Bin Hong

MicroRNAs 185, 96, and 223 Repress Selective High-Density Lipoprotein Cholesterol Uptake through Posttranscriptional Inhibition

ABSTRACT Hepatic scavenger receptor class B type I (SR-BI) plays an important role in selective high-density lipoprotein cholesterol (HDL-C) uptake, which is a pivotal step of reverse cholesterol transport. In this study, the potential involvement of microRNAs (miRNAs) in posttranscriptional regulation of hepatic SR-BI and selective HDL-C uptake was investigated. The level of SR-BI expression was repressed by miRNA 185 (miR-185), miR-96, and miR-223, while the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-HDL was decreased by 31.9% (P < 0.001), 23.9% (P < 0.05), and 15.4% (P < 0.05), respectively, in HepG2 cells. The inhibition of these miRNAs by their anti-miRNAs had opposite effects in these hepatic cells. The critical effect of miR-185 was further validated by the loss of regulation in constructs with mutated miR-185 target sites. In addition, these miRNAs directly targeted the 3′ untranslated region (UTR) of SR-BI with a coordinated effect. Interestingly, the decrease of miR-96 and miR-185 coincided with the increase of SR-BI in the livers of ApoE KO mice on a high-fat diet. These data suggest that miR-185, miR-96, and miR-223 may repress selective HDL-C uptake through the inhibition of SR-BI in human hepatic cells, implying a novel mode of regulation of hepatic SR-BI and an important role of miRNAs in modulating cholesterol metabolism.

Heat Stress Cognate 70 Host Protein as a Potential Drug Target against Drug Resistance in Hepatitis B Virus

ABSTRACT Heat stress cognate 70 (Hsc70) is a host protein associated with hepatitis B virus (HBV) replication. The goal of this study was to investigate whether Hsc70 could be an anti-HBV drug target. Our results showed that introducing Hsc70 increased HBV replication in HBV+ human hepatocytes (HepG2.2.15 cells). The coiled-coil region on Hsc70 (nucleotides 1533 to 1608; amino acids 511 to 536) was the key sequence for HBV replication. Knockdown of Hsc70 expression by RNA interference (RNAi) largely inhibited HBV replication with no cytotoxicity to the host. Using an Hsc70 mRNA screening assay, the natural compound oxymatrine (OMTR) was found to be a selective inhibitor for Hsc70 expression. Then, OMTR was used to investigate the potential of Hsc70 as an anti-HBV drug target. OMTR inhibited Hsc70 mRNA expression by 80% and HBV DNA replication by over 60% without causing cytotoxicity. The anti-HBV effect of OMTR appeared to be mediated by destabilizing Hsc70 mRNA. The half-life (T1/2) of Hsc70 mRNA decreased by 50% in OMTR-treated hepatocytes. The Hsc70 mRNA 3′-untranslated-region (UTR) sequence was the element responsible for OMTRs destabilization activity. OMTR suppressed HBV de novo synthesis at the reverse transcription stage from pregenomic RNA (pgRNA) to DNA and was active against either wild-type HBV or strains resistant to lamivudine, adefovir, and entecavir. Therefore, host Hsc70 could be a novel drug target against HBV, and OMTR appears to inhibit HBV replication by destabilizing Hsc70 mRNA. As the target is not a viral protein, OMTR is active for either wild-type HBV or strains resistant to reverse transcriptase (RT) inhibitors.

Identification of Upregulators of Human ATP-Binding Cassette Transporter A1 via High-Throughput Screening of a Synthetic and Natural Compound Library

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by mediating the cellular efflux of cholesterol and phospholipids to lipid-poor apolipoprotein A-I. Therefore, identification of a novel upregulator of ABCA1 would be beneficial for atherosclerosis prevention and/or therapy because of its pivotal role in cholesterol homeostasis and HDL metabolism. In this study, a high-throughput assay method for ABCA1 upregulators was developed and used for screening a synthetic and natural compound library. The cell-based high-throughput screen is conducted in a 96-well format using the human hepatoma HepG2 cells stably transfected with ABCA1 promoter-luciferase construct and calibrated with reference ABCA1 upregulators (oxysterols, 9-cis-retinoic acid, thiazolidinediones, cyclic adenosine monophosphate, verapamil, fenofibrate, and oncostatin M). Among 2600 compounds, 4 microbial compounds (pyrromycin, aclarubicin, daidzein, and pratensein) were picked up as hits by the high-throughput screening assay, and those compounds were further identified as upregulators of ABCA1 expression by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. (Journal of Biomolecular Screening 2008:648-656)

Salvianolic acid B inhibits macrophage uptake of modified low density lipoprotein (mLDL) in a scavenger receptor CD36-dependent manner

CD36, a class B scavenger receptor, has been implicated in the pathogenesis of a host of vascular inflammatory diseases. Through a high-throughput screening (HTS) assay for CD36 antagonist, we previously identified salvianolic acid B (SAB), a hydrophilic component derived from the herb Danshen, as a potential candidate. Danshen, the dried roots of Salvia miltiorrhiza, has been widely used in China for the prevention and treatment of atherosclerosis-related disorders. Previous studies showed that SAB acted as an anti-oxidant by preventing lipid peroxidation and oxidized LDL (oxLDL) formation. The present study was to investigate the specificity and efficacy of SAB in the inhibition of CD36-mediated lipid uptake. SAB reduced modified LDL (mLDL) uptake in a dose-dependent manner in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 and RAW 264.7 cells. In the CD36 silenced THP-1 cells, SAB had no effect in reducing mLDL uptake, whereas its overexpression in CHO cells reinstates the effect, indicating a specific involvement of SAB in antagonizing the CD36s function. Surface plasmon resonance (SPR) analysis revealed a direct binding of SAB to CD36 with a high affinity (K(D) = 3.74 μM), confirming physical interactions of SAB with the receptor. Additionally, SAB reduced oxLDL-induced CD36 gene expression in the cultured cell lines and primary macrophages. In ApoE KO mice fed a high fat diet, SAB reduced CD36 gene expression and lipid uptake in macrophages, showing its ability to antagonize CD36 pathways in vivo. These results demonstrate that SAB is an effective CD36 antagonist and suggest SAB as a potential anti-atherosclerotic agent.

Identification of trichostatin A as a novel transcriptional up-regulator of scavenger receptor BI both in HepG2 and RAW 264.7 cells

Scavenger receptor class B type I (SR-BI) and its human homologue CLA-1 plays an important role in reverse cholesterol transport (RCT). Using a previously established cell-based CLA-1 up-regulator screening assay, one of the positive strains, 04-9179, presented potent activity in elevating CLA-1 transcriptional level. We report here the identification of an active compound 9179A as a known compound trichostatin A (TSA), and its effects on CLA-1/SR-BI expression both in HepG2 human hepatoma cells and RAW 264.7 murine macrophage cells in vitro. The results showed that the mRNA and protein level of CLA-1/SR-BI were significantly up-regulated by 9179A both in HepG2 and RAW 264.7 cells. Corresponding to this, the uptake of DiI-HDL by both cells and the efflux of [(3)H]cholesterol by RAW 264.7 cells were increased by 9179A in dose-dependent manner. ABCA1 was also increased but SR-A decreased by 9179A in RAW 264.7 cells. Using a combination of reporter assays with various deletion in CLA-1 promoter and electrophoretic mobility shift assay, we demonstrated that -419/-232 bp fragment of the CLA-1 promoter mediated the effects of 9179A (i.e., TSA). Together, these studies identified TSA as a novel up-regulator of CLA-1/SR-BI both in HepG2 and RAW 264.7 cells.

Discovery of Antagonists for Human Scavenger Receptor CD36 via an ELISA-Like High-Throughput Screening Assay

CD36, a member of the class B scavenger receptor, is a high-affinity receptor for oxidatively modified low-density lipoprotein (oxLDL). Extensive evidence points to a significant role of CD36 in atherosclerosis and suggests that CD36 could be a potential target for treatment of atherosclerosis. Here, the extracellular domain of human CD36 (Gly30-Asn439) was expressed in Escherichia coli as His6-tagged soluble CD36 (sCD36), which could bind oxLDL specifically and effectively inhibit the uptake of oxLDL by murine macrophage RAW 264.7 cells. An enzyme-linked immunosorbent assay (ELISA)–like high-throughput screening (HTS) assay was developed for the discovery of CD36 antagonists, based on the competition of sCD36 binding to immobilized oxLDL and detection with a monoclonal antibody against His-tag. This assay was suitable for HTS in a 96-well format and was robust and reliable according to the evaluation parameter Z′ value of 0.82. The developed HTS assay was applied to both pure chemical compounds and microbial secondary metabolite crude extracts to identify CD36 antagonists. Three active compounds—sodium danshensu (DSS), rosmarinic acid (RA), and salvianolic acid B (SAB)—were shown to be antagonistic to sCD36-oxLDL binding and further validated by their inhibition of oxLDL uptake in RAW 264.7 cells. These results suggest that the ELISA-like assay represents a promising screening for identifying bioactive molecules targeting atherosclerosis at the level of CD36-ligand binding.

Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade.

In Streptomyces coelicolor, AtrA is an activator of transcription of the actinorhodin cluster-situated regulator gene actII-ORF4. In previous work, we showed that S. coelicolor AtrA binds in vitro to the promoter of S. griseusstrR, the streptomycin cluster-situated regulator. We show here that S. griseus carries a single close homologue of atrA and that expression of S. coelicolor AtrA in S. griseus causes a DNA binding-dependent reduction in streptomycin production and in the mRNA levels of strR and genes of streptomycin biosynthesis. However, there is no effect on the level of the mRNA of adpA, which is the only transcription factor that has so far been characterised for strR. The adpA gene is directly regulated by ArpA, the receptor protein for the γ-butyrolactone signalling molecule A-factor. Therefore, to our knowledge, our results provide the first in vivo evidence that A-factor-ArpA-AdpA-StrR regulatory cascade represents only part of the full complexity of regulation of streptomycin biosynthesis in S. griseus. The potential biotechnological application of our findings is discussed.

SsaA, a Member of a Novel Class of Transcriptional Regulators, Controls Sansanmycin Production in Streptomyces sp. Strain SS through a Feedback Mechanism

Sansanmycins, produced by Streptomyces sp. strain SS, are uridyl peptide antibiotics with activities against Pseudomonas aeruginosa and multidrug-resistant Mycobacterium tuberculosis. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turn-helix (HTH) motif. The disruption of ssaA abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN₂TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.

Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system

Mycobacterium tuberculosis kills about 2 million people annually and antibiotic resistance is a cause of increased mortality. Therefore, development of new antituberculosis drugs is urgent for the control of widespread tuberculosis infections. For this purpose, we performed an innovative screen to identify new agents that disrupt the function of ribosomes in M. tuberculosis. Two bacterial ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors (EFs) during translation. Therefore, the L12–L10 interaction should be essential for ribosomal function and protein synthesis. We established a yeast two-hybrid system to identify small molecules that block the interaction between L12 and L10 proteins from M. tuberculosis. Using this system, we identified two compounds T766 and T054 that show strong bactericidal activity against tuberculosis but with low toxicity to mice and other bacterial strains. Moreover, using surface plasmon resonance (SPR) assay, we have demonstrated that these compounds bind specifically to L12 to disrupt L12–L10 interaction. Overproduction of L12 protein, but not L10, lowers the antibacterial activity of T766 and T054, indicating that the ribosome is likely the cellular target. Therefore, our data demonstrate that this yeast two-hybrid system is a useful tool to identify unique antituberculosis agents targeting the ribosomal protein L12–L10 interaction.

Rutaecarpine suppresses atherosclerosis in ApoE-/- mice through upregulating ABCA1 and SR-BI within RCT.

ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE−/−) mice treated with RUT for 8 weeks showed ∼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE−/− mice. Meanwhile, RUT treatment significantly increased the fecal 3H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE−/− mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.