Alteration of Neuropilin-1 and Heparan Sulfate Interaction Impairs Murine B16 Tumor Growth.

Neuropilin-1 acts as a coreceptor with vascular endothelial growth factor receptors to facilitate binding of its ligand, vascular endothelial growth factor. Neuropilin-1 also binds to heparan sulfate, but the functional significance of this interaction has not been established. A combinatorial library screening using heparin oligosaccharides followed by molecular dynamics simulations of a heparin tetradecasaccharide suggested a highly conserved binding site composed of amino acid residues extending across the b1 and b2 domains of murine neuropilin-1. Mutagenesis studies established the importance of arginine513 and lysine514 for binding of heparin to a recombinant form of Nrp1 composed of the a1, a2, b1, and b2 domains. Recombinant Nrp1 protein bearing R513A,K514A mutations showed a significant loss of heparin-binding, heparin-induced dimerization, and heparin-dependent thermal stabilization. Isothermal calorimetry experiments suggested a 1:2 complex of heparin tetradecasaccharide:Nrp1. To study the impact of altered heparin binding in vivo, a mutant allele of Nrp1 bearing the R513A,K514A mutations was created in mice (Nrp1D) and crossbred to Nrp1+/- mice to examine the impact of altered heparan sulfate binding. Analysis of tumor formation showed variable effects on tumor growth in Nrp1D/D mice, resulting in a frank reduction in tumor growth in Nrp1D/- mice. Expression of mutant Nrp1D protein was normal in tissues, suggesting that the reduction in tumor growth was due to the altered binding of heparin/heparan sulfate to neuropilin-1. These findings suggest that the interaction of neuropilin-1 with heparan sulfate modulates its stability and its role in tumor formation and growth.

tLyp-1: A peptide suitable to target NRP-1 receptor.

Targeting vascular endothelial growth factor receptor (VEFGR) and its co-receptor neuropilin-1 (NRP-1) is an interesting vascular strategy. tLyp-1 is a tumor-homing and penetrating peptide of 7 amino acids (CGNKRTR). It is a truncated form of Lyp-1 (CGNKRTRGC), which is known to target NRP-1 receptor, with high affinity and specificity. It is mediated by endocytosis via C-end rule (CendR) internalization pathway. The aim of this study is to evaluate the importance of each amino acid in the tLyp-1 sequence through alanine-scanning (Ala-scan) technique, during which each of the amino acid in the sequence was systematically replaced by alanine to produce 7 different analogues. In silico approach through molecular docking and molecular dynamics are employed to understand the interaction between the peptide and its analogues with the NRP-1 receptor, followed by in vitro ligand binding assay study. The C-terminal Arg is crucial in the interaction of tLyp-1 with NRP-1 receptor. Substituting this residue dramatically reduces the affinity of this peptide which is clearly seen in this study. Lys-4 is also important in the interaction, which is confirmed via the in vitro study and the MM-PBSA analysis. The finding in this study supports the CendR, in which the presence of R/K-XX-R/K motif is essential in the binding of a ligand with NRP-1 receptor. This presented work will serve as a guide in the future work pertaining the development of active targeting agent towards NRP-1 receptor.

Neuropilin (NRPs) Related Pathological Conditions and Their Modulators.

Neuropilin 1 (NRP1) represents one of the two homologous neuropilins (NRP, splice variants of neuropilin 2 are the other) found in all vertebrates. It forms a transmembrane glycoprotein distributed in many human body tissues as a (co)receptor for a variety of different ligands. In addition to its physiological role, it is also associated with various pathological conditions. Recently, NRP1 has been discovered as a coreceptor for the SARS-CoV-2 viral entry, along with ACE2, and has thus become one of the COVID-19 research foci. However, in addition to COVID-19, the current review also summarises its other pathological roles and its involvement in clinical diseases like cancer and neuropathic pain. We also discuss the diversity of native NRP ligands and perform a joint analysis. Last but not least, we review the therapeutic roles of NRP1 and introduce a series of NRP1 modulators, which are typical peptidomimetics or other small molecule antagonists, to provide the medicinal chemistry community with a state-of-the-art overview of neuropilin modulator design and NRP1 druggability assessment.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

The N93D mutation of the human T-cell leukemia virus type 1 envelope glycoprotein found in symptomatic patients enhances neuropilin-1 b1 domain binding.

Human T-cell leukemia virus type 1 (HTLV-1) infection of host cells is mainly mediated by interactions with the viral envelope glycoprotein surface unit (SU) and three host receptors: heparan sulfate proteoglycan, neuropilin-1 (Nrp1), and glucose transporter type 1. Residues 90-94 of SU are considered as a Nrp1 binding site, and our previous results show that an SU peptide consisting of residues 85-94 can bind directly to the Nrp1 b1 domain with a binding affinity of 7.4 µM. Therefore, the SU peptide is expected to be a good model to investigate the SU-Nrp1 interaction. Recently, the N93D mutation in the Nrp1 b1 binding region of the SU was identified in symptomatic patients with HTLV-1 infections in the Brazilian Amazon. However, it remains unclear how the SU-N93D mutation affects Nrp1 b1 binding. To elucidate the impact of the substituted Asp93 of SU on Nrp1 b1 binding, we analyzed the interaction between the SU-N93D peptide and Nrp1 b1 using isothermal titration calorimetry and nuclear magnetic resonance. The SU-N93D peptide binds directly to Nrp1 b1 with a binding affinity of 3.5 µM, which is approximately two-fold stronger than wild-type. This stronger binding is likely a result of the interaction between the substituted residue Asp93 of the N93D peptide and the four residues Trp301, Lys347, Glu348, and Thr349 of Nrp1 b1. Our results suggest that the interaction of SU Asp93 with the four residues of Nrp1 b1 renders the high affinity of the N93D mutant for Nrp1 b1 binding during HTLV-1 entry.

Neuropilin-1 is a host factor for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

Does Cysteine Rule (CysR) Complete the CendR Principle? Increase in Affinity of Peptide Ligands for NRP-1 Through the Presence of N-Terminal Cysteine.

The structure-activity relationship of branched H-Lys(hArg)-Dab-Dhp-Arg-OH sequence analogues, modified with Cys-Asp or Cys at N-terminal amino acids (Lys, hArg), in VEGF-A165/Neuropilin-1 complex inhibition is presented. The addition of Cys residue led to a 100-fold decrease in the IC50 value, compared to the parent peptide. The change occurred regardless of coupling Cys to the free N-terminal amino group present in the main or the side chain. A few analogues extended by the attachment of Cys at the N-terminus of several potent NRP-1 peptide ligands documented in the literature are also presented. In all studied cases, the enhancement of inhibitory properties after the addition of Cys at the N-terminus is observed. It is particularly evident for the tetrapeptide derived from the C-terminus of VEGF-A165 (KPRR), suggesting that extending the K/RXXK/R motif (CendR) with the Cys moiety can significantly improve affinity to NRP-1 of CendR peptides.

Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Luminescent silicon nanoparticles for distinctive tracking of cellular targeting and trafficking.

Developing therapeutic nanoparticles that actively target disease cells or tissues by exploiting the binding specificity of receptors presented on the cell surface has extensively opened up biomedical applications for drug delivery and imaging. An ideal nanoparticle for biomedical applications is required to report confirmation of relevant targeting and the ultimate fate in a physiological environment for further verification, e.g. to adapt dosage or predict response. Herein, we demonstrate tracking of silicon nanoparticles through intrinsic photoluminescence (PL) during the course of cellular targeting and uptake. Time-resolved analysis of PL characteristics in cellular microenvironments provides dynamic information on the physiological conditions where the silicon nanoparticles are exposed. In particular, the PL lifetime of the silicon nanoparticles is in the order of microseconds, which is significantly longer than the nanosecond lifetimes exhibited by fluorescent molecules naturally presented in cells, thus allowing discrimination of the nanoparticles from the cellular background autofluorescence in time-gated imaging. The PL lifetime is a physically intensive property that reports the inherent characteristics of the nanoparticles regardless of surrounding noise. Furthermore, we investigate a unique means to inform the lifespan of the biodegradable silicon nanoparticles responsive to local microenvironment in the course of endocytosis. A multivalent strategy of nanoparticles for enhanced cell targeting is also demonstrated with complementary analysis of time-resolved PL emission imaging and fluorescence correlation spectroscopy. The result presents the promising potential of the photoluminescent silicon nanoparticles toward advanced cell targeting systems that simultaneously enable tracking of cellular trafficking and tissue microenvironment monitoring.

68 Ga-labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators.

The dual interaction with integrins and neuropilin-1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with 68 GaCl3 . Negatively charged 68 Ga-DOTAGA-iRGD and neutral 68 Ga-NODAGA-iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral 68 Ga-NODAGA-iRGD, but 68 Ga-DOTAGA-iRGD exhibited better tumor-to-blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.

Neuropilin-1 drives tumor-specific uptake of chlorotoxin.

BACKGROUND: Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (131I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study. METHODS: A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models. RESULTS: Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy. CONCLUSIONS: NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin-1.

BACKGROUND: Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. METHODS: We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. RESULTS: The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. CONCLUSION: Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

Neuropilins in the Context of Tumor Vasculature.

Neuropilin-1 and Neuropilin-2 form a small family of plasma membrane spanning receptors originally identified by the binding of semaphorin and vascular endothelial growth factor. Having no cytosolic protein kinase domain, they function predominantly as co-receptors of other receptors for various ligands. As such, they critically modulate the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. This review highlights the diverse neuropilin ligands and interacting partners on endothelial cells, which are relevant in the context of the tumor vasculature and the tumor microenvironment. In addition to tumor cells, the latter contains cancer-associated fibroblasts, immune cells, and endothelial cells. Based on the prevalent neuropilin-mediated interactions, the suitability of various neuropilin-targeted substances for influencing tumor angiogenesis as a possible building block of a tumor therapy is discussed.

Interactions between NRP1 and VEGFR2 molecules in the plasma membrane.

Here we use a quantitative FRET approach, specifically developed to probe membrane protein interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A (VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the plasma membrane. Our results underscore the complex nature of the interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the need for new methodologies that capture this complexity, and the need for precise physiological measurements of local receptor surface densities in the membrane of cells. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Architecture and hydration of the arginine-binding site of neuropilin-1.

Neuropilin-1 (NRP1) is a transmembrane co-receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C-terminal arginine of VEGF and residues in the NRP1-binding site including Tyr297, Tyr353, Asp320, Ser346 and Thr349. We obtained several complexes of the synthetic ligands and the NRP1-b1 domain and used X-ray crystallography and computational methods to analyse atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the six new high-resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand-binding site hydration map of NRP1 was in agreement with the experimentally derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein-ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands' C-terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design and validates the utility of the computational hydration map prediction method in the context of neuropilin. DATABASE: The structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.

A novel neuropilin-1-binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 µM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

A computational analysis of pro-angiogenic therapies for peripheral artery disease.

Inducing therapeutic angiogenesis to effectively form hierarchical, non-leaky networks of perfused vessels in tissue engineering applications and ischemic disease remains an unmet challenge, despite extensive research and multiple clinical trials. Here, we use a previously-developed, multi-scale, computational systems pharmacology model of human peripheral artery disease to screen a diverse array of promising pro-angiogenic strategies, including gene therapy, biomaterials, and antibodies. Our previously-validated model explicitly accounts for VEGF immobilization, Neuropilin-1 binding, and weak activation of VEGF receptor 2 (VEGFR2) by the "VEGFxxxb" isoforms. First, we examine biomaterial-based delivery of VEGF engineered for increased affinity to the extracellular matrix. We show that these constructs maintain VEGF close to physiological levels and extend the duration of VEGFR2 activation. We demonstrate the importance of sub-saturating VEGF dosing to prevent angioma formation. Second, we examine the potential of ligand- or receptor-based gene therapy to normalize VEGF receptor signaling. Third, we explore the potential for antibody-based pro-angiogenic therapy. Our model supports recent observations that improvement in perfusion following treatment with anti-VEGF165b in mice is mediated by VEGF-receptor 1, not VEGFR2. Surprisingly, the model predicts that the approved anti-VEGF cancer drug, bevacizumab, may actually improve signaling of both VEGFR1 and VEGFR2 via a novel 'antibody swapping' effect that we demonstrate here. Altogether, this model provides insight into the mechanisms of action of several classes of pro-angiogenic strategies within the context of the complex molecular and physiological processes occurring in vivo. We identify molecular signaling similarities between promising approaches and key differences between promising and ineffective strategies.

[Preparation and characterization of monoclonal antibodies against human neuropilin 1 b1b2 domain (HuNRP1b)].

Objective To prepare recombinant protein of human neuropilin 1 b1b2 domain (HuNRP1b) and monoclonal antibodies (mAbs) against the recombinant HuNRP1b (rHuNRP1b). Methods The coding sequence of HuNRP1b was amplified and cloned into vector pET22b to construct recombinant plasmid pET-HuNRP1b. After analyzed by restriction enzyme digestion and DNA sequencing, pET-HuNRP1b was transformed into Escherichia coli and induced to express rHuNRP1b with histidine tag (His-HuNRP1b), which was identified by SDS-PAGE analysis. Then His-HuNRP1b was used as immunogen to immune BALB/c mice. After the fusion of spleen cells and Sp2/0 cells, the positive hybridoma cells were screened by indirect ELISA that was established with recombinant HuNRP1b with a trigger factor tag (TF-HuNRP1b). The specificity of mAbs against HuNRP1b was identified by ELISA and Western blotting. The ability of mAbs to bind native HuNRP1 was revealed by indirect fluorescence assay (IFA). Results His-HuNRP1b was expressed and obtained. Western blotting showed that mAbs 1A10, 6A2 against rHuNRP1b could bind His-HuNRP1b with the band of 34 kDa and TF-HuNRP1b with 89-kDa band, respectively, but not the proteins from bacteria with empty vectors. IFA revealed that the mAbs could bind native HuNRP1 expressed on breast cancer cell line MDA-MB-231. Conclusion The mAbs specific to rHuNRP1b were generated successfully.

Ultrasmall AGuIX theranostic nanoparticles for vascular-targeted interstitial photodynamic therapy of glioblastoma.

Despite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall.

Neuropilin-1 promotes Hedgehog signaling through a novel cytoplasmic motif.

Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggests that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.