Nuclear localization signal in nuclear receptor VDR facilitates the mitotic genome bookmarking by involving distinct amino acid residues.

Mitotic genome-bookmarking preserves epigenetic information, re-establishing progenitor's gene expression profile through transcription factors, chromatin remodelers, and histone modifiers, thereby regulating cell fate and lineage commitment post-mitotically in progeny cells. Our recent study revealed that the constitutive association of VDR with mitotic chromatin involves its DNA-binding domain. However, amino acid residues in this domain, crucial for genome bookmarking, remain elusive. This study demonstrates that nuclear localization signal (NLS) residues between 49 and 55 amino acids in VDR are essential for receptor-chromatin interaction during mitosis. Furthermore, it is revealed that both bipartite nature of VDR-NLS region and N-terminally located positively charged arginine residues are critical for its 'genome-bookmarking' property. Since mitotic chromatin association of heterodimeric partner RXR depends on VDR-chromatin association, interventions in VDR binding also abort RXR-chromatin interaction. Overall, this study documents the mechanistic details underlying VDR-chromatin interactions in genome-bookmarking behavior, potentially aiding in comprehending VDR-mediated diseases attributed to certain SNPs.

Truncated variants of thyroid hormone receptor beta display disease-inflicting malfunctioning at cellular level.

Thyroid hormone receptor ß (THRß) is a member of the nuclear receptor superfamily of ligand-modulated transcription factors. Upon ligand binding, THRß sequentially recruits the components of transcriptional machinery to modulate target gene expression. In addition to regulating diverse physiological processes, THRß plays a crucial role in hypothalamus-pituitary-thyroid axis feedback regulation. Anomalies in THRß gene/protein structure are associated with onset of diverse disease states. In this study, we investigated disease-inflicting truncated variants of THRß using in-silico analysis and cell-based assays. We examined the THRß truncated variants on multiple test parameters, including subcellular localization, ligand-receptor interactions, transcriptional functions, interaction with heterodimeric partner RXR, and receptor-chromatin interactions. Moreover, molecular dynamic simulation approaches predicted that shortened THRß-LBD due to point mutations contributes proportionally to the loss of structural integrity and receptor stability. Deviant subcellular localization and compromised transcriptional function were apparent with these truncated variants. Present study shows that 'mitotic bookmarking' property of some THRß variants is also affected. The study highlights that structural and conformational attributes of THRß are necessary for normal receptor functioning, and any deviations may contribute to the underlying cause of the inflicted diseases. We anticipate that insights derived herein may contribute to improved mechanistic understanding to assess disease predisposition.

Mitotic genome-bookmarking by nuclear hormone receptors: A novel dimension in epigenetic reprogramming and disease assessment.

Arrival of multi-colored fluorescent proteins and advances in live cell imaging has immensely contributed to our understanding of intracellular trafficking of nuclear receptors and their roles in gene regulatory functions. These regulatory events need to be faithfully propagated from progenitor to progeny cells. This is corroborated by multiple converging mechanisms that include histone modifications and lately, the phenomenon of 'mitotic genome-bookmarking' by specific transcription factors. This phenomenon refers to the retention and feed-forward transmission of progenitor's architectural blueprint of active transcription status which is silenced and preserved during mitosis. Upon mitotic exit, this phenomenon ensures accurate reactivation of transcriptome, proteome, cellular traits and phenotypes in the progeny cells. In addition to diverse modes of genome-bookmarking by nuclear receptors, a correlation between disease-associated receptor polymorphism and disruption of this phenomenon is apparent. However, breakthrough technologies shall reveal finer details of this phenomenon to help achieve normalcy in receptor-specific diseases.

Hereditary Vitamin D-Resistant Rickets (HVDRR) associated SNP variants of vitamin D receptor exhibit malfunctioning at multiple levels.

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. It is a primary regulator of calcium and phosphate homeostasis required for skeleton and bone mineralization. Vitamin D in active form 1α,25 dihydroxyvitamin-D3 mediates its cellular functions by binding to VDR. Active VDR forms heterodimers with partner RXR (retinoid X receptor) to execute its physiological actions. HVDRR (Hereditary Vitamin D-Resistant Rickets) is a rare genetic disorder that occurs because of generalized resistance to the 1α,25(OH)2D3. HVDRR is caused by the polymorphic variations in VDR gene leading to defective intestinal calcium absorption and mineralization of newly forming bones. Using point and deletion SNPs of VDR we have studied several HVDRR-associated SNP variants for their subcellular dynamics, transcriptional functions, 'genome bookmarking', heterodimeric interactions with RXR, and receptor stability. We previously reported that VDR is a 'mitotic bookmarking factor' that remains constitutively associated with the mitotic chromatin to inherit 'transcriptional memory', however the mechanistic details remained unclear. We document that 'genome bookmarking' property by VDR is critically impaired by naturally occurring HVDRR-associated point and deletion variants found in patients. Furthermore, these HVDRR-associated SNP variants of VDR were found to be compromised in transcriptional function, nuclear translocation, protein stability and intermolecular interactions with its heterodimeric partner RXR. Intriguingly, majority of these disease-allied functional defects failed to be rescued by RXR. Our findings suggest that the HVDRR-associated SNP variations influence the normal functioning of the receptor, and this derived understanding may help in the management of disease with precisely designed small molecule modulators.

Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging.

Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples.HighlightsDiverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk.Nuclear receptors serve as 'xenosensors' for assessing the presence of xenobiotics in milk.Nuclear import of steroid receptors with (ant)agonist can be examined in live cells.Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction.A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.

Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay.

The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay via AR response can be conducted for assessing the (anti)androgenic ligands present in food chain including milk. Therefore, the cell line was further used to screen the (anti)androgenic activity of a total of 40 milk fat samples procured as raw or commercial milk. Some of the raw and commercial milk fat samples distinctly showed antiandrogenic activities. Subsequently, some commonly used environmental chemicals were also evaluated for their (anti)androgenic activities. Initial observations with molecular docking studies of experimental compounds were performed to assess their interaction with AR ligand binding domain. Furthermore, (anti)androgenic activities of these compounds were confirmed by performing luciferase assay using the HepARE-Luc cell line. None of the test compounds showed androgenic activities rather some of them like Bisphenol A (BPA) and rifamycin showed antiandrogenic activities. In conclusion, our results provide a valuable information about the assessment of (anti)androgenic activities present in milk samples. Overall, it is proposed that a robust cell-based CALUX assay can be used to assess the (anti)androgenic activities present in milk which can be attributed to different environmental chemicals present therein.

Mitotic genome bookmarking by nuclear receptor VDR advocates transmission of cellular transcriptional memory to progeny cells.

Mitosis is an essential process for the self-renewal of cells that is accompanied by dynamic changes in nuclear architecture and chromatin organization. Despite all the changes, the cell manages to re-establish all the parental epigenetic marks, post-mitotically. Recent reports suggest that some sequence-specific transcription factors remain attached to mitotic chromatin during cell division to ensure timely reactivation of a subset of transcription factors necessary to maintain cell identity. These mitotically associated factors are suggested to act as 'genome bookmarking factors' and the phenomenon is termed 'genome bookmarking'. Here, we studied this phenomenon with Vitamin D Receptor (VDR), a key regulator of calcium and phosphate homeostasis and a member of the nuclear receptor superfamily. This study, for the first time, has confirmed VDR as a mitotic bookmarking factor that may be playing a crucial role in the maintenance of cell identity and genome bookmarking. Full 'DNA binding domain (DBD)' present in VDR was identified as essential for enrichment of VDR on mitotic chromatin. Furthermore, the study also demonstrates that VDR evokes mitotic chromatin binding behaviour in its heterodimeric partner Retinoid X receptor (RXR). Interestingly, for promoting bookmarking behaviour in RXR, both DBD and/or ligand-binding domain (LBD) in conjunction with hinge region of VDR were required. Additionally, ChIP analysis showed that VDR remains associated with DR3 (direct repeat 3) region of its specific target gene promoter CYP24A1(Cytochrome P450 family 24 subfamily A member1), during mitosis. Altogether, our study illustrates a novel function of VDR in the epigenetic transmission and control of expression of target proteome for maintenance of cell identity and traits in progeny cells.

Global footprints of organochlorine pesticides: a pan-global survey.

Organochlorine pesticides (OCPs) are ubiquitous environmental contaminants widely used all over the world. These chlorinated hydrocarbons are toxic and often cause detrimental health effects because of their long shelf life and bioaccumulation in the adipose tissues of primates. OCP exposure to humans occurs through skin, inhalation and contaminated foods including milk and dairy products, whereas developing fetus and neonates are exposed through placental transfer and lactation, respectively. In 1960s, OCPs were banned in most developed countries, but because they are cheap and easily available, they are still widely used in most third world countries. The overuse or misuse of OCPs has been rising continuously which pose threats to environmental and human health. This review reports the comparative occurrence of OCPs in human and bovine milk samples around the globe and portrays the negative impacts encountered through the long history of OCP use.

Current Insights and Advancements in Head and Neck Cancer: Emerging Biomarkers and Therapeutics with Cues from Single Cell and 3D Model Omics Profiling.

Head and neck cancer (HNC) is among the ten leading malignancies worldwide, with India solely contributing one-third of global oral cancer cases. The current focus of all cutting-edge strategies against this global malignancy are directed towards the heterogeneous tumor microenvironment that obstructs most treatment blueprints. Subsequent to the portrayal of established information, the review details the application of single cell technology, organoids and spheroid technology in relevance to head and neck cancer and the tumor microenvironment acknowledging the resistance pattern of the heterogeneous cell population in HNC. Bioinformatic tools are used for study of differentially expressed genes and further omics data analysis. However, these tools have several challenges and limitations when analyzing single-cell gene expression data that are discussed briefly. The review further examines the omics of HNC, through comprehensive analyses of genomics, transcriptomics, proteomics, metabolomics, and epigenomics profiles. Patterns of alterations vary between patients, thus heterogeneity and molecular alterations between patients have driven the clinical significance of molecular targeted therapies. The analyses of potential molecular targets in HNC are discussed with connotation to the alteration of key pathways in HNC followed by a comprehensive study of protein kinases as novel drug targets including its ATPase and additional binding pockets, non-catalytic domains and single residues. We herein review, the therapeutic agents targeting the potential biomarkers in light of new molecular targeted therapies. In the final analysis, this review suggests that the development of improved target-specific personalized therapies can combat HNC's global plight.

Upsurge in autophagy, associated with mifepristone-treated polycystic ovarian condition, is reversed upon thymoquinone treatment.

Polycystic ovarian syndrome (PCOS) is a multi-factorial gynecological endocrine disorder. It affects fertility in women and also predisposes to insulin resistance, type 2 diabetes mellitus, obesity etc. Earlier, significance of autophagy has been explored in PCOS-related metabolic disorders and during normal folliculogenesis. Increasing evidences reveal connection of autophagy with chronic inflammatory behaviour, an associated phenomena in polycystic ovaries. However, understanding of the association of autophagy with PCOS is still obscure. This study reveals that increased autophagy in mifepristone (RU486) treated KK-1 cells and in vivo PCO rat model is characterized by upregulated Androgen Receptor (AR) expression and downregulated PCO biomarker aromatase. The prevalence of autophagy has been observed to be concomitant with increased expression of two autophagic markers Beclin1 and MAP-LC3-II while the autophagy substrate p62/SQSTM1 was downregulated. Immunohistochemical staining revealed increased localization of MAP-LC3 in the compacted granulosa layers of the follicular cysts in the PCO model. The PCO rat models also demonstrated augmented levels of p65, the active subunit of NF-κB, which acts as a transcriptional regulator of several pro-inflammatory factors. NF-κB repressor and anti-inflammatory herbal drug thymoquinone, known to alleviate PCO condition, downregulated autophagy modules substantially. Pre-treatment with thymoquinone upregulated aromatase, reduced AR levels and decreased autophagic markers as well as p65 levels, simulating super-ovulated condition. In conclusion, the anti-inflammatory phytochemical thymoquinone alleviated PCO condition.

Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions.

Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in 'gene-bookmarking'. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.

Prototype of a Smart Microfluidic Platform for the Evaluation of SARS-Cov-2 Pathogenesis, Along with Estimation of the Effectiveness of Potential Drug Candidates and Antigen-Antibody Interactions in Convalescent Plasma Therapy.

Originating in China during December 2019, the novel corona-virus, SARS-CoV-2, has created mayhem worldwide in a very short time. The outbreak has been so rapid and widespread that the only option to treat the patients was administering drugs already available in the market like chloroquine/hydroxychloroquine (an antimalarial drug) and remedesivir. A large number of patients have been cured but the attribution to survival by these drugs has been controversial. Till date, we do not have any specific drug or vaccine available for COVID-19 and the pandemic seems to be far from over. To handle the current challenges posed by the outbreak effectively, we need to employ innovative interdisciplinary approaches. Organ-on-chip (OOC), particularly lung-on-chip, is one such approach which combines the potential of microfluidics, cell culture and molecular biology into a single miniaturised platform. The device is realized to be capable of simulating in-vivo physiological responses of an organ. In the current study, an OOC, which is a multichannel 3D cell culture microfluidic device, is made via soft lithography technique, using polydimethylsiloxane-polymer and diverse polymeric porous/semipermeable membranes. Several polymer membranes i.e. PDMS, polyvinylidene fluoride (PVDF), nitrocellulose, polyester etc., integrated into the microdevices, were efficiently explored to realize their better cell-adhesion and viability property. We also propose for the application of a simple, smart and cost-effective lung-on-chip platform to study the SARS-CoV-2 pathogenesis in humans, drug toxicity testing and provide insights into antigen-antibody interactions. This platform will enable us to study multiple phenomena at a micro-level generating more reliable data and a better understanding of the underlying mechanisms of SARS-CoV-2 infection and pathogenesis.

Influence of signaling kinases on functional dynamics of nuclear receptor CAR.

Constitutive androstane receptor (CAR) is a xenobiotic nuclear receptor known to regulate genes involved in key physiological processes like drug metabolism, maintenance of energy homeostasis, and cell proliferation. Owing to the diverse regulatory roles played by the receptor, it is critical to understand the precise cellular signals that dictate functional dynamics of CAR. With the objective of exploring the hitherto unknown regulatory pathways modulating CAR, we subjected the CAR protein sequence to a kinase prediction tool and identified several kinases recognizing CAR as a substrate. Using fluorescence live cell imaging and specific inhibitors it was observed that CAR functions under the regulation of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) signaling cascade. Additionally, insulin-like growth factor 1 (IGF1)-mediated inhibition of GSK3 also induced nuclear translocation of CAR linking CAR to the Akt signaling pathway. Identification of T38 residue of CAR as the GSK3 target site further substantiated our observations. Taking cues from these findings, we propose a hypothetical model elucidating the GSK3-mediated regulation of CAR dynamics through the involvement of Akt pathway. Further research into this area is expected to provide novel therapeutic targets in disease conditions like type 2 diabetes and hepatocellular carcinoma.

A comprehensive evaluation of anti-diabetic drugs on nuclear receptor PXR platform.

Pregnane & Xenobiotic Receptor (PXR), one of the members of nuclear receptor superfamily, acts as a 'master-regulator' of drug metabolism and disposition machinery (DMD). Activation of PXR enables detoxification and elimination of toxic xenobiotics/endobiotics, and defends our body against chemical insults. On the contrary, PXR activation also imposes a serious concern for drug-drug interactions (DDIs). Such DDIs could either decrease the efficacy or lead to accumulation of co-administered drugs at toxic level. Therefore, it is desirable that during drug development process the small drug molecules are screened on PXR-platform prior to their clinical trial and prevent late stage failures. In view of this, we have selected a group of anti-diabetic drug molecules to examine if the success and potential failure of small molecule modulators can be pre-assessed and judiciously correlated on PXR platform. For this purpose, we have examined the PXR activation potential of the selected anti-diabetic drugs. Subsequent to screening of these anti-diabetic drugs, we elaborated the study further with rosiglitazone and pioglitazone (thiazolidinediones, TZDs) which are oral anti-diabetic formulations and have been in controversy owing to their association with cardiotoxicity and bladder cancer respectively. Our study revealed that some of the selected anti-diabetic drugs possess PXR activation potential, implying that these can up-regulate the expression of CYP3A4, UGT1A1, MDR1 and thereby can be predicted to inflict undesirable consequences.

Phosphorylation of HSP90 by protein kinase A is essential for the nuclear translocation of androgen receptor.

The androgen receptor (AR) is often activated in prostate cancer patients undergoing androgen-ablative therapy because of the activation of cellular pathways that stimulate the AR despite low androgen levels. In many of these tumors, the cAMP-dependent protein kinase A (PKA) pathway is activated. Previous studies have shown that PKA can synergize with low levels of androgen to enhance androgen signaling and consequent cell proliferation, leading to castration-resistant prostate cancer. However, the mechanism by which PKA causes AR stimulation in the presence of low/no androgen is not established yet. Here, using immunofluorescence immunoblotting assays, co-immunoprecipitation, siRNA-mediated gene silencing, and reporter gene assays, we demonstrate that PKA activation is necessary for the phosphorylation of heat shock protein (HSP90) that binds to unliganded AR in the cytoplasm, restricting its entry into the nucleus. We also found that PKA-mediated phosphorylation of the Thr89 residue in HSP90 releases AR from HSP90, enabling AR binding to HSP27 and its migration into the nucleus. Substitution of the Thr89 in HSP90 prevented its phosphorylation by PKA and significantly reduced AR transactivation and cellular proliferation. We further observed that the transcription of AR target genes, such as prostate-specific antigen (PSA), is also lowered in the HSP90 Thr89 variant. These results suggest that using a small-molecule inhibitor against the HSP90 Thr89 residue in conjunction with existing androgen-ablative therapy may be more effective than androgen-ablative therapy alone in the treatment of prostate cancer patients.

Nuclear localization signal region in nuclear receptor PXR governs the receptor association with mitotic chromatin.

In recent years, some transcription factors have been observed to remain associated with mitotic chromatin. Based on these observations, it is suggested that these chromatin-bound transcription factors may serve as 'epigenetic marks' for transmission of pattern of gene expression from progenitor to progeny cells. In this context, our laboratory has reported that nuclear receptor PXR, a master regulator of xenobiotic metabolism, remains constitutively associated with mitotic chromatin. However, the region responsible for this interaction with chromatin remained unknown. In this study, we have shown, for the first time, that mitotic chromatin association of this factor is mediated by the combined action of two zinc fingers present in the DNA-binding domain of PXR. Overall, the nuclear localization signal (NLS) region appears to play a major role in this interaction with mitotic chromatin. Also, we have identified a sub-region of 11 amino acid residues within NLS region of PXR (R66-76R) essential for receptor interaction with the mitotic chromatin. Interestingly, this minimal region is sequence-specific and independent of its basic charge. We have termed this minimal sub-region as 'mitotic chromatin binding-determining region' (MCBR). It is suggested that this receptor region is essential for activation of its target genes. Additionally, we have shown that PXR remains associated with the everted repeat (ER6) region of its major target gene, CYP3A4 promoter during mitosis implying its suggested role in 'gene bookmarking'.

Dynamic Hyaluronan drives liver endothelial cells towards angiogenesis.

BACKGROUND: Angiogenesis, the formation of new blood vessels from pre-existing vasculature is essential in a number of physiological processes such as embryonic development, wound healing as well as pathological conditions like, tumor growth and metastasis. Hyaluronic acid (HA), a high molecular weight polysaccharide, major component of extracellular matrix is known to associate with malignant phenotypes in melanomas and various other carcinomas. Hyaluronic acid binding protein 1 (HABP1) has been previously reported to trigger enhanced cellular proliferation in human liver cancer cells upon its over-expression. In the present study, we have identified the HA mediated cellular behaviour of liver endothelial cells during angiogenesis. METHODS: Endothelial cells have been isolated from perfused liver of mice. Cell proliferation was studied using microwell plates with tetrazole dye. Cell migration was evaluated by measuring endothelial monolayer wound repair as well as through transwell migration assay. Alterations in proteins and mRNA expression were estimated by immunobloting and quantitative real time PCR using Applied Biosystems. The paraformaldehyde fixed endothelial cells were used for immuno- florescence staining and F-actin detection with conjugated antibodies. The images were captured by using Olympus florescence microscope (IX71). RESULTS: We observed that administration of HA enhanced cell proliferation, adhesion, tubular sprout formation as well as migration of liver endothelial cells (ECs). The effect of HA in the rearrangement of the actins confirmed HA -mediated cytoskeleton re-organization and cell migration. Further, we confirmed enhanced expression of angiogenic factors like VEGF-A and VEGFR1 in endothelial cells upon HA treatment. HA supplementation led to elevated expression of HABP1 in murine endothelial cells. It was interesting to note that, although protein levels of ß- catenin remained unaltered, but translocation of this protein from membrane to nucleus was observed upon HA treatment, suggesting its role not only in vessel formation but also its involvement in angiogenesis signalling. CONCLUSIONS: The elucidation of molecular mechanism (s) responsible for HA mediated regulation of endothelial cells and angiogenesis contributes not only to our understanding the mechanism of disease progression but also offer new avenues for therapeutic intervention.

Stable cellular models of nuclear receptor PXR for high-throughput evaluation of small molecules.

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the ligand-modulated transcription factors belonging to nuclear receptor superfamily. Though PXR is now well-established as a 'xenosensor', regulating the central detoxification and drug metabolizing machinery, it has also emerged as a key player in several metabolic disorders. This makes PXR attractive to both, researchers and pharmaceutical industry since clinical success of small drug molecules can be pre-evaluated on PXR platform. At the early stages of drug discovery, cell-based assays are used for high-throughput screening of small molecules. The future success or failure of a drug can be predicted by this approach saving expensive resources and time. In view of this, we have developed human liver cell line-based, dual-level screening and validation protocol on PXR platform having application to assess small molecules. We have generated two different stably transfected cell lines, (i) a stable promoter-reporter cell line (HepXREM) expressing PXR and a commonly used CYP3A4 promoter-reporter i.e. XREM-luciferase; and (ii) two stable cell lines integrated with proximal PXR-promoter-reporter (Hepx-1096/+43 and Hepx-497/+43). Employing HepXREM, Hepx-1096/+43 and Hepx-497/+43 stable cell lines > 25 anti-cancer herbal drug ingredients were screened for examining their modulatory effects on a) PXR transcriptional activity and, b) PXR-promoter activity. In conclusion, the present report provides a convenient and economical, dual-level screening system to facilitate the identification of superior therapeutic small molecules.

Heterodimerization of Retinoid X Receptor with Xenobiotic Receptor partners occurs in the cytoplasmic compartment: Mechanistic insights of events in living cells.

Retinoid X Receptor (RXR) serves as the heterodimeric partner of two major xenobiotic nuclear receptors, Pregnane and Xenobiotic Receptor (PXR) and Constitutive Androstane Receptor (CAR). These receptors are primarily involved in the metabolism and clearance of endobiotics and xenobiotics (including clinical drugs) from the body. Here, we report for the first time that intermolecular interactions between RXR-PXR and RXR-CAR occurs in the cytoplasmic compartment of the cell in a ligand-independent manner. These interactions lead to nuclear import of the heterodimeric complex thereby making them competent for chromatin binding and transactivation of target genes. To explore the cellular site involved in the process of heterodimerization we created various RFP- and GFP-tagged receptor chimeras and also the mutants of their nuclear localization signal (NLS). From the study it is apparent that NLS of PXR/CAR/RXR play a major role in the import of the heterodimeric complex from the cytoplasm to the nucleus in a ligand-independent manner. We observed that along with the heterodimeric partner and/or respective ligand a functional NLS is necessary for activation of target gene. The data suggests that RXR is the major driving force to import the heterodimeric complex into the nucleus since the mutation in the NLS region of RXR weakens this import process dramatically, whereas mutations in the NLS regions of PXR and CAR have little or no significant effect. This RXR-dependent nuclear translocation of the heterodimeric complex also modulates the individual transcriptional activity of PXR and CAR by increasing the basal transcriptional activity. Finally, it is documented that the heterodimerization of RXR with both the partners (PXR, CAR) occurs in the cytoplasm and implies that these dynamic interactions have functional and regulatory attributes in gene expression. In addition, this RXR-dependent enhancement of the transcriptional activity of PXR and CAR may be utilized for evaluating the receptor-drug interactions.

Analyzing the role of CagV, a VirB8 homolog of the type IV secretion system of Helicobacter pylori.

The type IV secretion system of Helicobacter pylori (Cag-T4SS) is composed of ~ 27 components including a VirB8 homolog, CagV. We have characterized CagV and reported that it is an inner membrane protein and, like VirB8, forms a homodimer. Its stability is not dependent on the other Cag components and the absence of cagV affects the stability of only CagI, a protein involved in pilus formation. CagV is not required for the stability and localization of outer membrane subcomplex proteins, but interacts with them through CagX. It also interacts with the inner membrane-associated components, CagF and CagZ, and is required for the surface localization of CagA. The results of this study might help in deciphering the mechanistic contributions of CagV in the Cag-T4SS biogenesis and function.