Candidate Receptors Research Articles

AXL, an Important Host Factor for DENV and ZIKV Replication.

Flaviviruses, as critically important pathogens, are still major public health problems all over the world. For instance, the evolution of ZIKV led to large-scale outbreaks in the Yap island in 2007. DENV was considered by the World Health Organization (WHO) as one of the 10 threats to global health in 2019. Enveloped viruses hijack a variety of host factors to complete its replication cycle. Phosphatidylserine (PS) receptor, AXL, is considered to be a candidate receptor for flavivirus invasion. In this review, we discuss the molecular structure of ZIKV and DENV, and how they interact with AXL to successfully invade host cells. A more comprehensive understanding of the molecular mechanisms of flavivirus-AXL interaction will provide crucial insights into the virus infection process and the development of anti-flavivirus therapeutics.

Demystifying functional role of cocaine- and amphetamine-related transcript (CART) peptide in control of energy homeostasis: A twenty-five year expedition

Cocaine- and amphetamine-related transcript (CART) is a neuropeptide first discovered in the striatum of the rat brain. Later, the genetic sequence and function of CART peptide (CARTp) was found to be conserved among multiple mammalian species. Over the 25 years, since its discovery, CART mRNA (Cartpt) expression has been reported widely throughout the central and peripheral nervous systems underscoring its role in diverse physiological functions. Here, we review the localization and function of CARTp as it relates to energy homeostasis. We summarize the expression changes of central and peripheral Cartpt in response to metabolic states and make use of available large data sets to gain additional insights into the anatomy of the Cartpt expressing vagal neurons and their expression patterns in the gut. Furthermore, we provide an overview of the role of CARTp as an anorexigenic signal and its effect on energy expenditure and body weight control with insights from both pharmacological and transgenic animal studies. Subsequently, we discuss the role of CARTp in the pathophysiology of obesity and review important new developments towards identifying a candidate receptor for CARTp signalling. Altogether, the field of CARTp research has made rapid and substantial progress recently, and we review the case for considering CARTp as a potential therapeutic target for stemming the obesity epidemic.

Early Diagnosis of Cerebral Ischemia Reperfusion Injury and Revelation of Its Regional Development by a H3R Receptor-Directed Probe.

In vivo imaging of cerebral hydrogen peroxide (H2O2) may facilitate early diagnosis of cerebral ischemia reperfusion injury (CIRI) and a revelation of its pathological progression. In this study, we report our rational design of a brain-targeting fluorescent probe using the basis of a pyridazinone scaffold. A structure-activity relationship study reveals that PCAB is the best candidate (Ki = 15.8 nM) for a histamine H3 receptor (H3R), which is highly expressed in neurons of the central nervous system. As a two-photon fluorescent probe, PCAB exhibits a fast, selective reaction toward both extra- and intracellular H2O2 in SH-SY5Y cells under oxygen glucose deprivation and resupply. In vivo fluorescent imaging of a middle cerebral artery occlusion mouse confirms that PCAB is an ultrasensitive probe with potent blood-brain barrier penetration, precise brain targeting, and fast detection of CIRI.

Pulmonary midkine inhibition ameliorates sepsis induced lung injury

BackgroundMidkine is a multi-functional molecule participating in a various key pathological process. We aimed to evaluate the change of midkine in sepsis and its association with angiotensin-converting enzyme (ACE) system, as well as the mechanism by which midkine induced in sepsis and lung injury.MethodsThe peripheral blood sample of septic patients on admission was obtained and measured for midkine, ACE and angiotensin II. Cecal ligation and puncture (CLP) mouse model was used, and adeno-associated virus (AAV) was stilled trans-trachea for regional targeting midkine expression, comparing the severity of lung injury. Furthermore, we studied the in vitro mechanism of midkine activates ACE system by using inhibitors targeting candidate receptors of midkine, and its effects on the vascular endothelial cells.ResultsPlasma midkine was significantly elevated in sepsis, and was closely associated with ACE system. Both circulating and lung midkine was increased in CLP mouse, and was related to severe lung injury. Regional interfering midkine expression in lung tissue by AAV could alleviate acute lung injury in CLP model. In vitro study elucidated that Notch 2 participated in the activation of ACE system and angiotensin II release, induced by midkine and triggered vascular endothelial injury by angiotensin II induced reactive oxygen species production.ConclusionsMidkine inhibition ameliorates sepsis induced lung injury, which might via ACE/Ang II pathway and the participation of Notch 2 in the stimulation of ACE.Trial registrationClinicaltrials.gov NCT02605681. Registered 12 November 2015

Identifying Receptors for Neuropeptides and Peptide Hormones: Challenges and Recent Progress.

Intercellular signaling events mediated by neuropeptides and peptide hormones represent important targets for both basic science and drug discovery. For many bioactive peptides, the protein receptors that transmit information across the receiving cell membrane are not known, severely limiting these signaling pathways as potential therapeutic targets. Identifying the receptor(s) for a given peptide of interest is complicated by several factors. Most notably, cell-cell signaling peptides are generated through dynamic biosynthetic pathways, can act on many different families of receptor proteins, and can participate in complex ligand-receptor interactions that extend beyond a simple one-to-one archetype. Here, we discuss recent methodological advances to identify signaling partners for bioactive peptides. Recent efforts have centered on methods to identify candidate receptors via transcript expression, methods to match peptide-receptor pairs through high throughput screening, and methods to capture direct ligand-receptor interactions using chemical probes. Future applications of the receptor identification approaches discussed here, as well as technical advancements to address their limitations, promise to lead to a greater understanding of how cells communicate to deliver complex physiologies. Importantly, such advancements will likely provide novel targets for the treatment of human diseases within the central nervous and endocrine systems.

AXL is a candidate receptor for SARS-CoV-2 that promotes infection of pulmonary and bronchial epithelial cells

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.

Receptor-Like Kinase (RLK) as a candidate gene conferring resistance to Hemileia vastatrix in coffee

The biotrophic fungus Hemileia vastatrix causes coffee leaf rust (CLR), one of the most devastating diseases in Coffea arabica . Coffee, like other plants, has developed effective mechanisms to recognize and respond to infections caused by pathogens. Plant resistance gene analogs (RGAs) have been identified in certain plants as candidates for resistance ( R ) genes or membrane receptors that activate the R genes. The RGAs identified in different plants possess conserved domains that play specific roles in the fight against pathogens. Despite the importance of RGAs, in coffee plants these genes and other molecular mechanisms of disease resistance are still unknown. This study aimed to sequence and characterize candidate genes from coffee plants with the potential for involvement in resistance to H. vastatrix . Sequencing was performed based on a library of bacterial artificial chromosomes (BAC) of the coffee clone ‘Híbrido de Timor’ (HdT) CIFC 832/2 and screened using a functional marker. Two RGAs, HdT_LRR_RLK1 and HdT_LRR_RLK2, containing the motif of leucine-rich repeat-like kinase (LRR-RLK) were identified. Based on the presence or absence of the HdT_LRR_RLK2 RGA in a number of differential coffee clones containing different combinations of the rust resistance gene, these RGAs did not correspond to any resistance gene already characterized (SH1-9). These genes were also analyzed using qPCR and demonstrated a major expression peak at 24 h after inoculation in both the compatible and incompatible interactions between coffee and H. vastatrix . These results are valuable information for breeding programs aimed at developing CLR-resistant cultivars, in addition to enabling a better understanding of the interactions between coffee and H. vastatrix .

Major ascaroside pheromone component asc-C5 influences reproductive plasticity among isolates of the invasive species pinewood nematode.

Pheromones are communication chemicals and regulatory signals used by animals and represent unique tools for organisms to mediate behaviors and make "decisions" to maximize their fitness. Phenotypic plasticity refers to the innate capacity of a species to tolerate a greater breadth of environmental conditions across which it adapts to improve its survival, reproduction, and fitness. The pinewood nematode, Bursaphelenchus xylophilus, an invasive nematode species, was accidentally introduced from North America into Japan, China, and Europe; however, few studies have investigated its pheromones and phenotypic plasticity as a natural model. Here, we demonstrated a novel phenomenon, in which nematodes under the condition of pheromone presence triggered increased reproduction in invasive strains (JP1, JP2, CN1, CN2, EU1, and EU2), while it simultaneously decreased reproduction in native strains (US1 and US2). The bidirectional effect on fecundity, mediated by presence/absence of pheromones, is henceforth termed pheromone-regulative reproductive plasticity (PRRP). We further found that synthetic ascaroside asc-C5 (ascr#9), the major pheromone component, plays a leading role in PRRP and identified 2 candidate receptor genes, Bxydaf-38 and Bxysrd-10, involved in perceiving asc-C5. These results suggest that plasticity of reproductive responses to pheromones in pinewood nematode may increase its fitness in novel environments following introduction. This opens up a new perspective for invasion biology and presents a novel strategy of invasion, suggesting that pheromones, in addition to their traditional roles in chemical signaling, can influence the reproductive phenotype among native and invasive isolates. In addition, this novel mechanism could broadly explain, through comparative studies of native and invasive populations of animals, a potential underlying factor behind of the success of other biological invasions.

Signaling in rat brainstem via Gpr160 is required for the anorexigenic and antidipsogenic actions of cocaine- and amphetamine-regulated transcript peptide.

Recent work identified Gpr160 as a candidate receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp) and described its role in pain modulation. The aims of the present study were to determine if Gpr160 is required for the CARTp's ability to reduce food intake and water intake and to initially identify the distribution of Gpr160-like immunoreactivity (Gpr160ir) in the rat brain. A passive immunoneutralization approach targeting Gpr160 was used to block the behavioral effects of a pharmacological dose of CARTp in the fourth cerebroventricle (4V) of rats and to determine the importance of endogenously produced CARTp in the control of ingestive behaviors. Passive immunoneutralization of Gpr160 in the 4V blocked the actions of CARTp to inhibit food intake and water intake. Blockade of Gpr160 in the 4V, independent of pharmacological CART treatment, caused an increase in both overnight food intake and water intake. The decrease in food intake, but not water intake, caused by central injection of CARTp was demonstrated to be interrupted by prior administration of a glucagon-like peptide 1 (GLP-1) receptor antagonist. Gpr160ir was observed in several, distinct sites throughout the rat brain, where CARTp staining has been described. Importantly, Gpr160ir was observed to be present in both neuronal and nonneuronal cell types. These data support the hypothesis that Gpr160 is required for the anorexigenic actions of central CARTp injection and extend these findings to water drinking. Gpr160ir was observed in both neuronal and nonneuronal cell types in regions known to be important in the multiple pharmacological effects of CARTp, identifying those areas as targets for future compromise of function studies.

Effect of a Neuropilin-1-Derived Virus Receptor Trap on Enterovirus A71 Infection In Vitro.

We discovered that neuropilin 1 (NRP1) is a new receptor candidate to mediate enterovirus A71 (EVA71) into cells. In the engineered form as a decoy receptor, NRP1 was able to recognize and neutralize EVA71 but not enterovirus D68 or coxsackievirus B3 (CVB3). NRP1 recognizes EVA71 through a novel domain on the VP3 capsid protein. The principle in the design, engineering, and refinement of the NRP1-based decoy receptor described in this study represents a general and well-suited antiviral strategy.

Nicotine sensitization (part 1): estradiol or tamoxifen is required during the induction phase and not the expression phase to enable locomotor sensitization to nicotine in female rats

Nicotine sensitization involves two functionally distinct phases: induction and expression. Estradiol enhances nicotine sensitization in female rats, but it is not known whether this enhancement is specific to one or both phases. We investigated the effects of estradiol selectively during the induction and the expression of nicotine sensitization. Ovariectomy (OVX) rats were administered E2 during the induction (2 injection days) and/or the expression phase (9 days later) of nicotine sensitization. The selective estrogen receptor modulator tamoxifen (agonist of ERα and ERß, agonist of the g-coupled estradiol receptor GPER1) also was used to elucidate receptor candidates for the effects of E2 on nicotine sensitization. Gonadally intact female rats exhibited expression of nicotine sensitization after a 9-day delay, whereas OVX females did not. Administration of E2 limited to the induction phase of nicotine sensitization rescued expression of nicotine sensitization in OVX females. Tamoxifen during induction did not alter expression of sensitization in gonadally intact female rats, and, like E2, was sufficient to reverse the dampening effects of OVX on expression of sensitization. The enhancing effects of E2 on nicotine sensitization occur during the induction phase of nicotine sensitization, although require a delay to produce the effects on locomotor activity to nicotine, and may involve non-canonical estrogen pathways (e.g., activation of GPER1).

Potential role of Methoprene-tolerant (Met) in methyl farnesoate-mediated vitellogenesis in the Chinese mitten crab (Eriocheir sinensis).

Methoprene-tolerant (Met) belongs to the basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of nuclear transcriptional regulators and is a leading candidate receptor for juvenile hormone (JH III) in insects. Methyl farnesoate (MF) is a de-epoxide form of JH III that regulates many developmental processes in crustaceans, including reproduction, molting, and morphogenesis, much like JH III in insects. In this study, the full-length cDNA for Met was cloned from the Chinese mitten crab (Eriocheir sinensis) (EsMet). The amino acid sequence of EsMet contains three conserved domains (bHLH, PAS-A, and PASB) characteristic of the bHLH-PAS family, having six conserved amino acid residues specifically responsible for JH or MF binding. Tissue distribution analysis revealed that EsMet mRNA is highly expressed in the hepatopancreas. In addition, EsMet and EsVg expression in the hepatopancreas were found to be significantly increased in early endogenous vitellogenic oocytes (stage II) during ovarian development, and the hemolymph MF titer was significantly increased in late exogenous vitellogenic oocytes (stage III), indicating that EsMet is involved in vitellogenesis regulation. In vitro, MF addition markedly upregulated EsMet and EsVg expression in hepatopancreatic tissue, but only EsVg was induced in ovarian tissue. In vivo, EsMet and EsVg expression in the hepatopancreas were both significantly and synchronously increased after MF injection, but not in the ovaries. In addition, EsMet and EsVg expression were upregulated in the hepatopancreas after eyestalk ablation, while only EsVg expression was induced in the ovaries. Thus, our results indicate that Met may act as a receptor for MF in MF-mediated vitellogenesis in crustaceans.

Identification of Neuropeptides and Their Receptors in the Ectoparasitoid, Habrobracon hebetor.

Neuropeptides are a group of signal molecules that regulate many physiological and behavioral processes by binding to corresponding receptors, most of which are G-protein-coupled receptors (GPCRs). Using bioinformatic methods, we screened genomic and transcriptomic data of the ectoparasitoid wasp, Habrobracon hebetor, and annotated 34 neuropeptide candidate precursor genes and 44 neuropeptide receptor candidate genes. The candidate neuropeptide genes were found to encode all known insect neuropeptides except allatotropin, neuropeptide F, pigment dispersing factor, and CCHamides. When compared with the endoparasitic wasp Pteromalus puparum and the ectoparasitic wasp Nasonia vitripennis, trissin and FMRFamide were found only in H. hebetor. A similar result held for the neuropeptide receptor genes, for the receptors were found in H. hebetor except the receptors of CCHamides and neuroparsin. Furthermore, we compared and analyzed the differences in neuropeptides in eight Braconidae wasps and identified natalisin in H. hebetor, Diachasma alloeum, Fopius arisanus and Microplitis demolitor, but not in the other wasps. We also analyzed the transcriptome data and qRT-PCR data from different developmental stages and tissues to reveal the expression patterns of the neuropeptides and their receptors. In this study, we revealed composition of neuropeptides and neuropeptide receptors in H. hebetor, which may contribute to future neurobiological studies.

Osteoprotegerin interacts with syndecan-1 to promote human endometrial stromal decidualization by decreasing Akt phosphorylation.

Does osteoprotegerin (OPG) promote human endometrial stromal decidualization? OPG is essential for human endometrial stromal decidualization through its interaction with syndecan-1 to decrease Akt phosphorylation. OPG (a cytokine receptor) levels are significantly increased in the circulation of pregnant women. However, the role and mechanism of OPG in human endometrial stromal cell (ESC) decidualization remain elusive. We analyzed the endometrial expression of OPG in endometrial tissue samples collected from women with regular menstrual cycles (ranging from 25 to 35 days), and decidual tissue samples collected from woman with normal early pregnancy or recurrent pregnancy loss (RPL) who visited the Department of Gynecology and Obstetrics at a tertiary care center from January to October 2018. None of the subjects had hormonal treatment for at least 3 months prior to the procedure. In total, 16 women with normal early pregnancy and 15 with RPL were selected as subjects for this study. The function of OPG in decidualization was explored in a human endometrial stromal cell (HESC) line and primary cultures of HESCs. We collected endometrial tissues (by biopsy) from the subjects during their menstrual cycle and decidual tissues from subjects with a normal early pregnancy and those with RPL at the time of dilation and curettage. The control group comprised randomly selected women who underwent termination of an apparently normal early pregnancy. The endometrial OPG expression was analyzed using immunohistochemical staining and quantitative RT-PCR (qRT-PCR). Immunofluorescence staining and western blot, and qRT-PCR were used to explore the mRNA and protein expression, respectively, of OPG in an immortalized HESC line and in primary cultures of HESC during proliferation and decidualization. siRNA-mediated knockdown experiments were performed to examine the function of OPG in HESC proliferation and decidualization. Flow cytometry and the cell proliferation MTS assay were performed to further examine the role of OPG in HESC proliferation. We also analyzed decidual marker gene expression by qRT-PCR to assess the consequences of OPG loss for HESC decidualization. A co-immunoprecipitation (IP) assay was used to determine the potential interaction between the OPG and Syndecan-1. Western blot analysis of the rescue experiments performed using the phosphatidylinositol 3-kinase (PI3K) signaling-specific inhibitor LY294002 was used to investigate the downstream signaling pathways through which OPG could mediate HESC decidualization. OPG was expressed in both the human endometrium and in vitro decidualized ESCs. Knockdown experiments revealed that OPG loss impaired the expression of IGF-binding protein-1 (IGFBP-1) (P < 0.05) and prolactin (PRL) (P < 0.05), two specific markers of decidualization, in HESC undergoing decidualization. We also uncovered that OPG knockdown induced the aberrant activation of Akt (protein kinase B) during HESC decidualization (P < 0.05). The inhibition of Akt activation could rescue the impaired expression of the decidual markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) in response to OPG knockdown. Syndecan-1 was considered a potential receptor candidate, as it was expressed in both the endometrium and in vitro cultured stromal cells. Subsequent co-IP experiments demonstrated the interaction between OPG and Syndecan-1 during decidualization. In addition, Syndecan-1 knockdown not only clearly attenuated the decidualization markers PRL (P < 0.05) and IGFBP-1 (P < 0.05) but also induced the aberrant enhancement of Akt phosphorylation in decidualized cells, consistent with the phenotype of OPG knockdown cells. Finally, we revealed that the transcript and protein expression of both OPG and Syndecan-1 was significantly lower in the decidual samples of women with RPL than in those of women with normal pregnancy (P < 0.05). N/A. In this study, based on a number of approaches, it was demonstrated that OPG mediated the repression of Akt that occurs during human stromal cell decidualization, however, the molecular link between OPG and Akt signaling was not determined, and still requires further exploration. OPG is required for decidualization, and a decrease in OPG levels is associated with RPL. These findings provide a new candidate molecule for the diagnosis and potential treatment of RPL. This work was supported in part by the National Natural Science Foundation of China U1605223 (to G.S.), 81701457 (to Y.J.) and 81601349 (to Y.J.). The authors have no conflicts of interest to disclose.

Identification of scavenger receptors and thrombospondin-type-1 repeat proteins potentially relevant for plastid recognition in Sacoglossa.

Functional kleptoplasty is a photosymbiotic relationship, in which photosynthetically active chloroplasts serve as an intracellular symbiont for a heterotrophic host. Among Metazoa, functional kleptoplasty is only found in marine sea slugs belonging to the Sacoglossa and recently described in Rhabdocoela worms. Although functional kleptoplasty has been intensively studied in Sacoglossa, the fundamentals of the specific recognition of the chloroplasts and their subsequent incorporation are unknown. The key to ensure the initiation of any symbiosis is the ability to specifically recognize the symbiont and to differentiate a symbiont from a pathogen. For instance, in photosymbiotic cnidarians, several studies have shown that the host innate immune system, in particular scavenger receptors (SRs) and thrombospondin‐type‐1 repeat (TSR) protein superfamily, is playing a major role in the process of recognizing and differentiating symbionts from pathogens. In the present study, SRs and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida, and Elysia chlorotica, were identified by translating available transcriptomes into potential proteins and searching for receptor specific protein and/or transmembrane domains. Both receptors classes are highly diverse in the slugs, and many new domain arrangements for each receptor class were found. The analyses of the gene expression of these three species provided a set of species‐specific candidate genes, that is, SR‐Bs, SR‐Es, C‐type lectins, and TSRs, that are potentially relevant for the recognition of kleptoplasts. The results set the base for future experimental studies to understand if and how these candidate receptors are indeed involved in chloroplast recognition.

Nociceptive afferent phenotyping reveals that transient receptor potential ankyrin 1 promotes cold pain through neurogenic inflammation upstream of the neurotrophic factor receptor GFRα3 and the menthol receptor transient receptor potential melastatin 8.

The proper detection and behavioral response to painfully cold temperatures is critical for avoiding potentially harmful tissue damage. Cold allodynia and hyperalgesia, pain associated with innocuous cooling and exaggerated pain with noxious cold, respectively, are common in patients with chronic pain. In peripheral somatosensory afferents, the ion channels transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) are candidate receptors for innocuous and noxious cold temperatures, respectively. However, the role of TRPA1 as a cold sensor has remained controversial, and recent evidence suggests that TRPM8 channels and afferents mediate the detection of both pleasant and painful cold. To determine the role of TRPA1 afferents in cold-induced mouse behaviors in vivo, we used functional phenotyping by targeted nerve conduction block with the cell-impermeant lidocaine derivative QX-314. Surprisingly, we find that injection of QX-314 with TRPA1 agonists reduces cold-induced behaviors in mice, but does so in a TRPM8-dependent manner. Moreover, this effect is sexually dimorphic and requires the glial cell line-derived neurotrophic factor receptor GFRα3, as does cold hypersensitivity produced by the activation of TRPA1 channels. Taken together, these results suggest that under conditions of neurogenic inflammation, TRPA1 works upstream of GFRα3 and TRPM8 to produce cold hypersensitivity, providing novel insights into the role of TRPA1 channels in cold pain.

Abstract 3372: VISTA interaction with PSGL1, a likely VISTA receptor in tumors, is effectively disrupted by K0401-020 anti-VISTA antibody

Abstract Background: VISTA (V-domain Ig suppressor of T cell Activation) is a transmembrane B7 family protein that is described as a negative checkpoint of T cell response. K01401-020 is a novel anti-VISTA antibody entering clinical trials (Loukili et al, AACR 2019). Unlike PD1 protein for which PDL1 and PDL2 are clearly demonstrated as its main ligands, several VISTA candidate receptors have been suggested with various degrees of evidence. The prominent candidate receptors published for VISTA are VSIG3/IGSF11 (Wang et al, Immunology, 2018; Mehta et al, Cell Rep, 2019), VSIG8 (WO2016090347), LRIG1 (WO2015187395), and VISTA itself (WO2015179799). PSGL1 recently joined the list based on in vitro biochemical experiments describing VISTA as an acidic pH selective ligand for PSGL1 (Johnston et al, 2019). Methods: Here we report a series of in vitro protein/protein interactions results aimed at testing the VISTA candidate receptors above. mRNA co-expression was analyzed via bioinformatics in a range of human cancer samples. The mRNA and protein of each candidate receptor were also precisely localized in tumor slices using amplified RNA-ISH or protein-IHC staining. Results: We found specific physicochemical conditions, in vitro, for each candidate, to show specific protein/protein interactions with VISTA, mostly occurring at acidic pH and being effectively disrupted by K01401-020 anti-VISTA antibody. In addition, K01401-020 was also confirmed to bind to VISTA whatever the pH conditions tested [6 to 7.4]. Despite the potential for biochemical interaction for all candidates, only VISTA and PSGL1 mRNAs were found strongly correlated across most cancer indications with PSGL1 being one of the best correlated genes to VISTA when compared to the whole transcriptome. In addition, when compared to the other proteins explored (VSIG3, LRIG1 and VSIG8), PSGL1 expression showed much greater correlation of expression with a myeloid-infiltrate identified by a transcriptomic signature. Dual amplified RNA-ISH on human tumors showed co-occurrence for VISTA and PSGL1, and only rare co-occurrence for VISTA and the other receptors. At the protein level, using dual IHC and proximity ligation staining, the proximity of VISTA and PSGL1 was confirmed in tumors, with metrics comparable to PD1 and PDL1 proximity. Potential interactions may still happen between circulating cells outside of tumors. Conclusions: VISTA interactions with other proteins (PSGL1, VISTA, VISG3, VSIG8 and LRIG1) were evidenced in vitro. However, based on RNA correlations and protein spatial distribution in a series of patient tumors, VISTA/PSGL1 may be a biologically relevant interaction in cancer. The likelihood that interactions with VISG3, VSIG8 and LRIG1play a meaningful biological role in tumors is relatively lower. Other potential candidate receptors to VISTA, including VISTA itself, may still need to be revealed. Citation Format: Francisco Cruzalegui, Noureddine Loukili, Olivier Delfour, Nicolas Boute, Céline Thuilliez, Thierry Champion, Martine Malissard, Pierre Launay, Eric Chetaille, Alexandre Passioukov, Nathalie Corvaïa, Pierre Ferré. VISTA interaction with PSGL1, a likely VISTA receptor in tumors, is effectively disrupted by K0401-020 anti-VISTA antibody [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3372.

Distinct evolutionary trajectories of V1R clades across mouse species

BackgroundMany animals rely heavily on olfaction to navigate their environment. Among rodents, olfaction is crucial for a wide range of social behaviors. The vomeronasal olfactory system in particular plays an important role in mediating social communication, including the detection of pheromones and recognition signals. In this study we examine patterns of vomeronasal type-1 receptor (V1R) evolution in the house mouse and related species within the genus Mus. We report the extent of gene repertoire turnover and conservation among species and clades, as well as the prevalence of positive selection on gene sequences across the V1R tree. By exploring the evolution of these receptors, we provide insight into the functional roles of receptor subtypes as well as the dynamics of gene family evolution.ResultsWe generated transcriptomes from the vomeronasal organs of 5 Mus species, and produced high quality V1R repertoires for each species. We find that V1R clades in the house mouse and relatives exhibit distinct evolutionary trajectories. We identify putative species-specific gene expansions, including a large clade D expansion in the house mouse. While gene gains are abundant, we detect very few gene losses. We describe a novel V1R clade and highlight candidate receptors for future study. We find evidence for distinct evolutionary processes across different clades, from largescale turnover to highly conserved repertoires. Patterns of positive selection are similarly variable, as some clades exhibit abundant positive selection while others display high gene sequence conservation. Based on clade-level evolutionary patterns, we identify receptor families that are strong candidates for detecting social signals and predator cues. Our results reveal clades with receptors detecting female reproductive status are among the most conserved across species, suggesting an important role in V1R chemosensation.ConclusionAnalysis of clade-level evolution is critical for understanding species’ chemosensory adaptations. This study provides clear evidence that V1R clades are characterized by distinct evolutionary trajectories. As receptor evolution is shaped by ligand identity, these results provide a framework for examining the functional roles of receptors.

The Role of Receptor Tyrosine Kinases in Lassa Virus Cell Entry

The zoonotic Old World mammarenavirus Lassa (LASV) causes severe hemorrhagic fever with high mortality and morbidity in humans in endemic regions. The development of effective strategies to combat LASV infections is of high priority, given the lack of a licensed vaccine and restriction on available treatment to off-label use of ribavirin. A better understanding of the fundamental aspects of the virus’s life cycle would help to improve the development of novel therapeutic approaches. Host cell entry and restriction factors represent major barriers for emerging viruses and are promising targets for therapeutic intervention. In addition to the LASV main receptor, the extracellular matrix molecule dystroglycan (DG), the phosphatidylserine-binding receptors of the Tyro3/Axl/Mer (TAM), and T cell immunoglobulin and mucin receptor (TIM) families are potential alternative receptors of LASV infection. Therefore, the relative contributions of candidate receptors to LASV entry into a particular human cell type are a complex function of receptor expression and functional DG availability. Here, we describe the role of two receptor tyrosine kinases (RTKs), Axl and hepatocyte growth factor receptor (HGFR), in the presence and absence of glycosylated DG for LASV entry. We found that both RTKs participated in the macropinocytosis-related LASV entry and, regardless of the presence or absence of functional DG, their inhibition resulted in a significant antiviral effect.

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family.

Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses.IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.