Interferon-induced Proteins With Tetratricopeptide Repeats Research Articles

Thymoquinone regulates gene expression levels in the estrogen metabolic and interferon pathways in MCF7 breast cancer cells

New drugs are continuously being developed for the treatment of patients with estrogen receptor-positive breast cancer. Thymoquinone is one of the drugs that exhibits anticancer characteristics based on in vivo and in vitro models. This study further investigates the effects of thymoquinone on human gene expression using cDNA microarray technology. The quantification of RNA samples was carried out using an Agilent 2100 Bioanalyser to determine the RNA integrity number (RIN). The Agilent Low Input Quick Amplification Labelling kit was used to generate cRNA in two-color microarray analysis. Samples with RIN >9.0 were used in this study. The universal human reference RNA was used as the common reference. The samples were labelled with cyanine-3 (cye-3) CTP dye and the universal human reference was labelled with cyanine-5 (cye-5) CTP dye. cRNA was purified with the RNeasy Plus Mini kit and quantified using a NanoDrop 2000c spectrophotometer. The arrays were scanned data analysed using Feature Extraction and GeneSpring software. Two-step qRT-PCR was selected to determine the relative gene expression using the High Capacity RNA-to-cDNA kit. The results from Gene Ontology (GO) analysis, indicated that 8 GO terms were related to biological processes (84%) and molecular functions (16%). A total of 577 entities showed >2-fold change in expression. Of these entities, 45.2% showed an upregulation and 54.7% showed a downregulation in expression. The interpretation of single experiment analysis (SEA) revealed that the cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and UDP glucuronosyltransferase 1 family, polypeptide A8 (UGT1A8) genes in the estrogen metabolic pathway were downregulated significantly by 43- and 11-fold, respectively. The solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11 (SLC7A11) gene in the interferon pathway, reported to be involved in the development of chemoresistance, was downregulated by 15-fold. The interferon-induced protein with tetratricopeptide repeats (IFIT)1, IFIT2, IFIT3, interferon, α-inducible protein (IFI)6 (also known as G1P3), interferon regulatory factor 9 (IRF9, ISGF3), 2′–5′-oligoadenylate synthetase 1, 40/46 kDa (OAS1) and signal transducer and activator of transcription 1 (STAT1) genes all showed changes in expression following treatment with thymoquinone. The caspase 10, apoptosis-related cysteine peptidase (CASP10) gene was activated and the protein tyrosine phosphatase, receptor type, R (PTPRR) and myocyte enhancer factor 2C (MEF2C) genes were upregulated in the classical MAPK and p38 MAPK pathways. These findings indicate that thymquinone targets specific genes in the estrogen metabolic and interferon pathways.

The TLR signalling adaptor TRIF/TICAM-1 has an N-terminal helical domain with structural similarity to IFIT proteins

TRIF/TICAM-1 (TIR domain-containing adaptor inducing interferon-β/TIR domain-containing adaptor molecule 1) is the adaptor protein in the Toll-like receptor (TLR) 3 and 4 signalling pathway that leads to the production of type 1 interferons and cytokines. The signalling involves TIR (Toll/interleukin-1 receptor) domain-dependent TRIF oligomerization. A protease-resistant N-terminal region is believed to be involved in self-regulation of TRIF by interacting with its TIR domain. Here, the structural and functional characterization of the N-terminal domain of TRIF (TRIF-NTD) comprising residues 1-153 is reported. The 2.22 Å resolution crystal structure was solved by single-wavelength anomalous diffraction (SAD) using selenomethionine-labelled crystals of TRIF-NTD containing two additional introduced Met residues (TRIF-NTDA66M/L113M). The structure consists of eight antiparallel helices that can be divided into two subdomains, and the overall fold shares similarity to the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both the recognition of viral RNA and modulation of innate immune signalling. Analysis of TRIF-NTD surface features and the mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or interaction with other signalling molecules or ligands. TRIF-NTD suppresses TRIF-mediated activation of the interferon-β promoter, as well as NF-κB-dependent reporter-gene activity. These findings thus identify opportunities for the selective targeting of TLR3- and TLR4-mediated inflammation.

Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein–protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5′-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5′-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.Supplementary informationThe online version of this article (doi:10.1038/nature11783) contains supplementary material, which is available to authorized users.

Abstract 3996: Genomic analysis in breast cancer cells after metallothionein-2A silencing

Abstract Introduction Breast cancer is the most prevalent type of cancer reported in women and mammoth effort has been put in to understand its etiology. Metallothionein (MT), a divalent cation binding protein, was previously shown to be associated with histopathological parameters of breast cancer and mediated cancer progression. MTs belong to a family of ubiquitous, low-molecular weight house-keeping proteins. In human, 10 known functional isoforms of MT, namely 1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4, encode 4 classes of MT proteins. MT-2A has been shown to be the most abundant isoform in breast cancers and was reported to promote cell proliferation and chemo-resistance. Screening for MT-2A and its associated genes may elucidate the role it plays in the progression of breast neoplasia and help devise therapeutic strategies to combat this disease. Methods Chemically synthesized small interfering RNA (siRNA) targeting the MT-2A isoform was employed to down-regulate its expression in MCF-7 breast cancer cells and validated via real-time Polymerase Chain Reaction (PCR) and immunocytochemistry. Cell viability and drug sensitivity of transfected cells were evaluated post silencing using a tetrazolium based (MTT) assay. RNAs extracted from MT-2A silenced cells were hybridized to Affymetrix gene chips for genome-wide screening and compared to control group treated with scrambled sequence siRNA. Analysis was performed across multiple platforms such as GeneSpring, dChip and GCOS to “fish” for genes associated with MT-2A down-regulation. Candidate genes that demonstrated at least 2 fold change statistically (p<0.05) were further screened via real-time PCR to confirm their association with MT-2A silencing. Results Silencing efficiencies of 88% and 90% were achieved through the use of two different MT-2A specific siRNA duplexes (siMT-2A_1 and siMT-2A_2) respectively. Cells exhibited reduced viability (∼10% lesser) and more evidently (up to 76% lesser) after Doxorubicin administration at IC50 post MT-2A silencing. Results obtained from the cDNA microarray indicate that interferon inducible genes such as interferon induced proteins with tetratricopeptide repeats (IFITs), interferon induced transmembrane protein 1 (IFITM1) and oligoadenylate synthetase (OAS) accounted for more than 54% (12/22) of the candidate gene list and these genes showed concordant fold changes in real-time PCR analysis. Conclusion The results suggest that MT-2A may regulate interferon signaling directly or indirectly. This set of candidate genes are known to play important roles in apoptosis and chemo-resistance. The molecular interaction between MT-2A and members of the interferon inducible proteins warrants further attention and we postulate that they may work co-operatively to contribute to tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3996.