Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity.

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide range of activities in eukaryotic cells, including antiviral effects that are at present incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting these coactivators, would block viral infection and gene expression. In cell culture assays, curcumin significantly decreased HSV-1 infectivity and IE gene expression. Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters was not affected by curcumin, but recruitment of RNA polymerase II to those promoters was significantly diminished. However, these effects were observed using lower curcumin concentrations than those required to substantially inhibit global H3 acetylation. No changes were observed in histone H3 occupancy or acetylation at viral IE gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was not affected by the presence of curcumin. Finally, disruption of p300 expression using a short hairpin RNA did not affect viral IE gene expression. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity.

The tumor suppressor DAL-1/4.1B modulates protein arginine N-methyltransferase 5 activity in a substrate-specific manner.

We previously identified DAL-1/4.1B as a growth suppression protein involved in the pathogenesis of lung, breast, and meningioma tumors. Using yeast two-hybrid interaction cloning, protein arginine N-methyltransferase 3 (PRMT3) was originally identified as a DAL-1/4.1B-interacting protein. PRMTs catalyze the sequential transfer of methyl groups from S-adeonsyl-l-methionine to the guanidino nitrogens of arginine residues in proteins, the effect of which can include regulation of signal transduction, transcription regulation, and RNA transport, suggesting that modulating this event may have far-reaching impact. In this study, we assessed the impact of DAL-1/4.1B binding on the activity of another family member, PRMT5, both in vitro and in cells. In contrast to PRMT3, DAL-1/4.1B was found to mediate PRMT5 by either inhibiting (Sm proteins) or enhancing (myelin basic protein) protein methylation. We propose that this interaction between a tumor suppressor and a post-translational methylation enzyme is of biological importance in controlling tumorigenesis.

DAL-1/4.1B tumor suppressor interacts with protein arginine N-methyltransferase 3 (PRMT3) and inhibits its ability to methylate substrates in vitro and in vivo.

DAL-1 (differentially expressed in adenocarcinoma of the lung)/4.1B is a tumor suppressor gene on human chromosome 18p11.3 whose expression is lost in >50% of primary non-small-cell lung carcinomas. Based on sequence similarity, DAL-1/4.1B has been assigned to the Protein 4.1 superfamily whose members interact with plasma membrane proteins through their N-terminal FERM (4.1/Ezrin/Radixin/Moesin) domain, and cytoskeletal components via their C-terminal SAB (spectrin-actin binding) region. Using the DAL-1/4.1B FERM domain as bait for yeast two-hybrid interaction cloning, we identified protein arginine N-methyltransferase 3 (PRMT3) as a specific DAL-1/4.1B-interacting protein. PRMT3 catalyses the post-translational transfer of methyl groups from S-adenosyl-L-methionine to arginine residues of proteins. Coimmunoprecipitation experiments using lung and breast cancer cell lines confirmed this interaction in mammalian cells in vivo. In vitro binding assays demonstrated that this was an interaction occurring via the C-terminal catalytic core domain of PRMT3. DAL-1/4.1B was determined not to be a substrate for PRMT3-mediated methylation but its presence inhibits the in vitro methylation of a glycine-rich and arginine-rich methyl-accepting protein, GST (glutathione-S-transferase-GAR (glycine- and arginine-rich), which contains 14 'RGG' consensus methylation sites. In addition, induced expression of DAL-1/4.1B in MCF-7 breast cancer cells showed that the DAL-1/4.1B protein significantly inhibits PRMT3 methylation of cellular substrates. These findings suggest that modulation of post-translational methylation may be an important mechanism through which DAL-1/4.1B affects tumor cell growth.

Allele-specific loss of heterozygosity at the DAL-1/4.1B (EPB41L3) tumor-suppressor gene locus in the absence of mutation.

DAL-1/4.1B (EPB41L3)is a member of the protein 4.1 superfamily, which encompasses structural proteins that play important roles in membrane processes via interactions with actin, spectrin, and the cytoplasmic domains of integral membrane proteins. DAL-1/4.1B localizes within chromosomal region 18p11.3, which is affected by loss of heterozygosity (LOH) in various adult tumors. Reintroduction of this protein into DAL-1/4.1B-null lung and breast tumor cell lines significantly reduced the number of cells, providing functional evidence that this protein possesses a growth suppressor function not confined to a single cell type. For characterization of the mutational mechanisms responsible for loss of DAL-1/4.1B function in tumors, the exon-intron structure of DAL-1/4.1B was examined for mutations in 15 normal/tumor pairs of non-small cell lung carcinoma by single-strand conformation polymorphism analysis. These studies revealed that small intragenic mutations are uncommon in DAL-1/4.1B. Furthermore, LOH analysis on 129 informative early-stage breast tumors utilizing a new intragenic C/T single-nucleotide polymorphism in exon 14 revealed that LOH resulted in preferential retention of the C-containing allele, suggesting that allele-specific loss is occurring. These studies indicate that mechanisms such as imprinting or monoallelic expression in combination with loss of heterozygosity may be responsible for loss of the DAL-1/4.1B protein in early breast disease.