Putative Receptor Binding Site Research Articles

H19 influenza A virus exhibits species-specific MHC class II receptor usage

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.

Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection

The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.

Design and Synthesis of Bovine Leukemia Virus-Associated Peptide-Based Qβ Conjugate Eliciting Long-Lasting Neutralizing Antibodies in Mice.

Bovine leukemia virus (BLV) is a C-type retrovirus of cattle that causes huge economic losses with high infection rates in the majority of countries worldwide. To develop an anti-BLV vaccine, we constructed a peptide conjugate using the envelope glycoprotein gp51-peptide epitope, a putative receptor-binding site. This highly antigenic peptide was covalently linked to a mutant bacteriophage carrier (mQβ) using two different linker strategies, isothiocyanate (NCS) and dinitrophenyl adipate. Both constructs elicited higher anti-BLV peptide IgG titers than the corresponding conjugate with keyhole limpet hemocyanin protein carrier (gold standard) in mice with the NCS linker strategy requiring less sample processing. The mQβ-gp51-peptide construct is the first BLV peptide-based vaccine candidate to generate durable immunity (>539 days), which recognized both native gp51 protein and BLV particles and significantly decreased fusion of a susceptible cell line exposed to infectious BLV. These results support the high translational and animal health potential of the vaccine construct.

Synthesis, antimicrobial and antioxidant evaluation with in silico studies of new thiazole Schiff base derivatives

A series of nineteen thiazole Schiff base derivatives 2a-2s were synthesized (Scheme 1) and elucidated by spectral analyses (IR, 1H NMR and HRMS). The evaluation of their antimicrobial activities against two gram-positive, two gram-negative, and two fungal strains revealed that some compounds displayed moderate antimicrobial activities compared to standard ciprofloxacin and miconazole in disc diffusion technique. Compound 2a displayed very strong antioxidant efficacy (IC50 = 3.52 ± 0.86 µg/mL) compared to standard ascorbic acid (IC50 = 27.34 ± 1.86 µg/mL) in DPPH free radical scavenging assay. Compounds 2j, 2k, and 2m displayed significant antioxidant activities in this assay as well. In silico analysis predicted that the synthesized compounds follow the Lipinski's rule of five and Veber's rule with one exception. The compounds displayed good drug-likeness and drug-score properties. The molecular docking studies predicted that the synthesized thiazole Schiff base derivatives are tolerable in putative receptor binding sites.

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins.

Identification of Peptide Binders to Truncated Recombinant Chikungunya Virus Envelope Protein 2 Using Phage Display Technology and Their In Silico Characterization.

To identify and characterize peptide binders to truncated recombinant chikungunya virus envelope protein 2. Despite extensive research on the chikungunya virus (CHIKV), the specific antiviral treatment's unavailability has stressed the need for the urgent development of therapeutics. The Envelope protein 2 (E2) of CHIKV that displays putative receptor binding sites and specific epitopes for virus neutralizing antibodies is a critical target for the therapeutic intervention. The study aims to identify the unique peptides that can bind to truncated E2 protein of CHIKV and further explore their properties as potential therapeutic candidate. A stretch of CHIKV-E2 (rE2), which is prominently exposed on the surface of virion, was used as bait protein to identify peptide binders to the CHIKV-rE2 using a 12-mer phage display peptide library. Three rounds of biopanning yielded several peptide binders to CHIKV-rE2 and their binding affinities were compared by phage ELISA. Additionally, a fully flexible-blind docking simulation investigated the possible binding modes of the selected peptides. Furthermore, the selected peptides were characterized and their ADMET properties were explored in silico. Five peptides were identified as potential binders based on their robust reactivity to the bait protein. The selected peptides appeared to interact with the crucial residues that were notably exposed on the surface of E1-E2 trimeric structure. The explored in silico studies suggested their non-allergenicity, non-toxicity and likeliness to be antiviral. The potential binding peptides of CHIKV-rE2 protein were identified using phage display technology and characterized in silico. The selected peptides could be further used for the development of therapeutics against the CHIKV infection.>.

COVID-19 and comorbidities: A role for dipeptidyl peptidase 4 (DPP4) in disease severity?

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome (MERS-CoV), which cause acute respiratory distress syndrome and case fatalities. COVID-19 disease severity is worse in older obese patients with comorbidities such as diabetes, hypertension, cardiovascular disease, and chronic lung disease. Cell binding and entry of betacoronaviruses is via their surface spike glycoprotein; SARS-CoV binds to the metalloprotease angiotensin-converting enzyme 2 (ACE2), MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4), and recent modeling of the structure of SARS-CoV-2 spike glycoprotein predicts that it can interact with human DPP4 in addition to ACE2. DPP4 is a ubiquitous membrane-bound aminopeptidase that circulates in plasma; it is multifunctional with roles in nutrition, metabolism, and immune and endocrine systems. DPP4 activity differentially regulates glucose homeostasis and inflammation via its enzymatic activity and nonenzymatic immunomodulatory effects. The importance of DPP4 for the medical community has been highlighted by the approval of DPP4 inhibitors, or gliptins, for the treatment of type 2 diabetes mellitus. This review discusses the dysregulation of DPP4 in COVID-19 comorbid conditions; DPP4 activity is higher in older individuals and increased plasma DPP4 is a predictor of the onset of metabolic syndrome. DPP4 upregulation may be a determinant of COVID-19 disease severity, which creates interest regarding the use of gliptins in management of COVID-19. Also, knowledge of the chemistry and biology of DPP4 could be utilized to develop novel therapies to block viral entry of some betacoronaviruses, potentially including SARS-CoV-2.

A simple and rapid pipeline for identification of receptor-binding sites on the surface proteins of pathogens

Ligand-receptor interactions play a crucial role in the plethora of biological processes. Several methods have been established to reveal ligand-receptor interface, however, the majority of methods are time-consuming, laborious and expensive. Here we present a straightforward and simple pipeline to identify putative receptor-binding sites on the pathogen ligands. Two model ligands (bait proteins), domain III of protein E of West Nile virus and NadA of Neisseria meningitidis, were incubated with the proteins of human brain microvascular endothelial cells immobilized on nitrocellulose or PVDF membrane, the complex was trypsinized on-membrane, bound peptides of the bait proteins were recovered and detected on MALDI-TOF. Two peptides of DIII (~916 Da and ~2003 Da) and four peptides of NadA (~1453 Da, ~1810 Da, ~2051 Da and ~2433 Da) were identified as plausible receptor-binders. Further, binding of the identified peptides to the proteins of endothelial cells was corroborated using biotinylated synthetic analogues in ELISA and immunocytochemistry. Experimental pipeline presented here can be upscaled easily to map receptor-binding sites on several ligands simultaneously. The approach is rapid, cost-effective and less laborious. The proposed experimental pipeline could be a simpler alternative or complementary method to the existing techniques used to reveal amino-acids involved in the ligand-receptor interface.

Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein

A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin–Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine.

Structures of Coxsackievirus A10 unveil the molecular mechanisms of receptor binding and viral uncoating

Coxsackievirus A10 (CVA10), a human type-A Enterovirus (HEV-A), can cause diseases ranging from hand-foot-and-mouth disease to polio-myelitis-like disease. CVA10, together with some other HEV-As, utilizing the molecule KREMEN1 as an entry receptor, constitutes a KREMEN1-dependent subgroup within HEV-As. Currently, there is no vaccine or antiviral therapy available for treating diseases caused by CVA10. The atomic-resolution structure of the CVA10 virion, which is within the KREMEN1-dependent subgroup, shows significant conformational differences in the putative receptor binding sites and serotype-specific epitopes, when compared to the SCARB2-dependent subgroup of HEV-A, such as EV71, highlighting specific differences between the sub-groups. We also report two expanded structures of CVA10, an empty particle and uncoating intermediate at atomic resolution, as well as a medium-resolution genome structure reconstructed using a symmetry-mismatch method. Structural comparisons coupled with previous results, reveal an ordered signal transmission process for enterovirus uncoating, converting exo-genetic receptor-attachment inputs into a generic RNA release mechanism.

Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition

The ability of phages to infect specific bacteria has led to their exploitation as bio-tools for bacterial remediation and detection. Many phages recognize bacterial hosts via adhesin tips of their long tail fibers (LTFs). Adhesin sequence plasticity modulates receptor specificity, and thus primarily defines a phage's host range. Here we present the crystal structure of an adhesin (gp38) attached to a trimeric β-helical tip (gp37) from the Salmonella phage S16 LTF. Gp38 contains rare polyglycine type II helices folded into a packed lattice, herein designated "PGII sandwich." Sequence variability within the domain is limited to surface-exposed helices and distal loops that form putative receptor-binding sites. In silico analyses revealed a prevalence of the adhesin architecture among T-even phages, excluding the archetypal T4 phage. Overall, S16 LTF provides a valuable model for understanding binding mechanisms of phage adhesins, and for engineering of phage adhesins with expandable or modulated host ranges.

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis against Enterotoxigenic Escherichia coli Infection.

Enterotoxigenic Escherichia coli (ETEC) causes diarrheal illness in infants in the developing world and travelers to countries where the disease is endemic, including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion, leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC infection. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have the potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 antibodies identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Over 300 unique anti-CfaE IgG1 HuMAbs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or sIgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A 2- to 4-log decrease in CFU was observed in comparison to mice challenged with irrelevant isotype controls. We identified fully human monoclonal antibodies against the CfaE adhesion domain that can be potentially employed as an immunoprophylactic to prevent ETEC-related diarrhea.

Abstract 3882: Functional validation of a genome-wide association study (GWAS) SNP associated with anthracycline-induced congestive heart failure

Abstract Anthracyclines are commonly used chemotherapies for the treatment of breast cancer, but they can cause dose-related cardiotoxicities and lead to congestive heart failure (CHF) in ~2% of patients. There are no clinically available predictive biomarkers for this toxicity. Previously we have identified and validated the association of a single nucleotide polymorphism (SNP), rs28714259, with CHF in three large adjuvant phase III breast cancer trials. rs28714259 locates in a putative glucocorticoid receptor (GR) binding site and the risk (minor) allele is predicted to disrupt GR binding. Interestingly, activation of GR-signaling by dexamethasone has been shown to protect cardiomyocytes from doxorubicin-induced apoptosis in rat. To test whether the risk allele affects GR binding efficiency in vitro, we performed electrophoretic mobility shift assay (EMSA) in MCF-7 and A549 cells. 100nM of dexamethasone for 4 hours induced a prominent band shift in either the major- or risk-allele probes. Notably, we observed a ~50% reduction in the shifted band intensity of risk-allele probes compared to major-allele probes, suggesting decreased binding in risk-allele probes. No additional supershift was detected after the binding reaction was co-incubated with a GR antibody. However, the previously shifted band was significantly diminished in both the major- and risk-allele probes, probably due to a conformational change induced by GR antibody and leading to the disruption of GR/DNA binding, which in turn suggests the observed shift was specifically caused by GR. Further validations are ongoing using cardiomyocytes derived from human induced pluripotent stem cells (iPSCs). These studies provide further insight into the function and role of rs28714259 in anthracycline-induced CHF. Citation Format: Xi Wu, Gloria Xue, Laura Gardner, Fei Shen, Guanglong Jiang, Santosh Philips, Geneva Cunningham, Bryan P. Schneider. Functional validation of a genome-wide association study (GWAS) SNP associated with anthracycline-induced congestive heart failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3882.

Virion Structure of Iflavirus Slow Bee Paralysis Virus at 2.6-Angstrom Resolution.

ABSTRACTThe western honeybee (Apis mellifera) is the most important commercial insect pollinator. However, bees are under pressure from habitat loss, environmental stress, and pathogens, including viruses that can cause lethal epidemics. Slow bee paralysis virus (SBPV) belongs to the Iflaviridae family of nonenveloped single-stranded RNA viruses. Here we present the structure of the SBPV virion determined from two crystal forms to resolutions of 3.4 Å and 2.6 Å. The overall structure of the virion resembles that of picornaviruses, with the three major capsid proteins VP1 to 3 organized into a pseudo-T3 icosahedral capsid. However, the SBPV capsid protein VP3 contains a C-terminal globular domain that has not been observed in other viruses from the order Picornavirales. The protruding (P) domains form “crowns” on the virion surface around each 5-fold axis in one of the crystal forms. However, the P domains are shifted 36 Å toward the 3-fold axis in the other crystal form. Furthermore, the P domain contains the Ser-His-Asp triad within a surface patch of eight conserved residues that constitutes a putative catalytic or receptor-binding site. The movements of the domain might be required for efficient substrate cleavage or receptor binding during virus cell entry. In addition, capsid protein VP2 contains an RGD sequence that is exposed on the virion surface, indicating that integrins might be cellular receptors of SBPV. IMPORTANCE Pollination by honeybees is needed to sustain agricultural productivity as well as the biodiversity of wild flora. However, honeybee populations in Europe and North America have been declining since the 1950s. Honeybee viruses from the Iflaviridae family are among the major causes of honeybee colony mortality. We determined the virion structure of an Iflavirus, slow bee paralysis virus (SBPV). SBPV exhibits unique structural features not observed in other picorna-like viruses. The SBPV capsid protein VP3 has a large C-terminal domain, five of which form highly prominent protruding “crowns” on the virion surface. However, the domains can change their positions depending on the conditions of the environment. The domain includes a putative catalytic or receptor binding site that might be important for SBPV cell entry.

Binding specificity of P[8] VP8* proteins of rotavirus vaccine strains with histo-blood group antigens

RotaTeq® and Rotarix™ are two common human rotavirus (RV) vaccines currently on the market worldwide. Recent studies indicate histo-blood group antigens (HBGAs) may be attachment factors for RVs. The P[8] VP8* proteins of RotaTeq and Rotarix were expressed and purified, and their binding specificities were evaluated. Saliva-based binding assays showed that the VP8* proteins bound to the saliva samples of secretors irrespective of ABO blood types. However, in the oligosaccharide binding assay, the VP8* proteins displayed no specific binding to the HBGAs tested, including Lewis b and H1. The structure of RotaTeq P[8] VP8* was solved at 1.9Å. Structural comparisons revealed that the putative receptor binding site was different to that of other genotypes and displayed a novel potential binding region. These findings indicate RotaTeq and Rotarix may have better efficiency in areas with a high percentage of secretors.

Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

The human endometrium undergoes regular cycles of synchronous tissue shedding (wounding) and repair that occur during menstruation before estrogen-dependent regeneration. Endometrial repair is normally both rapid and scarless. Androgens regulate cutaneous wound healing, but their role in endometrial repair is unknown. We used a murine model of simulated menses; mice were treated with a single dose of the nonaromatizable androgen dihydrotestosterone (DHT; 200 µg/mouse) to coincide with initiation of tissue breakdown. DHT altered the duration of vaginal bleeding and delayed restoration of the luminal epithelium. Analysis of uterine mRNAs 24 h after administration of DHT identified significant changes in metalloproteinases (Mmp3 and -9; P < 0.01), a snail family member (Snai3; P < 0.001), and osteopontin (Spp1; P < 0.001). Chromatin immunoprecipitation analysis identified putative androgen receptor (AR) binding sites in the proximal promoters of Mmp9, Snai3, and Spp1. Striking spatial and temporal changes in immunoexpression of matrix metalloproteinase (MMP) 3/9 and caspase 3 were detected after DHT treatment. These data represent a paradigm shift in our understanding of the role of androgens in endometrial repair and suggest that androgens may have direct impacts on endometrial tissue integrity. These studies provide evidence that the AR is a potential target for drug therapy to treat conditions associated with aberrant endometrial repair processes.-Cousins, F. L., Kirkwood, P. M., Murray, A. A., Collins, F., Gibson, D. A., Saunders, P. T. K. Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

Whole genome sequencing reveals novel non-synonymous mutation in ectodysplasin A (EDA) associated with non-syndromic X-linked dominant congenital tooth agenesis.

Congenital tooth agenesis in human is characterized by failure of tooth development during tooth organogenesis. 300 genes in mouse and 30 genes in human so far have been known to regulate tooth development. However, candidature of only 5 genes viz. PAX9, MSX1, AXIN2, WNT10A and EDA have been experimentally established for congenitally missing teeth like hypodontia and oligodontia. In this study an Indian family with multiple congenital tooth agenesis was identified. Pattern of inheritance was apparently autosomal dominant type with a rare possibility to be X-linked. Whole genome sequencing of two affected individuals was carried out which revealed 119 novel non-synonymous single nucleotide variations (SNVs) distributed among 117 genes. Out of these only one variation (c.956G>T) located at exon 9 of X-linked EDA gene was considered as pathogenic and validated among all the affected and unaffected family members and unrelated controls. This variation leads to p.Ser319Ile change in the TNF homology domain of EDA (transcript variant 1) protein. In silico analysis predicts that this Ser319 is well conserved across different vertebrate species and a part of putative receptor binding site. Structure based homology modeling predicts that this amino acid residue along with four other amino acid residues nearby, those when mutated known to cause selective tooth agenesis, form a cluster that may have functional significance. Taken together these results suggest that c.956G>T (p.Ser319Ile) mutation plausibly reduces the receptor binding activity of EDA leading to distinct tooth agenesis in this family.

OP0193 The Rheumatoid Arthritis -Risk Locus CCR6 and Its Snp-Dependent Response to Estrogen: A Possible Genomic Link between Sex Hormones and the IL-17 Inflammatory Pathway

BackgroundThe CCR6-CCL20–mediated migration of Th17 cells to inflamed tissues may represent an important mechanism in the etiology of rheumatoid arthritis (RA). The CCR6 polymorphism rs3093023 is associated with RA disease...

Crystal Structure of the Avian Astrovirus Capsid Spike

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.

Abstract 1315: Regulation of Nodal expression by estrogen in hormone responsive human breast cancer cell lines.

Abstract Breast cancer is one of the most common cancers among Canadian women. Estrogen plays a key role in the in the normal growth and development of the breast and is also a major contributor to breast cancer growth. Recent studies have demonstrated that embryonic morphogen, Nodal is expressed in metastatic breast cancers and that its expression is correlated with breast cancer progression. In silico analysis of the Nodal promoter has revealed putative estrogen receptor binding sites (ERE) in proximity to the transcription start site. Our goal in this study is to investigate the role of estrogen on Nodal expression in human breast cancer cell-lines. Stimulation of MCF-7 and T47D breast cancer cell-lines that are estrogen receptor positive and express low levels of Nodal with 10nM of estradiol (E2) for varying amounts of time revealed that Nodal protein expression increases at about 48 hours after E2 exposure and persists up to 120 hours. Treatment with the E2 antagonist, ICI-182,780 (ICI) abrogates the E2-enhanced Nodal protein expression. Conversely, treating the MCF- 7 and T47D cells with recombinant Nodal resulted in a decreased expression of estrogen receptor in these cells both at the transcript and protein levels. These preliminary results suggest that there is a bidirectional interaction between estrogen receptor and Nodal in these cell-lines. Future experiments are aimed at understanding in depth this bidirectional phenomenon to provide better insight into hormonal regulation in metastatic breast cancer. Citation Format: Padmalaya Das, Daniela Quail, Lynne-Marie Postovit. Regulation of Nodal expression by estrogen in hormone responsive human breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1315. doi:10.1158/1538-7445.AM2013-1315