Screening Of Expression Library Research Articles

Experimental Strategies for Cloning or Identifying Genes Encoding DNA-Binding Proteins

This article describes experimental strategies for cloning or identifying genes encoding DNA-binding proteins. DNA-binding proteins are most commonly identified by electrophoretic mobility-shift assay (EMSA) or DNase I footprinting. To identify the gene encoding a protein detected by EMSA or DNase footprinting, the protein often needs to be purified and its sequence analyzed, as described here. Other methods are also available which do not resort to protein purification, including the one-hybrid screen, in vitro expression library screen, and mammalian expression cloning. These methods are outlined, and their advantages and disadvantages are discussed.

Microarray-based method for screening of immunogenic proteins from bacteria

BackgroundDetection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.ResultsFour proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity.We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.ConclusionsThe method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Absence of markers of betaretrovirus infection in human pulmonary adenocarcinoma

A proportion of human pulmonary adenocarcinomas has been shown previously to express an antigen related to the Gag protein of a betaretrovirus, Jaagsiekte sheep retrovirus, that causes ovine pulmonary adenocarcinoma. To investigate further the hypothesis that a retrovirus might be present in human lung adenocarcinoma, we examined specimens from patients with lung cancer for evidence of retroviral infection by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting and cDNA library screening. Thirty-eight percent of the tumor samples analyzed were positive by immunohistochemistry for Gag-related antigen of Jaagsiekte sheep retrovirus. However, this antigen was not detected by immunoblotting using the same antiserum. In addition, plasma samples from the patients did not contain antibodies reacting with Gag proteins from Jaagsiekte sheep retrovirus or other betaretroviruses on immunoblots. Reverse transcriptase-polymerase chain reaction identified the expression of endogenous betaretroviruses in tumor tissue and in normal lung tissue, but no specific provirus was associated with tumor. Expression library screening did not identify the Gag-reactive antigen. This study has confirmed the expression of a Jaagsiekte sheep retrovirus Gag-related antigen in some human lung tumors but additional evidence of betaretroviral infection was not obtained. While these data do not rule out a role for a retrovirus in human pulmonary adenocarcinomas, they suggest that, if such a virus is present, it is unrelated to known betaretroviruses.

Cryptic Epitopes Induce High-Titer Humoral Immune Response in Patients with Cancer

In search of novel markers for diagnosis, prognosis, and therapy of cancer, screening of rcDNA expression libraries with patient's sera has been established as a valuable tool for identification of cancer-specific Ags. Interestingly, besides the expected humoral responses to annotated proteins, patients with cancer were frequently found to have serum Abs that bind to peptides without homology to known proteins. So far, the nature of these unconventional epitopes and their possible significance in tumor immunology have never been thoroughly investigated. In our study, we specifically analyzed humoral immune response toward such peptides in patients with pancreatic or breast cancer using yeast-displayed cDNA expression libraries derived from tumor tissue. A detailed analysis of the identified peptides revealed that they originated from translation of sequences outside annotated open reading frames and may derive from the use of alternative start codons or from DNA indel mutations. In several cases, the corresponding mRNA templates have a known association with cancer. In a final analysis, we were able to detect one of these tumor Ags in cancer tissue arrays by a selected Fab-Ab. We conclude that cryptic epitopes may elicit specific humoral immune responses in patients with cancer and thus play a role in immunologic surveillance. Due to the high prevalence of immune responses against some of the peptides, they may also be valuable markers for cancer diagnosis, prognosis, or therapy monitoring.

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.

Tumor antigen analysis in neuroblastoma by serological interrogation of bioinformatic data

The identification of tumor antigens remains a major objective in tumor immunology, especially in pediatric malignancies where solid tumors often do not express a single dominant antigen. Methods such as the Serological Screening of Recombinant cDNA Expression Libraries (SEREX) have been used in the discovery of tumor-expressed proteins by virtue of their ability to induce an antibody response. To focus and accelerate this approach, we first identified candidate antigens by gene expression profiling data from clinical neuroblastoma specimens and then used an animal model to generate an antibody response to an engineered cell-based vaccine. Candidate tumor antigens were expressed as recombinant proteins in a mammalian system and screened for antibody recognition using serum from mice vaccinated with a neuroblastoma cell-based vaccine engineered to express CD80 and CD86, with or without Treg depletion. Through this procedure, the never in mitosis A (NIMA)-related kinase NEK2 was identified as a tumor-associated antigen. Direct testing of serum from patients newly diagnosed with neuroblastoma showed specific serological responses in two of 20 patients. Although NEK2 was not universally recognized, it may serve as a tumor antigen for some patients.

A Viral Vaccine Encoding Prostate-Specific Antigen Induces Antigen Spreading to a Common Set of Self-Proteins in Prostate Cancer Patients

We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding prostate-specific antigen (PSA) with radiotherapy in patients with localized prostate cancer. Here, we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome. Pretreatment and posttreatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serologic screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses. Western blotting revealed treatment-associated autoantibody responses in 15 of 33 (45.5%) patients treated with vaccine + EBRT versus 1 of 8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that 7 of 33 patients (21.2%) treated with vaccine + EBRT showed a vaccine-associated autoantibody response to four ubiquitously expressed self-antigens: DIRC2, NDUFS1, MRFAP1, and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend toward decreased biochemical-free survival. Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self-antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self-proteins, which may influence the efficacy of vaccination.

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

BackgroundFluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.ResultsHere we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M−1 cm−) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.ConclusionsThe spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

Baculovirus for Eukaryotic Protein Display

The baculovirus Autographa californica nuclear polyhedrosis virus is an attractive candidate for eukaryotic virus display. A variety of strategies exists to incorporate and present target proteins on the surface of infected insect cells as well as on budded virions. Native baculovirus proteins such as the major envelope protein and the capsid protein, but also foreign scaffolds such as the vesicular stomatitis virus G-1 protein or the influenza A virus hemagglutinin serve as fusion partners for surface presentation of target proteins. The purposes of surface display are manifold; efficient presentation of antigenic epitopes on budded virions serves to induce specific immune response, designed surface odifications alter the binding properties and host specificities of the baculovirus to e.g. mammalian cells and help to enhance mammalian cell transduction. Eukaryotic surface display libraries based on the baculovirus system allow selecting for specific binding proteins while providing post translational modifications. Here we describe the different possibilities and strategies to accessibly present foreign proteins and peptides on the surface of insect cells and baculoviruses, as well as the applications thereof, including vector design, cloning strategies and construction and screening of surface expression libraries.

Abstract 2742: Identification of tumor antigens in neu transgenic mice may provide biomarkers useful for the early detection and diagnosis of human breast cancer

Abstract Early detection of breast cancer is crucial for better prognosis and successful treatment of the patients. Development of novel methods for screening for breast cancer is needed for early detection and diagnosis. The human immune system responds to tumor-specific antigens in the pre-malignant stage of breast cancer and produces specific antibodies, which can be detected as potential breast cancer biomarkers. Due to the limited availability of well-annotated pre-cancer and post-cancer sera from human subjects, we performed serial serum collection in a neu transgenic mouse model from the age of 10 weeks to the terminal stage of disease. Using a technique called serological screening of cDNA expression library (SEREX), a high-throughput method to rapidly screen recombinant proteins expressed in a bacteriophage-based cDNA library, we have previously identified tumor antigens in the late stage of neu breast cancers. The mouse tumors have similar immunogenicity as their human counterparts due to the fact that some of these antigens are also immunogenic in human. Indeed, we have found that one mouse antigen called gelsolin, shares 93% homology with human gelsolin and has the highest response to human serum antibody among other antigens. In an ELISA analysis using the serum samples from 50 breast cancer cases and 50 normal donors, the area-under-curve (AUC) value of the receiver-operating-characteristic (ROC) curve of gelsolin ELISA detects more than 70% of cancer patients. Using the SEREX technique, we sought to further identify the early-stage serum antibody biomarkers by comparing the pre-cancer, early stage, and late stage sera from the same mouse. We have identified three early-stage biomarkers, A20/TNFaip3, Rpl5, and Pdhx, in neu mice. In summary, we conclude that identification of tumor antigens in the neu transgenic animal model is useful for the discovery of serum antibody biomarkers for early detection and diagnosis. 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 2742.

FOXK2 transcription factor is a novel G/T-mismatch DNA binding protein

DNA mismatch repair is an important mechanism in the prevention of mutations. We reported the existence in the HL60 cell line of a novel G/T-mismatch DNA-binding protein (nGTBP) requiring strict DNA sequences. In this report, we identify the FOXK2 transcription factor as the nGTBP. FOXK2 fragments were obtained as the only clones with specifically binding activity to the G/T-mismatch DNA by screening of a human brain expression library. The recombinant forkhead domain of FOXK2 specifically recognized the G/T-mismatch DNA. The forkhead domain also recognized hypoxanthine/T and G/uracil, derived from the deamination of the exocyclic amino groups of A/T and G/C, respectively. An electrophoretic mobility shift assay (EMSA) analysis using HL60 cell nuclear extract and antibody raised against FOXK2 resulted in the exclusive binding of FOXK2 to G/T-mismatch DNA. Furthermore, FOXK2 bound to G/T-mismatch DNA with higher affinity than 'match' FOXK2 consensus DNA. We therefore propose that FOXK2 is a G/T-mismatch DNA-binding protein and a deaminated DNA-binding protein.

Tpl2/AP-1 Enhances Murine Gammaherpesvirus 68 Lytic Replication

How cellular factors regulate gammaherpesvirus lytic replication is not well understood. Here, through functional screening of a cellular kinase expression library, we identified mitogen-activated protein kinase kinase kinase 8 (MAP3K8/Tpl2) as a positive regulator of murine gammaherpesvirus 68 (MHV-68 or gammaHV-68) lytic gene expression and replication. Tpl2 enhances MHV-68 lytic replication by upregulating lytic gene expression and promoter activities of viral lytic genes, including RTA and open reading frame 57 (ORF57). By screening a cellular transcription factor library, we identified the Fos AP-1 transcription factor as a downstream factor that is both necessary and sufficient for mediating the enhancement of MHV-68 lytic replication by Tpl2. In addition, Tpl2 stimulates the promoter activities of key viral lytic genes, including RTA and ORF57, in an AP-1-dependent manner. We identified an AP-1-responsive element on the MHV-68 RTA promoter as the cis element mediating the upregulation of RTA promoter activity by Tpl2. MHV-68 lytic infection upregulates Fos expression, AP-1 activity, and RTA promoter activity in a Tpl2-dependent manner. We constructed a mutant MHV-68 virus that abolished this AP-1-responsive element. This mutant virus exhibited attenuated lytic replication kinetics, indicative of a critical role of this AP-1-responsive element during lytic replication. Moreover, Tpl2 knockdown inhibited the lytic replication of wild-type MHV-68 (MHV-68-WT) but not that of the MHV-68 mutant virus, indicating that endogenous Tpl2 promotes efficient virus lytic replication through AP-1-dependent upregulation of RTA expression. In summary, through tandem functional screens, we identified the Tpl2/AP-1 signaling transduction pathway as a positive regulator of MHV-68 lytic replication.

RHAMM (CD168) Is Overexpressed at the Protein Level and May Constitute an Immunogenic Antigen in Advanced Prostate Cancer Disease

Localized prostate cancer (CaP) can be cured using several strategies. However, the need to identify active substances in advanced tumor stages is tremendous, as the outcome in such cases is still disappointing. One approach is to deliver human tumor antigen-targeted therapy, which is recognized by T cells or antibodies. We used data mining of the Cancer Immunome Database (CID), which comprises potential immunologic targets identified by serological screening of expression libraries. Candidate antigens were screened by DNA microarrays. Genes were then validated at the protein level by tissue microarrays, representing various stages of CaP disease. Of 43 targets identified by CID, 10 showed an overexpression on the complementary DNA array in CaP metastases. The RHAMM (CD168) gene, earlier identified by our group as an immunogenic antigen in acute and chronic leukemia, also showed highly significant overexpression in CaP metastases compared with localized disease and benign prostatic hyperplasia. At the protein level, RHAMM was highest in metastatic tissue samples and significantly higher in neoplastic localized disease compared with benign tissue. High RHAMM expression was associated with clinical parameters known to be linked to better clinical outcome. Patients with high RHAMM expression in the primaries had a significantly lower risk of biochemical failure. The number of viable cells in cell cultures was reduced in blocking experiments using hormone-sensitive and hormone-insensitive metastatic CaP cell lines. Acknowledging the proven immunogenic effects of RHAMM in leukemia, this antigen is intriguing as a therapeutic target in far-advanced CaP.

Castration induces autoantibody and T cell responses that correlate with inferior outcomes in an androgen‐dependent murine tumor model

We recently reported that hormone therapy induces antigen-specific autoantibody responses in prostate cancer patients. However, the contribution of autoantibody responses to clinical outcomes is unknown. We used an animal model to test the hypothesis that hormone therapy-induced immune responses may be associated with delayed tumor recurrence. Male DD/S mice bearing established tumors from the androgen-dependent Shionogi carcinoma line were castrated to induce tumor regression. Tumor-specific autoantibody responses were measured by immunoblot, and the underlying antigen was identified by serological screening of a cDNA expression library. T cell responses were assessed by immunohistochemistry and IFN-gamma ELISPOT. Following castration, 97% of mice underwent complete tumor regression. Of these, 72% experienced tumor recurrence 18-79 days postcastration, whereas the remaining 28% remained tumor-free for the duration of the experiment. In 55% of mice, castration induced autoantibody responses to an antigen identified as poly(A) binding protein nuclear 1 (PABPN1). Castration also induced PABPN1-specific T cell responses, which were highly correlated to autoantibody responses, and this was accompanied by dense infiltration of tumors by CD3+ T cells 1-2 weeks after castration. Unexpectedly, mice that developed autoantibody and T cell responses to PABPN1 showed a higher rate and shorter latency of tumor recurrence. In mice with recurrent tumors, T cell responses to PABPN1 were still detectable; however, T cell infiltrates were restricted to the peripheral stroma of tumors. In conclusion, castration-induced immune responses are associated with inferior outcomes in the Shionogi carcinoma model, raising concerns about the influence of treatment-induced immune responses on clinical outcomes in humans.

Optimierung der Proteinexpression im Hochdurchsatz

to shaken cultures, and an equal growth in parallel cultures, in a number of examples also the amount of the soluble active form of the target product was significantly increased per cell unit. EnBaseTM is a valuable method for screening of bioprocess conditions in microor deepwell plates and allows a direct scaling to the bioreactor scale. Based on the positive experiences of a number of laboratories with EnBaseTM various ready-to-use kits have been developed for micro-well plate, deep-well plate, tube, and shake flask formats to facilitate the production of recombinant proteins, DNA, and the screening of expression libraries.

Identification of a functional Antigen5-related allergen in the saliva of a blood feeding insect, the tsetse fly

Our previous screening of a Glossina morsitans morsitans λgt11 salivary gland expression library with serum of a tsetse fly exposed rabbit identified a cDNA encoding Tsetse Antigen5 (TAg5, 28.9 kDa), a homologue of Antigen5 sting venom allergens. Recombinant TAg5 was produced in Sf9 cells in order to assess its immunogenic properties in humans. Plasma from a patient that previously exhibited anaphylactic reactions against tsetse fly bites contained circulating anti-TAg5 and anti-saliva IgEs. In a significant proportion of plasma samples of African individuals, TAg5 and saliva binding IgEs (respectively 56 and 65%) can be detected. Saliva, harvested from flies that were subjected to TAg5-specific RNA interference (RNAi), displayed significantly reduced IgE binding potential. Allergenic properties of TAg5 and tsetse fly saliva were further illustrated in immunized mice, using an immediate cutaneous hypersensitivity and passive cutaneous anaphylaxis assay. Collectively, TAg5 was illustrated to be a tsetse fly salivary allergen, demonstrating that Antigen5-related proteins are represented as functional allergens not only in stinging but also in blood feeding insects.

Transcriptional regulation of the human gene coding for proline-rich nuclear receptor coactivator (PNRC) by regulatory factor X (RFX1)

PNRC (Proline-rich Nuclear Receptor Coactivator) is a novel coactivator for multiple nuclear receptors. PNRC was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, functional analysis of the 5' flanking region of the human PNRC gene revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Gel shift and ChIP analyses demonstrated the specific binding of RFX1 (Regulatory Factor X) protein to the human PNRC promoter region. In co-transfection experiments RFX1 was shown to repress promoter activity of PNRC gene in a dose-dependent manner. These results indicate that r RFX1 specifically bind to promoter region and negatively regulate the transcription of the human PNRC gene.

Mucin 1 (MUC1) is a novel partner for MAL2 in breast carcinoma cells.

BackgroundThe MAL2 gene, encoding a four-transmembrane protein of the MAL family, is amplified and overexpressed in breast and other cancers, yet the significance of this is unknown. MAL-like proteins have trafficking functions, but their molecular roles are largely obscure, partly due to a lack of known binding partners.MethodsYeast two-hybrid screening of a breast carcinoma cDNA expression library was performed using a full-length MAL2 bait, and subsequent deletion mapping experiments were performed. MAL2 interactions were confirmed by co-immunoprecipitation analyses and confocal microscopy was employed to compare protein sub-cellular distributions. Sucrose density gradient centrifugation of membranes extracted in cold Triton X-100 was employed to compare protein distributions between Triton X-100-soluble and -insoluble fractions.ResultsThe tumor-associated protein mucin 1 (MUC1) was identified as a potential MAL2 partner, with MAL2/MUC1 interactions being confirmed in myc-tagged MAL2-expressing MCF-10A cells using co-immunoprecipitation assays. Deletion mapping experiments demonstrated a requirement for the first MAL2 transmembrane domain for MUC1 binding, whereas the MAL2 N-terminal domain was required to bind D52-like proteins. Confocal microscopy identified cytoplasmic co-localisation of MUC1 and MAL2 in breast cell lines, and centrifugation of cell lysates to equilibrium in sucrose density gradients demonstrated that MAL2 and MUC1 proteins were co-distributed between Triton X-100-soluble and -insoluble fractions. However co-immunoprecipitation analyses detected MAL2/MUC1 interactions in Triton X-100-soluble fractions only. Myc-MAL2 expression in MCF-10A cells was associated with both increased MUC1 detection within Triton X-100-soluble and -insoluble fractions, and increased MUC1 detection at the cell surface.ConclusionThese results identify MUC1 as a novel MAL2 partner, and suggest a role for MAL2 in regulating MUC1 expression and/or localisation.