Protein Sigma Research Articles

14-3-3 protein sigma isoform co-localizes with phosphorylated α-synuclein in lewy bodies and lewy neurites of patients with lewy body disease

14-3-3 protein sigma isoform co-localizes with phosphorylated α-synuclein in lewy bodies and lewy neurites of patients with lewy body disease

Past, present, and future of acute heart failure clinical trials-a high-risk population in search of a strategy.

Past, present, and future of acute heart failure clinical trials-a high-risk population in search of a strategy.

NKp46 Recognizes the Sigma1 Protein of Reovirus: Implications for Reovirus-Based Cancer Therapy

The recent approval of oncolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK cell-activating receptor NKp46 and the orthologous mouse protein NCR1 recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify sites of NKp46/NCR1 binding to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo Collectively, we have identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed both for clearance of reovirus infection and for reovirus-based tumor therapy.IMPORTANCE Reovirus infects much of the population during childhood, causing mild disease, and hence is considered to be efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We report here that natural killer (NK) cells recognize a viral protein named sigma1 through the NK cell-activating receptor NKp46. Using several mouse tumor models, we demonstrate the importance of NK cells in protection from reovirus infection and in reovirus killing of tumors in vivo Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in the control of reovirus infections and in reovirus-based cancer therapy.

Role of Sigma-1 Receptor/p38 MAPK Inhibition in Acupoint Catgut Embedding-Mediated Analgesic Effects in Complete Freund's Adjuvant-Induced Inflammatory Pain.

The endoplasmic reticulum chaperone protein Sigma-1 receptor (Sig-1 R) and mitogen-activated protein kinases (MAPKs) are involved in the mechanism of pain. Acupoint stimulation exerts an exact antihyperalgesic effect in inflammatory pain. However, whether Sig-1 R and MAPKs are associated with the acupoint stimulation-induced analgesic effects is not clear. This study investigated the analgesic effect of acupoint catgut embedding (ACE) and the inhibition of Sig-1 R and MAPKs in ACE analgesia. Rats were prepared with intrathecal catheter implantation. ACE was applied to bilateral "Kunlun" (BL60), "Zusanli" (ST36), and "Sanyinjiao" (SP6) acupoints in the rat model of inflammatory pain (complete Freund's adjuvant [CFA] intraplantar injection). Then, Sig-1R agonist PRE084 or saline was intrathecally given daily. The paw withdrawal thresholds and paw edema were measured before CFA injection and at 1, 3, and 5 day after CFA injection. Western bolt was used to evaluate the protein expression of spinal Sig-1R, p38MAPK, and extracellular signal-regulated kinase (ERK), and immunohistochemistry of Sig-1R was detected at 1, 3, and 5 days after CFA injection. ACE exhibited specific analgesic effects. ACE increased paw withdrawal thresholds and markedly decreased CFA-induced paw edema at 1, 3, and 5 days. ACE downregulated the protein expression of Sig-1R, which was increased significantly at 1, 3, and 5 days after CFA injection. ACE decreased the expression of p38 MAPK and ERK at 1 and 3 days but not at 5 days. However, an injection of Sig-1R agonist PRE084 markedly reversed these alterations, except ERK expression. The present study demonstrated that ACE exhibited antihyperalgesic effects via the inhibition of the Sig-1R that modulated p38 MAPK, but not ERK, expression in the CFA-induced inflammatory pain model in rats.

Genetic and pathogenic characterization of a novel reassortant mammalian orthoreovirus 3 (MRV3) from a diarrheic piglet and seroepidemiological survey of MRV3 in diarrheic pigs from east China

Mammalian orthoreoviruses (MRVs), which cause gastrointestinal and respiratory illness, have been isolated from a wide variety of mammalian species including bats, minks, pigs and humans. Here we report the isolation and genetic and pathogenic characterization of a novel MRV type 3 (MRV3), named MRV-ZJ2013, from the diarrheic feces of piglets in Zhejiang province, China. Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from reassortments among mink, bat, and pig MRVs, suggesting the hypothesis that interspecies transmission has occurred in pig herds. Neonatal piglets infected with MRV-ZJ2013 displayed mild clinical signs such as poor appetite and soft feces, but vomiting and diarrhea were not observed. Fecal virus shedding was detected only in three out of six piglets, each for one- or two-day post-infection. In contrast, piglets inoculated with a virulent porcine epidemic diarrhea virus (PEDV) strain as the control group had severe signs characterized by acute vomiting and watery diarrhea. These findings suggest that the virulence of MRV-ZJ2013, if any, was likely not significant compared to that of PEDV. A seroepidemiological survey of MRV by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant MRV3 capsid protein sigma1 as antigen revealed a high seroprevalence (77%) in 1037 samples from diarrheic pigs of different ages from 24 herds in seven provinces of east China between 2015 and 2016, indicating that MRV3 is endemic in pig herds in China, and may contribute collectively to enteric disease along with other porcine pathogens.

Synthesis and pharmacological evaluation of benzamide derivatives as potent and selective sigma-1 protein ligands

A series of novel benzamide-derived compounds was designed, synthesized and pharmacologically evaluated. Among all 37 synthesized compounds, two series were developed with the modulation of the nature, the position of atoms or groups on the benzamide scaffold, but also the nature of the amine group separated from the benzamide with 2, 3 or 4 methylene groups. In vitro competition binding assays against sigma proteins (sigma-1 S1R and sigma-2 S2R) revealed that most of them conferred S2R/S1R selectivity toward without cytotoxic effects on SY5Y cells, especially with the first series with compounds 7a-z. Some selected compounds were also evaluated for their agonist and antagonist activities on a panel of 40 receptors. Results showed the importance of the nature and the position with halogeno atom on the benzamide scaffold, the length chain but also the contribution of the hydrophobic part on the amine group. Among them, compounds 7i, w, y with Cl, CN or NO2 groups at the 4-position of the benzamide scaffold showed excellent affinity for S1R (Ki = 1.2–3.6 nM), selectivity for S2R (Ki up to 1400 nM) and high selectivity index (IC50(SY5Y)/Ki(S1R) ratio from 28 000 to 83 000). Futhermore, these compounds presented an excellent safety profile over 40 other receptors. These derivatives will be selected for further biological investigations.

The prominent proteins expressed in healthy gingiva: a pilot exploratory tissue proteomics study.

Gingiva is a unique tissue which protects the underlying periodontal tissues from consistent mechanical and bacterial aggressions. Molecular analysis of gingiva is likely to improve our understanding of the underlying biological processes at work. The aim of this preliminary exploratory study is to analyze the proteomic profile of healthy gingiva and to detect prominently expressed proteins. Gingival tissue samples were obtained from periodontally healthy individuals who underwent surgical crown lengthening procedure. After protein isolation, two dimensional gel electrophoresis (2DE) gels were prepared for each sample and only protein spots common to all gels were selected to eliminate the bias caused by the effect of individuals on proteomic profile. Following the 2DE; in-gel tryptic digestion and MALDI-TOF/TOF steps were performed for protein identifications. Forty-seven proteins were successfully identified. The identified proteins were classified based on their classes, molecular functions and involvements in biological processes and metabolic pathways. Among them, 14-3-3 protein sigma, Protein DJ-1, Alpha-enolase, Triosephosphate isomerase, Superoxide dismutase, Peroxiredoxin-1, Protein S100-A9, Galectin-7, Annexin A2/A4, Carbonic anhydrase 1 and chaperone proteins are worthy of attention. The proteomic profile of the gingiva reflected its highly dynamic characteristics. Despite complexity of the gingival tissue proteome, 2DE was an effective approach in studying the common protein expression profile of the gingiva. Considering the significance of gingiva in the formation of periodontal diseases, it is important to generate a detailed proteome map of gingival tissue to set up a bridge between molecular events and the disease formation. This study established an initial proteome map of the gingival tissue from healthy individuals.

Sigma1 Pharmacology in the Context of Cancer.

Sigma1 (also known as sigma-1 receptor, Sig1R, σ1 receptor) is a unique pharmacologically regulated integral membrane chaperone or scaffolding protein. The majority of publications on the subject have focused on the neuropharmacology of Sigma1. However, a number of publications have also suggested a role for Sigma1 in cancer. Although there is currently no clinically used anti-cancer drug that targets Sigma1, a growing body of evidence supports the potential of Sigma1 ligands as therapeutic agents to treat cancer. In preclinical models, compounds with affinity for Sigma1 have been reported to inhibit cancer cell proliferation and survival, cell adhesion and migration, tumor growth, to alleviate cancer-associated pain, and to have immunomodulatory properties. This review will highlight that although the literature supports a role for Sigma1 in cancer, several fundamental questions regarding drug mechanism of action and the physiological relevance of aberrant SIGMAR1 transcript and Sigma1 protein expression in certain cancers remain unanswered or only partially answered. However, emerging lines of evidence suggest that Sigma1 is a component of the cancer cell support machinery, that it facilitates protein interaction networks, that it allosterically modulates the activity of its associated proteins, and that Sigma1 is a selectively multifunctional drug target.

Sigma-1 Receptors Fine-Tune the Neuronal Networks.

The endoplasmic reticular (ER) protein sigma-1 receptor (Sig-1R) has been implicated in CNS disorders including but not limited to neurodegenerative diseases, depression , amnesia, and substance abuse. Sig-1Rs are particularly enriched in the specific domain where ER membranes make contacts with the mitochondria (MAM). Within that specific domain, Sig-1Rs play significant roles governing calcium signaling and reactive oxygen species homeostasis to maintain proper neuronal functions. Studies showed that the Sig-1R is pivotal to regulate neuroplasticity and neural survival via multiple aspects of mechanism. Numerous reports have been focusing on Sig-1R's regulatory effects in ER stress, mitochondrial function, oxidative stress and protein chaperoning. In this book chapter, we will discuss the emerging role of Sig-1R in balancing the populations of neuron and glia and their implications in CNS diseases.

Differential proteins among normal cervix cells and cervical cancer cells with HPV-16 infection, through mass spectrometry-based Proteomics (2D-DIGE) in women from Southern México.

BackgroundCervical cancer (CC) is the fourth most common cancer in women worldwide with an estimated 528,000 new cases in 2012. The same year México had an incidence of 13,960 and a mortality of 4769 cases. There are several diagnosis methods of CC; among the most frequents are the conventional Pap cytology (Pap), colposcopy, and visual inspection with acetic acid (VIA), histopathological examination, tests of imaging and detection of high-risk papilloma virus (HR-HPV) with molecular tests (PCR, hybridization, sequencing). Proteomics is a tool for the detection of new biomarkers that can be associated with clinical stage, histological type, prognosis, and/or response to treatment. In this study we performed a comparative analysis of CC cells with normal cervical cells. The proteomic analysis was carried out with the fluorescent two-dimensional electrophoresis (2D-DIGE) technique to subsequently identify differential protein profiles using Decyder Software, and the selected proteins were identified by Mass Spectrometry (MALDI-TOF).ResultsThe proteins that showed an increased expression in cervical cancer in comparison with normal cervix cells were: Mimecan, Actin from aortic smooth muscle and Lumican. While Keratin, type II cytoskeletal 5, Peroxiredoxin-1 and 14-3-3 protein sigma showed a decrease in their protein expression level in cervical cancer in comparison with normal cervix cells.ConclusionsThus, this study was successful in identifying biomarker signatures for cervical cancer, and might provide new insights into the mechanism of CC progression.

Abstract 3023: Cytoplasmic sequestration and autophagic degradation of ErbB receptors in HER2-driven cancer cells by small molecule Sigma1 modulators

Abstract Epidermal growth factor receptors (EGFR/ErbB) drive cell growth, survival, metastasis, and resistance in a range of cancers. Heterodimerization of ErbB1/EGFR, ErbB2/HER2, and ErbB3/HER3 drives aggressive tumor growth through hyperactivation of cancer cell survival and growth signaling pathways. The increased protein production required to sustain these activities renders cancer cells acutely dependent on support factors, such as chaperones and scaffolds, that maintain protein homeostasis. ErbB receptors are integral membrane proteins and as such are synthesized in and transported through the secretory pathway, which comprises the endoplasmic reticulum (ER), Golgi, and associated compartments and vesicles. Nascent ErbB receptors are stabilized and chaperoned through this pathway, in part, by heat shock protein 90 family members, HSP90 and GRP94. Sigma1 (also known as sigma1 receptor) is a unique integral membrane protein found primarily in the ER. Emerging lines of evidence suggest that Sigma1 may function as a chaperone or possibly a scaffolding protein. We find that the levels of Sigma1 protein are elevated and aberrantly distributed in HER2-amplified breast tumor biopsies compared to benign breast tissue. These data indicate that the status and potentially the physiological role of Sigma1 are altered in malignancy and that Sigma1 may be a valid drug target in the treatment of HER2-driven breast cancers. Previously, we discovered that certain selective small molecule modulators of Sigma1 could be used to induce the unfolded protein response (UPR) and autophagy in a panel of cancer cell lines. Here, we demonstrate that these responses to Sigma1 modulators can be exploited to alter the trafficking, stability, and thus signaling of ErbB receptors in cancer cells. In vivo, Sigma1 modulators suppress the growth of xenografted HER2-amplified breast tumors. In the tumors, as well as in vitro cell culture, ErbB1-3 all are eliminated in response to treatment with prototypic small molecule Sigma1 modulators. This corresponds with suppression of downstream PI3K/Akt signaling and with induction of UPR and autophagy. Using high resolution microscopy and organelle fractionation techniques, we confirmed that the Sigma1 modulators induce cytoplasmic sequestration and subsequent degradation of ErbB receptors in ubiquitin-enriched autophagosomes. This process is blocked by cotreatment with autolysosome inhibitor, bafilomycin A1, suggesting that autophagy is the primary mechanism of Sigma1 modulator induced ErbB receptor degradation. Altogether, these data suggest that Sigma1 is a unique, ligand-operated scaffolding protein that contributes to the trafficking and stability of ErbB receptors in HER2-driven cancer cells. Furthermore, these data suggest that Sigma1 is a druggable component of the protein homeostasis regulatory apparatus of cancer cells. Citation Format: Christina M. Maher, Jane Y. Tong, Charles G. Longen, Mercedes I. Lioni, Jeffrey D. Thomas, Xing Tan, Logan Tyler, Fernando U. Garcia, Felix J. Kim. Cytoplasmic sequestration and autophagic degradation of ErbB receptors in HER2-driven cancer cells by small molecule Sigma1 modulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3023.

Sigma-1 receptors modulate neonatal Nav1.5 ion channels in breast cancer cell lines.

The main aim of this study was to investigate a possible functional connection between sigma-1 receptors and voltage-gated sodium channels (VGSCs) in human breast cancer cells. The hypothesis was that sigma-1 drugs could alter the metastatic properties of breast cancer cells via the VGSC. Evidence was found for expression of sigma-1 receptor and neonatal Nav1.5 (nNav1.5) expression in both MDA-MB-231 and MDA-MB-468 cells. Sigma-1 drugs (SKF10047 and dimethyltryptamine) did not affect cell proliferation or migration but significantly reduced adhesion to the substrate. Silencing sigma-1 receptor expression by siRNA similarly reduced the adhesion. Blocking nNav1.5 activity with a polyclonal antibody (NESOpAb) targeting an extracellular region of nNav1.5 also reduced the adhesion in both cell lines. Importantly, the results of combined treatments with NESOpAb and a sigma-1 drug or sigma-1 siRNA suggested that both treatments targeted the same mechanism. The possibility was tested, therefore, that the sigma-1 receptor and the nNav1.5 channel formed a physical, functional complex. This suggestion was supported by the results of co-immunoprecipitation experiments. Furthermore, application of sigma-1 drugs to the cells reduced the surface expression of nNav1.5 protein, which could explain how sigma-1 receptor activation could alter the metastatic behaviour of breast cancer cells. Overall, these results are consistent with the idea of a sigma-1 protein behaving like either a "chaperone" or a regulatory subunit associated with nNav1.5.

Structural Insights into Sigma1 Function.

Sigma1 (also known as this sigma-1 receptor) is an unusual and enigmatic transmembrane protein implicated in a diverse array of biological processes ranging from neurodegenerative disease to cancer. Despite decades of research, the molecular architecture of Sigma1 is only beginning to become clear. Recent work has established that Sigma1 is an oligomer, and crystallographic studies have now offered the first high-resolution views of its molecular structure. For the first time, these results provide a detailed framework to understand mutagenesis data and the molecular pharmacology of Sigma1 ligands. Structural data also raise new questions surrounding the mechanisms of ligand activity and the molecular basis for interactions between Sigma1 and other proteins. As Sigma1 research enters the structural era, the field is poised for new discoveries and reevaluation of old data and old models.

Synthesis and pharmacological evaluation of benzannulated derivatives as potent and selective sigma-1 protein ligands

The σ1 proteins are considered to be a new class of target structures for several central nervous system disorders, including depression, anxiety, psychosis, and Parkinson's and Alzheimer's diseases. Recently, the involvement of these receptors in neuropathic pain and cancer has also been observed. So far, only a few ligands are in clinical trials. In a continuation of our previous studies on the development of σ1 ligands, a new series of benzannulated heterocycles was designed and synthesised. Invitro competition binding assays showed that many of them possessed high σ1 receptor affinity (Ki=0.6-10.3nM), and good σ2/σ1 subtype selectivity, without cytotoxic effects on SY5Y cells (human neuroblastoma cell line).

Kaempferol Induces DNA Damage and Inhibits DNA Repair Associated Protein Expressions in Human Promyelocytic Leukemia HL-60 Cells

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60 cells, which may be the factors for kaempferol induced cell death in vitro.

Effect of Saikokeishito, a Kampo medicine, on hydrogen peroxide-induced premature senescence of normal human dermal fibroblasts

Saikokeishito (TJ-10) is a Kampo (traditional Japanese herbal) medicine, clinically used for hundreds of years in East Asia. Among its various mechanisms elucidated so far, TJ-10 inhibits the production of transforming growth factor-β1 (TGF-β1) and development of pancreatic fibrosis in vivo. Oxidative damage of normal human dermal fibroblasts (NHDFs) in the corium is a cause of human dermal senescence. Our aim was to determine whether TJ-10 protects NHDFs from premature senescence by hydrogen peroxide (H₂O₂). Premature senescence was induced in NHDFs by 200 μmol/L H₂O₂ for 4 h. Cell viability and the expressions of p53, AMP-activated protein kinase α1 (AMPKα1), AMPKα2, and 14-3-3 protein sigma (14-3-3 σ) were measured in NHDFs treated with TJ-10 for 48 h before exposure to H₂O₂for 4 h. Cell viability after treatment with 200 μmol/L H₂O₂ for 4 h was similar (about 80%) to after pre-treatment with TJ-10. Ascorbic acid as a control did not protect NHDFs from damage by 200 μmol/L H₂O₂. Treatment with 200 μmol/L H₂O₂tended to up-regulate p53 and to down-regulate SIRT1 and AMPKα1, but had no effect on AMPKα2 and 14-3-3 σ expression. Pretreatment with TJ-10 inhibited H₂O₂-induced up-regulation of p53 and enhanced AMPKα1 expression. It is suggested that Saikokeishito has a protective effect on oxidative stress-induced senescence of NHDFs.

Carboline- and phenothiazine-derivated heterocycles as potent SIGMA-1 protein ligands.

Sigma 1 receptors are associated with neurodegenerative and psychiatric disorders. These receptors, via their chaperoning functions that counteract endoplasmic reticulum stress and block neurodegeneration, may serve as a target for a new generation of antidepressants or neuroprotective agents. The involvement of these receptors has also been observed in neuropathic pain and cancer. Only a few ligands, such as Igmesine and Anavex 2-73, have been involved in clinical trials. Thus the development of sigma 1 ligands is of interest to a new generation of drugs. Previous work in our lab underlined the potency of benzannulated bicyclic compounds as interesting ligands. Herein the work was extended to a series of novel tricyclic compounds. Carboline- and phenothiazine-derivated compounds were designed and synthesized. Invitro competition binding assays for sigma 1 and 2 receptors showed that most of them have high affinity for sigma 1 receptor (Ki=2.5-18nM), and selectivity toward sigma 2 receptor, without cytotoxic effects on SY5Y cells.

Identification of Novel Biomarker Candidates for the Immunohistochemical Diagnosis of Cholangiocellular Carcinoma

The aim of this study was the identification of novel biomarker candidates for the diagnosis of cholangiocellular carcinoma (CCC) and its immunohistochemical differentiation from benign liver and bile duct cells. CCC is a primary cancer that arises from the epithelial cells of bile ducts and is characterized by high mortality rates due to its late clinical presentation and limited treatment options. Tumorous tissue and adjacent non-tumorous liver tissue from eight CCC patients were analyzed by means of two-dimensional differential in-gel electrophoresis and mass-spectrometry-based label-free proteomics. After data analysis and statistical evaluation of the proteins found to be differentially regulated between the two experimental groups (fold change ≥ 1.5; p value ≤ 0.05), 14 candidate proteins were chosen for determination of the cell-type-specific expression profile via immunohistochemistry in a cohort of 14 patients. This confirmed the significant up-regulation of serpin H1, 14-3-3 protein sigma, and stress-induced phosphoprotein 1 in tumorous cholangiocytes relative to normal hepatocytes and non-tumorous cholangiocytes, whereas some proteins were detectable specifically in hepatocytes. Because stress-induced phosphoprotein 1 exhibited both sensitivity and specificity of 100%, an immunohistochemical verification examining tissue sections of 60 CCC patients was performed. This resulted in a specificity of 98% and a sensitivity of 64%. We therefore conclude that this protein should be considered as a potential diagnostic biomarker for CCC in an immunohistochemical application, possibly in combination with other candidates from this study in the form of a biomarker panel. This could improve the differential diagnosis of CCC and benign bile duct diseases, as well as metastatic malignancies in the liver.

A glycosylation‐specific interaction between the Sigma‐1 and Neuorkinin‐1 receptors (659.13)

The putative chaperone protein Sigma‐1 Receptor (S1R) has been implicated in chronic pain via pharmacological and gene knockout studies, however, how S1R affects pain is not known. Activation of the GPCR Neurokinin‐1 Receptor (NK1R) is implicated in pain signaling. Herein we demonstrate a novel robust interaction between S1R (FLAG‐tagged) and NK1R (HA‐tagged) using co‐immunoprecipitation (co‐IP) in transiently transfected HEK293 cells. NK1R are glycoproteins with several mobility‐shifted immunoreactive bands present on a Western blot. Co‐IP demonstrates specific interaction between S1R and only the non‐glycosylated NK1R. This specific interaction was confirmed by tunicamycin‐treatment to eliminate the glycosylation and by expression of a glycosylation‐deficient NQ‐NK1R mutant. Additionally, we assay a panel of GPCRs for S1R interaction, (including the NK2R, NK3R, MOR, DA1, and MRGR), and demonstrate that all of the interactions are restricted to the non‐glycosylated GPCR. We conclude that S1R acts as a protein chaperone by a selective interaction with the non‐glycosylated GPCR proteins possibly providing insight on how S1R may influence nociceptive transmission.Grant Funding Source: Supported by the National Institute of General Medical Sciences

Cantharidin induces DNA damage and inhibits DNA repair-associated protein levels in NCI-H460 human lung cancer cells

Cantharidin is one of the major compounds from mylabris and it has cytotoxic effects in many different types of human cancer cells. Previously, we found that cantharidin induced cell death through cell cycle arrest and apoptosis induction in human lung cancer NCI-H460 cells. However, cantharidin-affected DNA damage, repair, and associated protein levels in NCI-H460 cells have not been examined. In this study, we determined whether cantharidin induced DNA damage and condensation and altered levels of proteins in NCI-H460 cells in vitro. Incubation of NCI-H460 cells with 0, 2.5, 5, 10, and 15 μM of cantharidin caused a longer DNA migration smear (comet tail). Cantharidin also increased DNA condensation. These effects were dose-dependent. Cantharidin (5, 10, and 15 μM) treatment of NCI-H460 cells reduced protein levels of ataxia telangiectasia mutated (ATM), breast cancer 1, early onset (BRCA-1), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6) -methylguanine-DNA methyltransferase (MGMT), and mediator of DNA damage checkpoint protein 1 (MDC1). Protein translocation of p-p53, p-H2A.X (S140), and MDC1 from cytoplasm to nucleus was induced by cantharidin in NCI-H460 cells. Taken together, this study showed that cantharidin caused DNA damage and inhibited levels of DNA repair-associated proteins. These effects may contribute to cantharidin-induced cell death in vitro.