on the criteria necessary to obtain clear, interpretable results. We also include support protocols for generating a screening library and validating screening results, as well as an alternate protocol for RDIMIS enabling the profiling of naturally occurring extracellular vesicles. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Generating and isolating extracellular vesicles from cells Basic Protocol 2: Characterizing recombinant extracellular vesicles Support Protocol 1: Preparing the receptor screening library Basic Protocol 3: Performing the Receptor Display in Membranes Interaction Screen (RDIMIS) Support Protocol 2: Validating RDIMIS results using microscopy Alternate Protocol: Detecting unlabeled endogenous vesicles.

Structural basis for HCMV Pentamer receptor recognition and antibody neutralization.

Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.

Control of oviductal fluid flow by the G-protein coupled receptor Adgrd1 is essential for murine embryo transit.

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice.

The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology.

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

The homophilic receptor PTPRK selectively dephosphorylates multiple junctional regulators to promote cell-cell adhesion.

Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.