Marika Häkli

Identification of a Novel RING Finger Protein as a Coregulator in Steroid Receptor-Mediated Gene Transcription

ABSTRACT Using the DNA-binding domain of androgen receptor (AR) as a bait in a yeast two-hybrid screening, we have identified a small nuclear RING finger protein, termed SNURF, that interacts with AR in a hormone-dependent fashion in both yeast and mammalian cells. Physical interaction between AR and SNURF was demonstrated by coimmunoprecipitation from cell extracts and by protein-protein affinity chromatography. Rat SNURF is a highly hydrophilic protein consisting of 194 amino acid residues and comprising a consensus C3HC4 zinc finger (RING) structure in the C-terminal region and a bipartite nuclear localization signal near the N terminus. Immunohistochemical experiments indicated that SNURF is a nuclear protein. SNURF mRNA is expressed in a variety of human and rat tissues. Overexpression of SNURF in cultured mammalian cells enhanced not only androgen, glucocorticoid, and progesterone receptor-dependent transactivation but also basal transcription from steroid-regulated promoters. Mutation of two of the potential Zn2+coordinating cysteines to serines in the RING finger completely abolished the ability of SNURF to enhance basal transcription, whereas its ability to activate steroid receptor-dependent transcription was maintained, suggesting that there are separate domains in SNURF that mediate interactions with different regulatory factors. SNURF is capable of interacting in vitro with the TATA-binding protein, and the RING finger domain is needed for this interaction. Collectively, we have identified and characterized a ubiquitously expressed RING finger protein, SNURF, that may function as a bridging factor and regulate steroid receptor-dependent transcription by a mechanism different from those of previously identified coactivator or integrator proteins.

Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity

SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger‐dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin‐conjugating enzyme (E2)‐selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2‐25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2‐20K. In contrast, the well‐characterized RING E3, AO7, functions only with members of the UbcH5 family. Furthermore, depending on the E2 used, the ubiquitin modification manifests as mono‐ or multi‐ubiquitination. Mutation of conserved cysteine residues within the RING finger motif of SNURF abolishes the ubiquitination in vitro and in intact cells. Size fractionation of murine embryonal carcinoma F9 cell proteins shows that the majority of endogenous SNURF resides in salt‐resistant ≥500‐kDa complexes, suggesting that SNURF functions as a RING component in a multiprotein complex. Taken together, SNURF/RNF4 functions as an E3 ligase and this activity is closely linked to its transcription regulatory functions.

Effluxing ABC transporters in human corneal epithelium

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-6 (MRP1-6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile.

Impact of probe compound in MRP2 vesicular transport assays

MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-β-d-glucuronide (E217βG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used.

Monocarboxylate transport in human corneal epithelium and cell lines

Monocarboxylate transporters (MCTs) are transmembrane proteins capable of transferring lactate and other endogenous and exogenous monocarboxylates across the cell membrane. The aim of the present study was to assess the expression and transporter role of human MCT1, MCT3 and MCT4 in the corneal epithelium, corneal epithelial cell lines (primary HCEpiC and immortalized HCE cells) and isolated rabbit corneas. MCT1 and MCT4 were expressed in the human corneal epithelium and the cell lines at mRNA and protein levels. Cellular uptake studies showed saturable and pH-dependent l-lactic acid transport, which was inhibited by various monocarboxylates like diclofenac and flurbiprofen. The permeability of benzoic acid across the rabbit cornea was higher in absorptive direction and this directionality was diminished in the presence of monocarboxylate drug valproic acid. Monocarboxylate transport was functional in the human corneal epithelial cells and rabbit cornea and it may play a role in the ocular drug absorption.

The RING Finger Protein SNURF Is a Bifunctional Protein Possessing DNA Binding Activity

The small nuclear C3HC4 finger protein (SNURF), RNF4, acts as transcriptional coactivator for both steroid-dependent and -independent promoters such as those driven by androgen response elements and GC boxes, respectively. However, SNURF does not possess intrinsic transcription activation function, and the precise molecular mechanism of its action is unknown. We have studied herein the interaction of SNURF with DNA in vitro. SNURF binds to linear double-stranded DNA with no apparent sequence specificity in a cooperative fashion that is highly dependent on the length of the DNA fragment used. SNURF interacts efficiently with both supercoiled circular and four-way junction DNA, and importantly, it also recognizes nucleosomes. An intact RING structure of SNURF is not mandatory for DNA binding, whereas mutations of specific positively charged residues in the N terminus (amino acids 8–11) abolish DNA binding. Interestingly, the ability of SNURF to interact with DNA is associated with its capability to enhance transcription from promoters containing GC box elements. Because SNURF can interact with both DNA and protein (transcription) factors, it may promote assembly of nucleoprotein structures.

Chemokine-derived peptides as carriers for gene delivery to CXCR4 expressing cells

Cell and tissue‐specific DNA delivery can be achieved by derivatizing vehicles with a targeting ligand for certain receptor. CXCR4 is a receptor of chemokine stromal cell‐derived factor (SDF)‐1 and viral protein viral macrophage inflammatory protein (vMIP)‐II. It is expressed on some types of stem and cancer cells. The present study aimed to design and characterize the group of CXCR4 targeted peptides for receptor‐mediated gene delivery. We focused on bifunctional peptide carriers: two derived from N‐terminal sequences of SDF‐1 and one from vMIP‐II.