Structure and Function of Potential Glycosylation Sites of Dynactin-Associated Protein dynAP.

Overexpression of human dynactin-associated protein (dynAP) transforms NIH3T3 cells. DynAP is a single-pass transmembrane protein with a carboxy-terminal region (amino acids 135-210) exposed to the outside of the cell possessing one potential N-glycosylation site (position 143) and a distal C-terminal region (residues 173-210) harboring a Thr/Ser-rich (T/S) cluster that may be O-glycosylated. In SDS-PAGE, dynAP migrates anomalously at ~ 45 kDa, much larger than expected (22.5 kDa) based on the amino acid composition. Using dynAP mutants, we herein showed that the T/S cluster region is responsible for the anomalous migration. The T/S cluster region is required for transport to the cytoplasmic membrane and cell transformation. We produced and purified the extracellular fragment (dynAP135-210) in secreted form and analyzed the attached glycans. Asn143 displayed complex-type glycosylation, suggesting that oligosaccharide transferase may recognize the NXT/S sequon in the secretory form, but not clearly in full-length dynAP. Core I-type O-glycosylation (Gal-GalNAc) was observed, but the mass spectrometry signal was weak, clearly indicating that further studies are needed to elucidate modifications in this region.

Expression and cell transformation activity of dynactin-associated protein isoforms.

Overexpression of human dynactin-associated protein isoform a (dynAPa) transforms NIH3T3 cells. DynAPa is a single-pass transmembrane protein with a carboxy-terminal region exposed to the outside of cells. According to the NCBI RefSeq database, there may be two other splicing variants of the encoding gene (dynAPb and c). DynAPa and c differ in some amino-terminal residues (NH2 -MVA in dynAPa and NH2 -MEYQLL in dynAPc). DynAPb has the same amino-terminal residues as dynAPc, but lacks 55 residues in the intracellular region. All three isoforms have the same carboxy-terminal region, including the transmembrane domain. Expression of mRNAs of three splicing variants was found in human cancer cell lines ACHN and Caki-1. The subcellular localization and in vitro cell transformation ability of the three isoforms were examined using NIH3T3 cells overexpressing each respective isoform. All isoforms were found to be localized to the Golgi apparatus and plasma membrane, where the carboxy-terminal region was exposed to the outside of cells. Cell transformation was tested using focus formation due to loss of contact inhibition of cell proliferation, and colony formation was examined on soft agar and spheroid formation in ultralow U-bottomed wells. DynAPa robustly formed foci and colonies on soft agar and spheroid, whereas these abilities were considerably decreased for dynAPb and completely lost in dynAPc. These findings warrant dissection studies to identify the dynAP domain that is required for cell transformation.

Identification of ryuvidine as a KDM5A inhibitor.

KDM5 family members (A, B, C and D) that demethylate H3K4me3 have been shown to be involved in human cancers. Here we performed screening for KDM5A inhibitors from chemical libraries using the AlphaScreen method and identified a battery of screening hits that inhibited recombinant KDM5A. These compounds were further subjected to cell-based screening using a reporter gene that responded to KDM5A inhibition and 6 compounds were obtained as candidate inhibitors. When further confirmation of their inhibition activity on cellular KDM5A was made by immunostaining H3K4me3 in KDM5A-overexpressing cells, ryuvidine clearly repressed H3K4me3 demethylation. Ryuvidine prevented generation of gefitinib-tolerant human small-cell lung cancer PC9 cells and also inhibited the growth of the drug-tolerant cells at concentrations that did not affect the growth of parental PC9 cells. Ryuvidine inhibited not only KDM5A but also recombinant KDM5B and C; KDM5B was the most sensitive to the inhibitor. These results warrant that ryuvidine may serve as a lead compound for KDM5 targeted therapeutics.

Ridaifen-F conjugated with cell-penetrating peptides inhibits intracellular proteasome activities and induces drug-resistant cell death.

Ridaifen-F (RID-F) potently inhibits proteolytic activities of the 20S proteasome but poorly inhibits those of the 26S proteasome. Here, we report preparation of several conjugates in which various peptides are connected to RID-F. Conjugates with peptides consisting of seven amino acid residues significantly inhibited the 26S proteasome. Particularly, RID-F conjugated to an octaarginine peptide (R8, a so-called cell-penetrating peptide) inhibited intracellular proteasome activities and induced cell death in drug-resistant KMS-11 myeloma cells. RID-F conjugated to hydrophobic peptides also inhibited the 26S proteasome but failed to induce cell death, suggesting poor penetration into cells. We infer that the R8 peptide has dual functions: (1) rapid penetration of conjugates into the cell increases intracellular drug concentrations sufficient for exhibition of its effect, and (2) recognition of the conjugates by the 26S proteasome stimulates drug entry into the catalytic chamber. In the presence of ATPγS, RID-F conjugates containing R8 inhibited the 26S proteasome more potently than in the presence of ATP, suggesting efficient entry of drugs into the catalytic chamber in a similar fashion to the substrate. Taken together with docking simulations of RID-F conjugate interactions with proteasome active sites, the second function of R8 peptide is plausible. Thus, the conjugation of nonpeptidic proteasome inhibitors to a cell-penetrating peptide could represent a viable strategy for overcoming the drug-resistance of tumor cells.

Detection of rotavirus in clinical specimens using an immunosensor prototype based on the photon burst counting technique.

In this study, a sensitive fluorescence sensor was developed for the detection of small, fluorescence-labeled particles dispersed in a solution. The prototype system comprises of a laser confocal optical system and a mechanical sample stage to detect photon bursting of fluorescence-labeled small particles in sample volumes less than 5 µL within 3 minutes. To examine the feasibility of the prototype system as a diagnostic tool, assemblages of rotavirus and fluorescence-labeled antibody were analyzed. The detection sensitivity for rotavirus was 1 × 104 pfu/mL. Rotavirus in stool samples from patients with acute gastroenteritis was also detected. The advantages and disadvantages of this immunosensor with respect to ELISA and RT-PCR, the current gold standards for virus detection, are discussed.

Evaluation of phenylcyclopropylamine compounds by enzymatic assay of lysine-specific demethylase 2 in the presence of NPAC peptide.

Lysine-specific demethylase 2 (LSD2) demethylates mono- and dimethylated Lys-4 of histone H3 (H3K4me1 and H3K4me2). NPAC protein is known to interact with LSD2 and promote its H3K4 demethylase activity. In this study, we established a demethylation assay system that utilizes recombinant LSD2 in the presence of a synthetic NPAC peptide. Several phenylcyclopropylamine (PCPA)-based inhibitors were examined for their LSD2 inhibitory activity in the LSD2 enzymatic assay with the NPAC peptide. The assay results showed that the PCPA derivatives, including NCD41, selectively inhibited LSD1 in preference to LSD2.

Human Dynactin-Associated Protein Transforms NIH3T3 Cells to Generate Highly Vascularized Tumors with Weak Cell-Cell Interaction.

Human dynactin-associated protein (dynAP) is a transmembrane protein that promotes AktSer473 phosphorylation. Here, we report the oncogenic properties of dynAP. In contrast to control NIH3T3 cells expressing LacZ (NIH3T3LacZ), NIH3T3dynAP cells vigorously formed foci in two-dimensional culture, colonies on soft agar, and spheroids in anchorage-deficient three-dimensional culture. NIH3T3dynAP cells injected into nude mice produced tumors with abundant blood vessels and weak cell-cell contacts. Expression of dynAP elevated the level of rictor (an essential subunit of mTORC2) and promoted phosphorylation of FOXO3aSer253. FOXO3a is a transcriptional factor that stimulates expression of pro-apoptotic genes and phosphorylation of FOXO3a abrogates its function, resulting in promoted cell survival. Knockdown of rictor in NIH3T3dynAP cells reduced AktSer473 phosphorylation and formation of foci, colony in soft agar and spheroid, indicating that dynAP-induced activation of the mTORC2/AktSer473 pathway for cell survival contributes to cell transformation. E-cadherin and its mRNA were markedly reduced upon expression of dynAP, giving rise to cells with higher motility, which may be responsible for the weak cell-cell adhesion in tumors. Thus, dynAP could be a new oncoprotein and a target for cancer therapy.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

Identification of Jumonji AT-Rich Interactive Domain 1A Inhibitors and Their Effect on Cancer Cells.

Jumonji AT-rich interactive domain 1A (JARID1A), one of the jumonji C domain-containing histone demethylase (JHDM) family members, plays key roles in cancer cell proliferation and development of drug tolerance. Therefore, selective JARID1A inhibitors are potential anticancer agents. In this study, we searched for cell-active JARID1A inhibitors by screening hydroxamate compounds in our in-house library and the structural optimization based on docking study of the hit-compound to a homology model of JARID1A. As a result, we identified compound 6j, which selectively inhibits JARID1A over three other JHDM family members. Compound 7j, a prodrug form of compound 6j, induced a selective increase in the level of trimethylation of histone H3 lysine 4, a substrate of JARID1A. Furthermore, compound 7j synergistically enhanced A549 human lung cancer cell growth inhibition induced by vorinostat, a histone deacetylase inhibitor. These findings support the idea that JARID1A inhibitors have potential as anticancer agents.

Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2).

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.

A novel tamoxifen derivative, ridaifen-F, is a nonpeptidic small-molecule proteasome inhibitor.

In a survey of nonpeptide noncovalent inhibitors of the human 20S proteasome, we found that a novel tamoxifen derivative, RID-F (compound 6), inhibits all three protease activities of the proteasome at submicromolar levels. Structure-activity relationship studies revealed that a RID-F analog (RID-F-S*4, compound 25) is the smallest derivative compound capable of inhibiting proteasome activity, with a potency similar to that of RID-F. Kinetic analyses of the inhibition mode and competition experiments involving biotin-belactosin A (a proteasome inhibitor) binding indicated that the RID-F derivatives interact with the protease subunits in a different manner. Culturing of human cells with these compounds resulted in accumulation of ubiquitinated proteins and induction of apoptosis. Thus, the RID-F derivatives may be useful lead chemicals for the generation of a new class of proteasome inhibitors.

Identification of the KDM2/7 histone lysine demethylase subfamily inhibitor and its antiproliferative activity.

Histone N(ε)-methyl lysine demethylases KDM2/7 have been identified as potential targets for cancer therapies. On the basis of the crystal structure of KDM7B, we designed and prepared a series of hydroxamate analogues bearing an alkyl chain. Enzyme assays revealed that compound 9 potently inhibits KDM2A, KDM7A, and KDM7B, with IC50s of 6.8, 0.2, and 1.2 µM, respectively. While inhibitors of KDM4s did not show any effect on cancer cells tested, the KDM2/7-subfamily inhibitor 9 exerted antiproliferative activity, indicating the potential for KDM2/7 inhibitors as anticancer agents.

Establishment of true niacin deficiency in quinolinic acid phosphoribosyltransferase knockout mice.

Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice.

An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines.

Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.

Synthesis and biological activity of optically active NCL-1, a lysine-specific demethylase 1 selective inhibitor.

Optically active (1S,2R)-NCL-1 and (1R,2S)-NCL-1 were synthesized and evaluated for their lysine-specific demethylase 1 inhibitory activity and cell growth inhibitory activity. In enzyme assays, the (1S,2R)-isomer was approximately four times more potent than the (1R,2S)-isomer. In cell growth inhibition assays, the two isomers showed similar activity in HEK293 cells and SH-SY5Y cells, whereas the (1S,2R)-isomer showed approximately four times more potent activity than the (1R,2S)-isomer in HeLa cells.

Identification of SAP155 as the target of GEX1A (Herboxidiene), an antitumor natural product.

GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

A novel human dynactin-associated protein, dynAP, promotes activation of Akt, and ergosterol-related compounds induce dynAP-dependent apoptosis of human cancer cells.

There are several human genes that may encode proteins whose functions remain unknown. To find clues to their functions, we used the mutant yeast defective in Mad2, a component of the spindle checkpoint complex. Phenotypes that were provoked by the expression of a human C18orf26 protein in the mutant yeast encouraged further characterization of this protein in human cells. This protein was designated dynAP (dynactin-associated protein) because of its interaction with dynactin subunits that comprised a microtubule-based motor protein complex. The dynAP is a transmembrane protein localizing to Golgi apparatus and plasma membrane in a microtubule-dependent manner. This protein was expressed in half of human cancer cell lines but barely in normal human fibroblasts tested. The SV40-transformed fibroblasts expressed dynAP. Importantly, the expression of dynAP activated Akt (also known as protein kinase B) by promoting Ser47³ phosphorylation required for the full activation, whereas knockdown of dynAP abolished this activation. The ergosterol-related compounds identified by the yeast cell-based high-throughput screen abrogated activation of Akt and induced apoptosis in a dynAP-dependent manner. We propose a possible advantage of dynAP expression in cancer cells; the survival of cancer cells that express dynAP is supported by dynAP-induced activation of Akt, sustaining high rates of proliferation. The inactivation of dynAP by the selected compounds nullifies this advantage, and thereby, the apoptotic machinery is allowed to operate. Taken together, dynAP can be a new target for cancer therapy, and the selected chemicals are useful for developing a new class of anticancer drugs.

Design, synthesis, enzyme-inhibitory activity, and effect on human cancer cells of a novel series of jumonji domain-containing protein 2 histone demethylase inhibitors.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.