Transcriptional Landscape of PARs in Epithelial Malignancies

G protein-coupled receptors (GPCRs), the largest family of cell receptors, act as important regulators of diverse signaling pathways. Our understanding of the impact of GPCRs in tumors is emerging, yet there is no therapeutic platform based on GPCR driver genes. As cancer progresses, it disrupts normal epithelial organization and maintains the cells outside their normal niche. The dynamic and flexible microenvironment of a tumor contains both soluble and matrix-immobilized proteases that contribute to the process of cancer advancement. An example is the activation of cell surface protease-activated receptors (PARs). Mammalian PARs are a subgroup of GPCRs that form a family of four members, PAR1–4, which are uniquely activated by proteases found in the microenvironment. PAR1 and PAR2 play central roles in tumor biology, and PAR3 acts as a coreceptor. The significance of PAR4 in neoplasia is just beginning to emerge. PAR1 has been shown to be overexpressed in malignant epithelia, in direct correlation with tumor aggressiveness, but there is no expression in normal epithelium. In this review, the involvement of key transcription factors such as Egr1, p53, Twist, AP2, and Sp1 that control PAR1 expression levels specifically, as well as hormone transcriptional regulation by both estrogen receptors (ER) and androgen receptors (AR) are discussed. The cloning of the human protease-activated receptor 2; Par2 (hPar2) promoter region and transcriptional regulation of estrogen (E2) via binding of the E2–ER complex to estrogen response elements (ERE) are shown. In addition, evidence that TEA domain 4 (TEAD4) motifs are present within the hPar2 promoter is presented since the YAP oncogene, which plays a central part in tumor etiology, acts via the TEAD4 transcription factor. As of now, no information is available on regulation of the hPar3 promoter. With regard to hPar4, only data showing CpG methylation promoter regulation is available. Characterization of the PAR transcriptional landscape may identify powerful targets for cancer therapies

Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies

Immunolocalisation of <i>w</i>Bm-LigA in <i>Brugia malayi</i> microfilariae by confocal microscopy.

<p>(A–D, A′–D′) Microfilariae were incubated with anti-<i>w</i>Bm-LigA antibody followed by incubation with FITC conjugated secondary antibody and counterstaining with DAPI. (A, A′) Phase contrast image of microfilariae. (B, B′) Green fluorescence signal generated by FITC indicating distribution of <i>w</i>Bm-LigA. (C, C′) Blue signal produced by DAPI indicating presence of nuclear DNA. (D, D′) Merge image of the phase contrast and fluorescence emitted by DAPI and FITC. (A′–D′) are images of single microfilariae at higher magnification (40X). (E–H, E′–H′) Microfilariae incubated with BALB/c preimmune serum followed by incubation with FITC conjugated secondary antibody and counterstaining with DAPI served as control. (E, E′) Phase contrast image. (F, F′) No green fluorescence signal of FITC. (G, G′) Blue signal generated with DAPI. (H, H′) Merge image of the phase contrast and fluorescence emitted by DAPI. (E′–H′) are images of single microfilariae indicated in boxes in (E–H res.) at higher magnification (40X).</p

Cohesive end ligation activity of <i>w</i>Bm-DNA ligase at various temperature, pH and enzyme concentration.

<p>(A) Temperature dependence of cohesive end ligation activity of <i>w</i>Bm-LigA. Lane 1, 2, 3, 4, 5, 6 showed ligation at 10°C, 25°C, 30°C, 35°C, 40°C and 45°C temperature res.; lane C, control in which no enzyme was added. Enzyme showed maximum ligation at 25°C. Quantitation of the data is shown below as a bar diagram. (B) pH dependence on the cohesive end ligation. Lane 1, 2, 3, 4, 5 represents ligation at 6.5, 7.0, 7.5, 8.0, 8.5 pH res.; lane C, control. Maximum ligation activity was observed at 7.5 pH. Quantitation of the data is shown below as a bar diagram. (C) (Lanes 1–12), effect of enzyme concentration (0–25nM) on cohesive end ligation; Lane C, control. Quantitation of the data is shown to the right as a bar diagram. Ligation of cohesive ends of fragment 1 and fragment 4 of lamda DNA digested with BstEII into 14 kb ligated product was analyzed on a 1.0% agarose gel. Activity values were calculated by measuring the net intensity of the product band (indicated as 1+4 in the figures) relative to a non-substrate fragment (fragment 7).</p

Overexpression, purification and Western blot analysis of recombinant <i>w</i>Bm-LigA.

<p>(A) Overexpression of <i>w</i>Bm-LigA in Rosetta <i>E. coli</i> cells<b>.</b> lane M, standard protein molecular weight marker; lane1, total proteins extracts isolated from the uninduced <i>E. coli</i> cells; lanes 2–4, total proteins extracts isolated from the <i>E. coli</i> cells induced with 0.2mM, 0.5mM and 1.0mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG). ∼20 µg of proteins were loaded per lane. (B) Purification of <i>w</i>Bm-LigA by affinity chromatography using nickel column. lane 1, soluble <i>E. coli</i> proteins following induction with 1.0 mM IPTG at 25°C for 5 h; lane 2, flow through from the nickel column; (lanes 3–6), washes from column prior to elution; lane 7, elution of His-tagged purified recombinant <i>w</i>Bm-LigA) at 250 mM imidazole concentration. 5 µg of recombinant protein was loaded onto the gel; lane M, standard protein molecular weight marker. (C) Western blot analysis. lane1, confirmation of His-tagged <i>w</i>Bm-LigA expression with reaction of 20 µg of recombinant protein with anti-His antibody; lane 2, reaction of 10 µg of <i>w</i>Bm-LigA protein with anti-His antibody; lane M, standard protein molecular weight marker.</p

Stage-specific expression of <i>w</i>Bm-LigA by immunoblotting and immunolocalisation in female adult worm by confocal microscopy.

<p>(A) The stage-specific expression of <i>w</i>Bm-LigA. Western blot was done with anti-<i>w</i>Bm-LigA antibody to confirm presence/absence of <i>w</i>Bm-LigA. Lane 1, microfilariae; lane 2, infective larvae; lane 3, adult worms (both sexes) of <i>B. malayi</i>; lane 4, purified <i>w</i>Bm-LigA (positive control); lane M, standard protein molecular weight marker. <i>w</i>Bm-LigA was detected in all the three major life stages. (B–B″, C–C″) Immunolocalisation of <i>w</i>Bm-LigA in <i>B.malayi</i> female adult worm. (B–B″) Adult worm treated with pre-immune serum as primary antibody followed by fluorescein isothiocyanate (FITC, green) conjugated secondary antibody and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, blue). (B) Fluorescence signal generated with DAPI. (B′) No green signal in the control adult worm. (B″) Phase contrast image. (C–C″) Female adult worm incubated with mouse anti-<i>w</i>Bm-LigA serum followed with FITC conjugated secondary IgG and counterstained with DAPI. (C) Fluorescence signal generated with DAPI. (C′) Fluorescence signal generated by FITC conjugated secondary IgG indicating the distribution of <i>w</i>Bm-LigA in embryos (E) and stretched microfilariae (M) in female adult worm. (C″) Merge image of the fluorescence emitted by DAPI and FITC in C and C′. All the images were captured at 40X objective. Scale Bar: 5μm.</p

Clustal alignment of amino acid sequence of <i>w</i>Bm-LigA with other prokaryotic bacteria.

<p>Amino acid sequence of <i>w</i>Bm-LigA (GenBank:AAW71136.1) wasaligned with amino acid sequences of DNA ligases of other prokaryotic bacteria including alphaproteobacteria, <i>Rhizobium leguminosarum</i> (R.leg, reference sequence YP_002282076.1), <i>Rickettsia rickettsii</i> (R.ric, GenBank:ABY73051.1), <i>Wolbachia</i> endosymbiont of <i>Drosophilamelanogaster</i> (WDm, reference sequence NP_966531.1), <i>Drosophila simulans</i> (WDs, reference sequence ZP_00372215.1), <i>Culex quinquefasciatus</i> (WCq, reference sequence accession YP_001975247.1); Betaproteobacteria, <i>Bordetella pertussis</i>(B.per, reference sequence NP_882073.1.); Gamma proteobacteria, <i>Escherichia coli</i> (E.col, GenBank:AAN81395.1<b>)</b> and cyanobacteria <i>Prochlorococcus marinus</i> (P.mar, reference sequence NP_876232.1). Residues that are identical are highlighted in black, and the conserved amino acid changes are outlined in gray rectangular boxes.</p

Conserved domain architecture of <i>w</i>Bm-LigA and phylogenetic tree of DNA ligases from various organisms.

<p>(A) Conserved domain architecture of <i>w</i>Bm-LigA typical of DNA ligase superfamily includes adenylation domain (9–304) with KXDG catalytic site(114–117) involved in adenylation reaction, OB fold domain(307–387), HhH (helix hairpin helix) and BRCT domain (609–671). (B) Phylogenetic trees were constructed with Maximum likelihood (ML) methods using PROML programs of PHYLIP 3.69. All the prokaryotic NAD⁺-dependent DNA ligase bacteria formed a discrete cluster A which is divided into two branches, the first one comprising the subcluster A1 constituting <i>Nitrosomonas europaea</i> (reference sequence NP_841783.1), <i>Bordetella pertussis</i> (reference sequence NP_882073.1), <i>Gammaproteobacteria</i> (reference sequence ZP_05126247.1), <i>Pseudomonas aeruginosa</i> (reference sequence YP_791724.1), <i>Escherichia coli</i> (GenBank:AAN81395.1), <i>Mycobacterium tuberculosis</i> (reference sequence ZP_7441467.2), Frankia (reference sequence ZP_06413452.1), <i>Thermus thermophilus</i> (GenBank:AAD13190.1), <i>Prochlorococcus marinus</i> (reference sequence NP_876232.1<i>), Mycoplasma mycoides</i> (reference sequence NP_975740.1), <i>Bacillus subtilis</i> (reference sequence NP_388544.1), <i>Bradyrhizobium japonicum</i> (GenBank:BAC51856.1), <i>Rhizobium leguminosarum</i> (NCBI reference sequence YP_002282076.1), <i>Agrobacterium vitis</i> ( reference sequence YP_002550080.1) and the second one (subcluster A2) comprising the DNA ligase of <i>Wolbachia</i> endosymbiont present in <i>Brugia malayi</i> (GenBank:AAW71136.1) and insects as <i>Drosophila melanogaster</i> (reference sequence NP_966531.1), <i>Drosophila simulans</i> (reference sequence ZP_00372215.1) and <i>Culex quinquefasciatus</i> (reference sequence YP_001975247.1), <i>Wolbachia pipientis</i> (GenBank:AAY81980.1), <i>Anaplasma centrale</i> (reference sequence YP_003328904.1), <i>Ehrlichia chaffeensis</i> (NCBI reference sequence YP_507122.1) and <i>Rickettsia rickettsii</i> (GenBank:ABY73051.1). All the eukaryotic DNA ligase viz. <i>Homo sapiens</i> (reference sequence NP_000225.1), <i>Mus musculus</i> (reference sequence NP_001186239.1), <i>Brugia malayi</i> (NCBI reference sequence XP_001896804.1), <i>Plasmodium falciparum</i> (GenBank:AAN64156.1), <i>Plasmodium knowlesi</i> (reference sequence XP_002261933.1) and <i>Saccharomyces cerevisiae</i> (GenBank:CAA91582.1) are present in discrete cluster B indicating <i>Wolbachia</i> endosymbiont are distantly related to the ATP DNA ligase of either its host parasite <i>B. malayi or H.sapiens.</i></p