Two new geranyl flavanones from Tephrosia villosa.

Two new geranyl flavanones, (2S)-5,4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]flavanone and (2S)-5,4'-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octa-dienyl]-8-C-[(E)-3,7-dimethyl-2,6-octadienyl]flavanone, together with three known compounds, 7-O-methylglabranin, tephcalostan and 12a-dehydro-6-hydroxysumatrol, were isolated from the roots of Tephrosia villosa. The structures of these compounds were determined by extensive spectral studies.

Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin.

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Specific small-molecule activator of Aurora kinase A induces autophosphorylation in a cell-free system.

Aurora kinases are essential for chromosomal segregation and cell division and thereby important for maintaining the proper genomic integrity. There are three classes of aurora kinases in humans: A, B, and C. Aurora kinase A is frequently overexpressed in various cancers. The link of the overexpression and tumorigenesis is yet to be understood. By employing virtual screening, we have found that anacardic acid, a pentadecane aliphatic chain containing hydroxylcarboxylic acid, from cashew nut shell liquid could be docked in Aurora kinases A and B. Remarkably, we found that anacardic acid could potently activate the Aurora kinase A mediated phosphorylation of histone H3, but at a similar concentration the activity of aurora kinase B remained unaffected in vitro. Mechanistically, anacardic acid induces the structural changes and also the autophosphorylation of the aurora kinase A to enhance the enzyme activity. This data thus indicate anacardic acid as the first small-molecule activator of Aurora kinase, which could be highly useful for probing the function of hyperactive (overexpressed) Aurora kinase A.

Specific inhibition of p300-HAT alters global gene expression and represses HIV replication.

Reversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS, and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT-specific small-molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells, and consequently inhibit the multiplication of HIV.

Small molecule modulators in epigenetics: implications in gene expression and therapeutics.

Altered gene expression resulting from changes in the post-translational modification patterns of the histones and DNA is collectively termed epigenetics. Such changes are inherited albeit there are no alterations in the DNA sequence. Epigenetic regulation of gene expression is implemented by a wide repertoire of histone and DNA modifying enzymes including the acetyltransferases and deacetylases, the methyltransferases and kinases among others. Therefore, a regulation of these enzyme activities affords a tighter regulation of gene expression. Conversely, aberrant enzymatic activities lead to unregulated gene expression, resulting in several diseases such as RTS (loss of CBP HAT activity) and Spinal and Bulbar muscular atrophy (HATs and HMTases), apart from several forms of cancers, particularly myeloid leukemia (RAR-PML or RAR-PLZF fusion proteins resulting in the mistargeting of HDACs). Thus these enzymes have emerged as novel targets for the design of therapeutics. In this direction, several small molecule modulators (activators and inhibitors) of HATs, HDACs and HMTases are being reported in literature. This chapter introduces the different histone modifying enzymes involved in gene regulation, their connection to disease manifestation and focuses on the role of small molecule modulators in understanding enzyme function and also the design and the evolution of chromatin therapeutics

Surface-enhanced Raman scattering studies of human transcriptional coactivator p300.

We report for the first time the surface-enhanced Raman scattering (SERS) studies on p300, a large multidomain transcriptional coactivator protein. Vibration spectral analysis has been performed in an attempt to understand the structure of the p300 in the absence of its crystal structure. Strong Raman bands associated with amides I-III have been observed in the protein spectra. This has been confirmed by performing SERS on deuterated p300. We also observe Raman bands associated with the alpha-helix, tryptophan, phenylalanine, tyrosine, and histidine. These bands will provide an ideal tool to study the drug-protein interactions in therapeutics using SERS. We have successfully demonstrated the chloride ion effect on the SERS of p300. The Raman intensity increases in the SERS spectra upon addition of chloride ion along with appearance of new modes. We have developed a new method, namely, the "sandwich technique", which could be used to perform SERS experiments on proteins in dry conditions.

Infectious sequelae in the use of polyglycolic acid mesh for splenic salvage with intraperitoneal contamination.

Salvage of the injured spleen is important in the trauma patient. Loss of the spleen can result in both early and late infectious complications due to immunologic and phagocytic deficits. Splenic salvage techniques include the use of polyglycolic acid (PGA) mesh to wrap and tamponade the damaged and bleeding spleen. However, the use of mesh may increase the incidence of infection in the presence of intraperitoneal contamination. We examined whether mesh in the contaminated field increases the infection rate compared to splenectomy in a murine model. Sixty male Sprague-Dawley rats were divided into three groups of 20 each: splenectomy, splenic wrap with PGA, and control (with splenic mobilization). All rats were subjected to a standard inoculum of enteric bacteria at the time of celiotomy. Sixteen (80%) of the splenectomy rats, 10 (50%) of the PGA mesh wrapped rats, and four (20%) of the control rats expired (P < 0.5). In surviving rats, necropsy at 7 days demonstrated abscess formation in all four (100%) of splenectomy, four of 10 (40%) in PGA mesh wrapped, and two of 16 (13%) of control rats. All of the abscesses in the wrap group involved the mesh. Overall infection rates (including fatal peritonitis, abscess formation, and empyema) were 100% for splenectomy, 75% for PGA mesh wrapped, and 30% for control rats (P < 0.05). We conclude in this experimental model that the use of PGA mesh wrap does increase susceptibility to infection, but much less so than splenectomy in the presence of intraperitoneal contamination.

Two forms of Xenopus nuclear factor 7 have overlapping spatial but different temporal patterns of expression during development.

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functions in the determination of the dorsal-ventral body axis. We have cloned two xnf7 cDNAs, xnf7-O and xnf7-B, that have a different temporal pattern of expression. The cDNAs differ by 39 amino acid residues scattered throughout the molecule. Most of the changes were conservative in nature. Using gene specific probes we found that xnf7-O transcripts were abundant in oocytes and decreased until the neurula stage, after which they increased in abundance. Xnf7-B transcripts were in low abundance in oocytes and were expressed at high levels at the neurula stage and in adult brain. Both xnf7-O and xnf7-B transcripts at the neurula stage were localized in the dorsal region of the embryo, including the neural folds and somites. Xnf7 was not expressed in ventralized embryos that lacked dorsal structures, thereby substantiating its dorsal localization in the embryo. The promoter region of the xnf7-O gene does not possess a TATA box but does contain E2F, USF, Sp1-like and AP1 binding sites within the first 421 bp from the transcription initiation site. A 62 bp fragment of the xnf7-O promoter containing the Sp1-like and E2F sites can direct proper spatial expression of a transgene in embryos.

The association of Xenopus nuclear factor 7 with subcellular structures is dependent upon phosphorylation and specific domains.

The function of proteins is often regulated by their association with specific subcellular structures. Xenopus nuclear factor 7 (xnf7) is a putative transcription factor that is selectively retained in the cytoplasm from fertilization through the mid blastula transition (MBT). Cytoplasmic retention is dependent upon the presence of a 22-amino-acid cytoplasmic retention domain and the phosphorylation of two sites (site 1 and site 2) within the protein. We show that the N-terminal acidic domain of xnf7 transactivated a reporter gene in transfected cells, supporting its function as a transcription factor. During mitosis xnf7 was associated with the mitotic spindle and chromosomes, while during the short embryonic interphase it was associated with structures at the poles which were most likely centrosomes. The association with these structures was dependent upon the presence of protein domains and the phosphorylation of a specific phosphorylation site (site 2). In addition, we determined that association with the spindle or centrosomes was not necessary for cytoplasmic retention prior to the MBT. We suggest that the association of xnf7 with these structures is due to its interaction with other proteins that are colocalized.

Cytoplasmic retention of Xenopus nuclear factor 7 before the mid blastula transition uses a unique anchoring mechanism involving a retention domain and several phosphorylation sites.

Xenopus nuclear factor 7 (xnf7) is a maternally expressed protein that belongs to the B-box zinc finger gene family consisting of transcription factors, protooncogenes, and ribonucleoproteins. Its function is regulated by retention in the cytoplasm from oocyte maturation until the mid blastula transition (MBT) when it reenters the nucleus. We defined a 22-amino acid cytoplasmic retention domain (CRD) in xnf7 that functioned cooperatively with two phosphorylation sites within the xnf7 molecule to retain the protein in the cytoplasm until the MBT. Deletion of this region or mutations in the phosphorylation sites resulted in the early entry of xnf7 into the nucleus. A mutation changing one of the phosphorylation sites to a glutamic acid resulted in the prolonged retention of the xnf7 protein in the cytoplasm until stages 9-10, well past the MBT. Additionally, a mutant form of xnf7 possessing a second nuclear localization signal at the COOH terminus was retained in the cytoplasm. This suggests that retention of xnf7 was not due to the masking of its NLS as is the case with NFkB and dorsal but was due to a novel anchoring mechanism in which the CRD interacts with an anchor protein. The CRD sequence is also found in another B-box zinc finger protein that is also retained in the cytoplasm until the MBT in the newt. Therefore, we believe that this may be an important mechanism whereby the function of a number of nuclear proteins is regulated during development.

Characterisation of a novel cysteine/histidine-rich metal binding domain from Xenopus nuclear factor XNF7.

A 42 amino acid synthetic peptide corresponding to a newly defined cysteine/histidine-rich protein motif called B-box, from the Xenopus protein XNF7 has been characterised. The metal-binding stoichiometry and dissociation constant for zinc were determined by competition with the chromophoric chelator Br2BAPTA, demonstrating that one zinc atom binds per molecule of peptide despite the presence of seven putative metal ligands, and represents the first application of this method to measuring zinc stoichiometry of proteins and/or peptides. Cobalt binding studies indicate that the motif binds zinc more tightly than cobalt, that cysteines are used as ligands and that the cation is co-ordinated tetrahedrally. Circular dichroism and NMR studies both indicate that the B-box peptide is structured only in the presence of zinc, copper and to a lesser extent cobalt.

The cloning and characterization of a localized maternal transcript in Xenopus laevis whose zygotic counterpart is detected in the CNS.

We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.

Xlcaax-1 is localized to the basolateral membrane of kidney tubule and other polarized epithelia during Xenopus development.

Xlcaax-1 is a novel, maternally expressed, 110-kDa, CAAX box containing protein that undergoes isoprenylation and palmitoylation through which it associates with the plasma membrane. We report here the cellular and subcellular localization of the xlcaax-1 protein during development of Xenopus laevis. Whole-mount immunocytochemistry and immunoperoxidase staining of tissue sections show that during development the xlcaax-1 protein accumulation is coincident with the differentiation of the epidermis, pronephros, and mesonephros. In the pronephros and mesonephros the xlcaax-1 protein is localized to the basolateral membrane of differentiated tubule epithelial cells. Thus, the xlcaax-1 protein serves as a marker for tubule formation and polarization during Xenopus kidney development. Xlcaax-1 may also be used as a marker for the functional differentiation of the epidermis and the epidermally derived portions of the lens and some cranial nerves. Western blot analysis shows that in the adult the xlcaax-1 protein is most abundant in kidney. Immunogold EM analysis shows that the xlcaax-1 protein is highly enriched in the basal infoldings of the basolateral membrane of the epithelial cells in adult kidney distal tubules. In addition, immunoperoxidase staining of tissue sections detected low levels of xlcaax-1 protein in the epithelial cells of skin, urinary bladder, gall bladder, and parietal glands of the stomach. The localization pattern of xlcaax-1 suggests that the protein may function in association with an ion transport channel or pump.

The cloning and characterization of a maternally expressed novel zinc finger nuclear phosphoprotein (xnf7) in Xenopus laevis.

We report the cloning of a cDNA (xnf7) coding for a maternally expressed Xenopus protein that becomes highly enriched in nuclei of the central nervous system during later development and in nuclei of adult brain. The protein also shows stage-specific nuclear/cytoplasmic partitioning and phosphorylation that may be related to its function. In addition, it binds to double-stranded DNA in vitro. The conceptual protein produced by the xnf7 clone contains several acidic domains, a novel zinc finger domain, three putative p34cdc2 protein kinase phosphorylation sites, and a bipartite basic nuclear localization signal. The xnf7 mRNA was detected as a maternal transcript that decreased in abundance during development through the gastrula stage. It was reexpressed at the neural stage in mesoderm and neural tissues, and its reexpression was not dependent upon the normal juxtaposition of the mesoderm and ectoderm that occurs during neural induction as demonstrated by high titer in exogastrulae. In situ hybridization showed enrichment of the mRNA in the neural tube and a small amount in the mesoderm at the late neurula stage. Xnf7 is normally phosphorylated during oocyte maturation. The bacterially expressed xnf7 protein was phosphorylated in vitro by purified maturation-promoting factor at a threonine in a small N-terminal domain containing one of the p34cdc2 protein kinase phosphorylation sites, but not by several other protein kinases. The structural domains present in the protein and its localization in nuclei suggest that the xnf7 gene product performs an important nuclear function during early development, perhaps as a transcription factor or a structural component of chromatin.

The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development.

We describe the characterization in Xenopus laevis of a nuclear protein, xnf7, which is first detected in the oocyte GV and is eventually enriched in nuclei of cells of the adult brain. Previous studies have shown that this protein contains zinc-finger-like structures and acidic domains typical of transcriptional activators, and is phosphorylated in vitro by p34cdc2 protein kinase. The protein also binds to double-stranded DNA. These data suggest that xnf7 may function as a transcription factor. During oocyte maturation, xnf7 is released into the cytoplasm and is not detectable in nuclei until the mid-blastula-gastrula stage of development. Western blot analysis of xnf7 isolated from oocytes and eggs showed the existence of multiple bands or isoforms of the protein. Unique isoforms that are generated during oocyte maturation are the result of phosphorylation. The phosphorylated isoforms remain in the cytoplasm until the mid-blastula stage. The re-accumulation of protein in the embryonic nuclei at this time correlates with the increase in abundance of the less phosphorylated isoforms. The xnf7 protein possesses a nuclear localization signal (NLS) similar to the bipartite signal found in nucleoplasmin. Newly synthesized xnf7 accumulated in the oocyte GV to detectable levels within a few hours following synthesis suggesting that retention of the protein in the cytoplasm during early cleavage may be due to a process that interferes with the function of the NLS. These data suggest that compartmentalization and/or post-translational modification of the nuclear protein xnf7 may be involved in regulating its function during early development.

Identification of the cDNA for xlcaax-1, a membrane associated Xenopus maternal protein.

xlcaax-1 is a cDNA coding for a CAAX box containing protein in Xenopus laevis that undergoes isoprenylation and palmitoylation. Here we report on the confirmation that this clone (formerly xlgv7) codes for a 110 kDa membrane associated protein and not an 80 kDa nuclear protein as originally believed (1). The reason for the misidentification was the presence of a common epitope on these two proteins recognized by the monoclonal antibody 37-1A9. We clarified the discrepancy by raising polyclonal antibodies against the xlcaax-1 protein produced in a bacterial expression system and demonstrating that these antibodies only recognize the 110 kDa protein on western blots of oocyte extracts. During early development xlcaax-1 protein starts reaccumulating from the neurula stage. In the adult frog both the xlcaax-1 protein and its cognate mRNA are highly enriched in the kidney. Consistent with the presence of CAAX box at the C-terminus this protein is associated with the membranes in Xenopus tissue culture cells (XTC).

x121: a localized maternal transcript in Xenopus laevis.

We describe the cloning and characterization of a partial cDNA, x121, that represents an RNA, which is localized in the animal hemisphere of Xenopus oocytes. This RNA is also detected in an animal to vegetal gradient during early cleavage stages. The x121 RNA titer decreases from fertilization through the gastrula stage, after which it is not detectable on northern blots. The amino acid composition of the x121 conceptual protein derived from cDNA sequencing reveals a large number of acidic residues similar in distribution to proteins that function as transcriptional activators.