A widespread viral entry mechanism: The C-end Rule motif-neuropilin receptor interaction.

Many phylogenetically distant animal viruses, including the new coronavirus severe acute respiratory syndrome coronavirus 2, have surface proteins with polybasic sites that are cleaved by host furin and furin-like proteases. Other than priming certain viral surface proteins for fusion, cleavage generates a carboxy-terminal RXXR sequence. This C-end Rule (CendR) motif is known to bind to neuropilin (NRP) receptors on the cell surface. NRPs are ubiquitously expressed, pleiotropic cell surface receptors with important roles in growth factor signaling, vascular biology, and neurobiology, as well as immune homeostasis and activation. The CendR-NRP receptor interaction promotes endocytic internalization and tissue spreading of different cargo, including viral particles. We propose that the interaction between viral surface proteins and NRPs plays an underappreciated and prevalent role in the transmission and pathogenesis of diverse viruses and represents a promising broad-spectrum antiviral target.

Role of Neuropilin-2-mediated signaling axis in cancer progression and therapy resistance.

Neuropilins (NRPs) are transmembrane proteins involved in vascular and nervous system development by regulating angiogenesis and axon guidance cues. Several published reports have established their role in tumorigenesis. NRPs are detectable in tumor cells of several cancer types and participate in cancer progression. NRP2 is also expressed in endothelial and immune cells in the tumor microenvironment and promotes functions such as lymphangiogenesis and immune suppression important for cancer progression. In this review, we have taken a comprehensive approach to discussing various aspects of NRP2-signaling in cancer, including its regulation, functional significance in cancer progression, and how we could utilize our current knowledge to advance the studies and target NRP2 to develop effective cancer therapies.

The dual role of boron in vitro neurotoxication of glioblastoma cells via SEMA3F/NRP2 and ferroptosis signaling pathways.

Glioblastoma multiform (GBM) is a malignant tumor cancer that originates from the star-shaped glial support tissues, namely astrocytes, and it is associated with a poor prognosis in the brain. The GBM has no cure, and chemotherapy, radiation therapy, and immunotherapy are all ineffective. A certain dose of Boric acid (BA) has many biochemical effects, conspicuously over antioxidant/oxidant rates. This article sought to investigate the modifies of various doses of BA on the glioblastoma concerning cytotoxicity, ferroptosis, apoptosis, and semaphorin-neuropilin signaling pathway. The Cytotoxic activity and cell viability of BA (0.39-25 mM) in C6 cells were tested at 24, 48, and 72 h using 3-(4,5-dimethylthiazol, 2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The IC50 concentration of BA at 1.56 mM was found and cell lysate used for biochemical analysis. Glutathione peroxidase 4 (GPx4) and ACLS4 levels of ferroptosis, levels of total antioxidant (TAS) and oxidant (TAS) parameters, malondialdehyde (MDA), apoptotic proteins as caspase 3 (CASP3) and caspase 7 (CASP7) were measured. The ferroptosis, semaphoring-neuropilin, apoptotic pathway markers and cell counts were analyzed with flow cytometry, Q-PCR, Western and Elisa technique in the C6 cell lysate. BA triggered ferroptosis in the C6 cells dose-dependently, affecting the semaphorin pathway, so reducing proliferation with apoptotic compared with untreated cell as control group (p < .05). This study revealed that BA, defined as trace element and natural compound, incubated ferroptosis, total oxidant molecules, and caspase protein in a dose-dependently by disrupting SEMA3F in tumor cells.

Effective blocking of neuropilin-1activity using oligoclonal nanobodies targeting different epitopes.

Neuropilin-1 (NRP-1) is a non-tyrosine kinase receptor and when overexpressed, leads to angiogenesis. High expression of NRP-1 has been observed in various cancers. Unique characteristic of nanobodies (small size, high affinity and stability, and ease production) make them potential therapeutic tools. Oligoclonal nanobodies which detect multiple functional epitopes on the target antigen could be potential tools for inhibition of cancer resistance problems due to escape variant of tumor cells. In this study, oligoclonal anti-NRP-1 nanobodies were selected from camel immune library and their binding activities as well as in vitro functionality were evaluated. Anti-NRP-1 nanobodies were expressed in an Escherichia coli host, and purified using nickel affinity chromatography. The effect of each individual and oligoclonal nanobodies on human endothelial cells were evaluated by MTT, Tube formation, and migration assay as well. Results showed that oligoclonal anti-NRP-1 nanobodies detected different epitopes of NRP-1 antigen and inhibited in vitro angiogenesis of human endothelial cells better than each individual nanobody. Results indicate promising oligoclonal anti-NRP-1 nanobodies for inhibition of angiogenesis.

Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration.

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

An ancestral role for Semaphorin3F-Neuropilin signaling in patterning neural crest within the new vertebrate head.

The origin of the vertebrate head is one of the great unresolved issues in vertebrate evolutionary developmental biology. Although many of the novelties in the vertebrate head and pharynx derive from the neural crest, it is still unknown how early vertebrates patterned the neural crest within the ancestral body plan they inherited from invertebrate chordates. Here, using a basal vertebrate, the sea lamprey, we show that homologs of Semaphorin3F (Sema3F) ligand and its Neuropilin (Nrp) receptors show complementary and dynamic patterns of expression that correlate with key periods of neural crest development (migration and patterning of cranial neural crest-derived structures). Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that lamprey Sema3F is essential for patterning of neural crest-derived melanocytes, cranial ganglia and the head skeleton, but is not required for neural crest migration or patterning of trunk neural crest derivatives. Based on comparisons with jawed vertebrates, our results suggest that the deployment of Nrp-Sema3F signaling, along with other intercellular guidance cues, was pivotal in allowing early vertebrates to organize and pattern cranial neural crest cells into many of the hallmark structures that define the vertebrate head.

Neuropilins guide preganglionic sympathetic axons and chromaffin cell precursors to establish the adrenal medulla.

The adrenal medulla is composed of neuroendocrine chromaffin cells that secrete adrenaline into the systemic circulation to maintain physiological homeostasis and enable the autonomic stress response. How chromaffin cell precursors colonise the adrenal medulla and how they become connected to central nervous system-derived preganglionic sympathetic neurons remain largely unknown. By combining lineage tracing, gene expression studies, genetic ablation and the analysis of mouse mutants, we demonstrate that preganglionic axons direct chromaffin cell precursors into the adrenal primordia. We further show that preganglionic axons and chromaffin cell precursors require class 3 semaphorin (SEMA3) signalling through neuropilins (NRP) to target the adrenal medulla. Thus, SEMA3 proteins serve as guidance cues to control formation of the adrenal neuroendocrine system by establishing appropriate connections between preganglionic neurons and adrenal chromaffin cells that regulate the autonomic stress response.

A Potential Role of Semaphorin 3A during Orthodontic Tooth Movement.

BACKGROUND: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. METHODS: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, ß-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. RESULTS: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of ß-catenin and the expression of osteogenic marker genes. CONCLUSION: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and ß-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.

High neuropilin and tolloid-like 1 expression associated with metastasis and poor survival in epithelial ovarian cancer via regulation of actin cytoskeleton.

Abnormal expression of neuropilin and tolloid-like 1 (NETO1) has been detected in some human carcinomas. However, the expression of NETO1 and the underlying mechanism in epithelial ovarian cancer (EOC) remain unknown. In this study, we found that a higher NETO1 expression in EOC tissue samples compared to normal ovarian tissue samples was significantly correlated with worse overall survival. Additionally, Cox regression analysis suggested that NETO 1 was independently associated with overall survival. NETO1 overexpression enhanced the EOC cells' migration and invasion capability in vitro via regulation of actin cytoskeleton. Mechanistically, silencing NETO1 reduced the expression of ß-tubulin, F-actin and KIF2A. In conclusion, our results demonstrated the critical role of NETO1 in EOC invasion, and therapies aimed at inhibiting its expression or activity might significantly control EOC growth, invasion and metastatic dissemination.

Complexes of plexin-A4 and plexin-D1 convey semaphorin-3C signals to induce cytoskeletal collapse in the absence of neuropilins.

Class-3 semaphorin guidance factors bind to receptor complexes containing neuropilin and plexin receptors. A semaphorin may bind to several receptor complexes containing somewhat different constituents, resulting in diverse effects on cell migration. U87MG glioblastoma cells express both neuropilins and the four class-A plexins. Here, we show that these cells respond to Sema3A or Sema3B by cytoskeletal collapse and cell contraction but fail to contract in response to Sema3C, Sema3D, Sema3G or Sema3E, even when class-A plexins are overexpressed in the cells. In contrast, expression of recombinant plexin-D1 enabled contraction in response to these semaphorins. Surprisingly, unlike Sema3D and Sema3G, Sema3C also induced the contraction and repulsion of plexin-D1-expressing U87MG cells in which both neuropilins were knocked out using CRISPR/Cas9. In the absence of neuropilins, the EC50 of Sema3C was 5.5 times higher, indicating that the neuropilins function as enhancers of plexin-D1-mediated Sema3C signaling but are not absolutely required for Sema3C signal transduction. Interestingly, in the absence of neuropilins, plexin-A4 formed complexes with plexin-D1, and was required in addition to plexin-D1 to enable Sema3C-induced signal transduction.

Systemic Delivery of NAC-1 siRNA by Neuropilin-Targeted Polymersomes Sensitizes Antiangiogenic Therapy of Metastatic Triple-Negative Breast Cancer.

Antiangiogenic therapy with bevacizumab while being interesting for metastatic triple-negative breast cancer (mTNBC) is restrained by tumor hypoxia elevation and cancer stem cell enrichment. Here, we find that neuropilin-1 (NRP-1)-targeted delivery of nucleus accumbens-associated protein-1 (NAC-1) siRNA mediated by tLyP-1 peptide-functionalized chimaeric polymersomes (tLyP-1-Ps) effectively sensitizes antiangiogenic therapy of mTNBC in vivo. tLyP-1-Ps showed good encapsulation (up to 14.4 wt. %) of siNAC-1, giving robust tLyP-1-Ps-siNAC-1 nanoformulation with a defined size of 48.5 nm (PDI = 0.13) and a surface charge of -9.2 mV, and mediated efficient cytoplasmic transportation of siNAC-1 in MDA-MB-231 TNBC cells, resulting in significant silencing of NAC-1 mRNA and the corresponding oncoprotein. Transwell invasion and wound healing assays revealed that tLyP-1-Ps-siNAC-1 potently inhibited MDA-MB-231 cell invasion and migration. Intriguingly, tLyP-1-Ps-siNAC-1 was shown to markedly improve the bevacizumab therapy of mTNBC, significantly curbing lung metastasis and prolonging the survival time of the MDA-MB-231 metastatic model. The combination of targeted NAC-1 gene silencing and antiangiogenic therapy appears to be an innovative treatment for mTNBC.

Semaphorin/neuropilin binding specificities are stable over 400 million years of evolution.

Semaphorins are a large and important family of signaling molecules conserved in Bilateria. An important determinant of the biological function of their largest class, the secreted class 3 semaphorins, is the specificity of their binding to neuropilins, a key component of a larger holoreceptor complex. We compared these binding specificities in mice and zebrafish, species whose most recent common ancestor was more than 400 million years in the past. We also compared the binding specificities of zebrafish class 3 semaphorins that were duplicated very early within the teleost lineage. We found a surprising conservation of neuropilin binding specificities when comparing both paralogous zebrafish semaphorin pairs and orthologous zebrafish and mouse semaphorin pairs. This finding was further supported by a remarkable conservation of binding specificities in cross-species pairings of semaphorins and neuropilins. Our results suggest that the qualitative specificities with which particular semaphorins bind to particular neuropilins has remained nearly invariant over approximately 400 million years of evolution.

Peptides with the multibasic cleavage site of the hemagglutinin from highly pathogenic influenza viruses act as cell-penetrating via binding to heparan sulfate and neuropilins.

Cell-penetrating peptides (CPPs) show promise as an attractive delivery vehicle for therapeutic molecules-including nucleic acids, peptides, proteins, and even particulates-into several cell types. It is important to identify new CPPs and select the optimal CPP for each application, because CPPs differ in their internalized efficiency and internalization mechanisms. Here, we identified new CPPs derived from the peptides with the hemagglutinin cleavage site (pHACS) of highly pathogenic influenza viruses. We compared the potential of peptides from the pHACS of four subtypes of influenza A virus (H1, H3, H5, and H7) and an influenza B virus (H1-pHACS, H3-pHACS, H5-pHACS, H7-pHACS, and B-pHACS, respectively) to serve as CPPs. H5-pHACS and H7-pHACS, but not the other peptides, bound to mouse dendritic cells and human epithelial cells and were internalized efficiently into these cells. H5-pHACS and H7-pHACS required glycosaminoglycans, especially heparan sulfate and neuropilins, to bind to the cells. In addition, we designed a mutant H7-pHACS with superior cell-binding capability by changing a single amino acid. Furthermore, when conjugated with antigen, H5-pHACS and H7-pHACS induced antigen-specific antibody responses, demonstrating the usefulness of this antigen-delivery vehicle. Our results will improve our understanding of the mechanisms of CPPs and facilitate the development of novel drug-delivery vehicles designed to improve therapeutic efficacy.

Characterization of receptor binding kinetics for vascular endothelial growth factor-A using SPR.

Angiogenesis is a highly regulated process orchestrated, in large part, by the vascular endothelial growth factor-A (VEGF-A) system of ligands and receptors. Considerable effort has been invested in finding optimal ways to modulate VEGF-A activity to treat disease, however, the mechanisms by which the various components interact remain poorly understood. This is in part because of the difficulty of analyzing the various interactions in an intercomparable manner. In the present study, we established conditions to allow for the detailed characterization of the molecular interactions between VEGF and its receptors and the co-receptor NRP-1 using surface plasmon resonance (SPR). We found that VEGF dissociated 25-times faster from its major signaling receptor, VEGF receptor-2 (VEGFR-2) than from its "decoy" receptor, VEGF receptor-1 (VEGFR-1). Using a systematic approach, we obtained kinetic parameters for each individual interaction under a consistent set of experimental conditions allowing for comparison between various receptors. The set of quantitative kinetic parameters and experimental conditions reported herein will provide valuable tools for developing comprehensive models of the VEGF system.

Neuropilins are positive regulators of Hedgehog signal transduction.

The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

Neuropilins Controlling Cancer Therapy Responsiveness.

Neuropilins (NRPs) are cell surface glycoproteins, acting as co-receptors for secreted Semaphorins (SEMAs) and for members of the vascular endothelial growth factor (VEGF) family; they have been initially implicated in axon guidance and angiogenesis regulation, and more recently in cancer progression. In addition, NRPs have been shown to control many other fundamental signaling pathways, especially mediated by tyrosine kinase receptors (RTKs) of growth factors, such as HGF (hepatocyte growth factor), PDGF (platelet derived growth factor) and EGF (epidermal growth factor). This enables NRPs to control a range of pivotal mechanisms in the cancer context, from tumor cell proliferation and metastatic dissemination, to tumor angiogenesis and immune escape. Moreover, cancer treatment failures due to resistance to innovative oncogene-targeted drugs is typically associated with the activity of alternative RTK-dependent pathways; and neuropilins' capacity to control oncogenic signaling cascades supports the hypothesis that they could elicit such mechanisms in cancer cells, in order to escape cytotoxic stress and therapeutic attacks. Intriguingly, several studies have recently assayed the impact of NRPs inhibition in combination with diverse anti-cancer drugs. In this minireview, we will discuss the state-of-art about the relevance of NRPs as potential predictive biomarkers of drug response, and the rationale to target these proteins in combination with other anticancer therapies.

Class-3 Semaphorins and Their Receptors: Potent Multifunctional Modulators of Tumor Progression.

Abstract: Semaphorins are the products of a large gene family containing 28 genes of which 21 are found in vertebrates. Class-3 semaphorins constitute a subfamily of seven vertebrate semaphorins which differ from the other vertebrate semaphorins in that they are the only secreted semaphorins and are distinguished from other semaphorins by the presence of a basic domain at their C termini. Class-3 semaphorins were initially characterized as axon guidance factors, but have subsequently been found to regulate immune responses, angiogenesis, lymphangiogenesis, and a variety of additional physiological and developmental functions. Most class-3 semaphorins transduce their signals by binding to receptors belonging to the neuropilin family which subsequently associate with receptors of the plexin family to form functional class-3 semaphorin receptors. Recent evidence suggests that class-3 semaphorins also fulfill important regulatory roles in multiple forms of cancer. Several class-3 semaphorins function as endogenous inhibitors of tumor angiogenesis. Others were found to inhibit tumor metastasis by inhibition of tumor lymphangiogenesis, by direct effects on the behavior of tumor cells, or by modulation of immune responses. Notably, some semaphorins such as sema3C and sema3E have also been found to potentiate tumor progression using various mechanisms. This review focuses on the roles of the different class-3 semaphorins in tumor progression.

VEGF/Neuropilin Signaling in Cancer Stem Cells.

The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies.

Mechanisms of foxp3+ T regulatory cell-mediated suppression.

Foxp3(+) T regulatory (Treg) cells control all aspects of the immune response. Here, I will review the in vitro model systems that have been developed to define the mechanisms used by Treg cells to suppress a large number of distinct target cell types. These mechanisms can be broadly divided into those that target T cells (suppressor cytokines, IL-2 consumption, cytolysis) and those that primarily target antigen-presenting cells (decreased costimulation or decreased antigen presentation). Although multiple mechanisms for Treg cell suppression have been shown in vitro, it is unclear whether the same or different mechanisms are used by Treg cells in vivo. An increase in our understanding of Treg cell suppressor mechanisms will offer an insight into how Treg cell function can be manipulated either positively or negatively in vivo.