Preparation of cell-permeable Cre recombinase by expressed protein ligation.

BACKGROUND: Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli. RESULTS: Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC. CONCLUSIONS: Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Tunable heptamethine-azo dye conjugate as an NIR fluorescent probe for the selective detection of mitochondrial glutathione over cysteine and homocysteine.

Although a lot of mitochondria-targeting biothiol probes have been developed and applied to cellular imaging through thiol-induced disulfide cleavage or Michael addition reactions, relatively few probes assess mitochondrial GSH with high selectivity over Cys and Hcy and with NIR fluorescence capable of noninvasive imaging in biological samples. In order to monitor mitochondrial GSH with low background autofluorescence, we designed a heptamethine-azo conjugate as an NIR fluorescent probe by introducing a tunable lipophilic cation unit as the biomarker for mitochondria and a nitroazo group as the GSH-selective reaction site as well as the fluorescence quencher. The probe exhibited a dramatic off-on NIR fluorescence response toward GSH with high selectivity over other amino acids including Cys and Hcy. Further application to cellular imaging indicated that the probe was highly responsive to the changes of mitochondrial GSH in cells.

Intracellular protein delivery by hollow mesoporous silica capsules with a large surface hole.

We prepared cell membrane-permeable hollow mesoporous silica capsules (HMSCs) by a simple new method. CTAB micellar assembly in cholesterol emulsion gave rise to a novel capsular morphology of the HMSC particles. The HMSCs consisted of mesostructured silica walls with a large surface hole (25-50 nm) and the average particle dimension was 100-300 nm. They exhibited high surface areas of up to 719.3 m(2) g(-1) and a mesoporous range of pores of 2.4-2.7 nm. The surface-functionalized HMSCs could also be prepared by a similar co-condensation method using tetraethoxysilane with various organoalkoxysilane precursors in the presence of cholesterol. These organically modified HMSCs could be further modified on demand. For example, a carboxy-functionalized HMSC could be surface-functionalized by a green fluorescent 5-aminofluorescein (AFL) through an amidation reaction to afford a fluorescent AFL-HMSC. The hollow capsular morphology of the HMSCs with a large surface hole enabled us to develop very efficient intracellular delivery systems for membrane-impermeable ions, molecules, and various functional proteins. Non-covalent sequestration and delivery of proteins as well as covalent linkage of fluorescent molecules on the silica surface are effective for this system. The highly negatively charged green fluorescent probe mag-fluo-4 could be intracellularly delivered into HeLa cells by HMSC without any difficulty. The HMSCs could also effectively transport large functional proteins such as antibodies into HeLa cells. The efficiency of protein delivery by HMSC seems to be 3-22-fold higher than that of mesoporous silica nanospheres (MSNs) based on confocal laser scanning microscopy (CLSM) analysis.

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells.

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.

Expansion of chromosome territories with chromatin decompaction in BAF53-depleted interphase cells.

Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism.

Effects of Polygala tenuifolia root extract on proliferation of neural stem cells in the hippocampal CA1 region.

Neurogenesis persists in the adult mammalian brain and can be a target for modulation for therapeutic purposes. This study investigated the effect of a Polygala tenuifolia root extract on the proliferation of a stem cell population in the rat hippocampus. The root extract of P. tenuifolia (2 mg/kg/day, 14 times intraperitoneal injections) increased the incorporation of bromodeoxyuridine (BrdU) into cells in the hippocampal CA1 region. This activity was enriched in the saponin-containing fraction. The majority of cells labelled with BrdU were immunoreactive to nestin or Tuj1 and the percentages of nestin/BrdU- and Tuj1/BrdU-double positive cells were increased by the P. tenuifolia root extract, suggesting that the P. tenuifolia root extract promotes the proliferation of neural stem cells. In addition, this extract promoted the neurite outgrowth of rat neuronal precursor cells, HiB5. These activities of P. tenuifolia root extract may contribute to the therapeutic benefits of herbal medicines containing P. tenuifolia root for the treatment of patients with insomnia, neurosis and dementia.

Aucubin Promotes Differentiation of Neural Precursor Cells into GABAergic Neurons.

Aucubin is a small compound naturally found in traditional medicinal herbs with primarily anti-inflammatory and protective effects. In the nervous system, aucubin is reported to be neuroprotective by enhancing neuronal survival and inhibiting apoptotic cell death in cultures and disease models. Our previous data, however, suggest that aucubin facilitates neurite elongation in cultured hippocampal neurons and axonal regrowth in regenerating sciatic nerves. Here, we investigated whether aucubin facilitates the differentiation of neural precursor cells (NPCs) into specific types of neurons. In NPCs cultured primarily from the rat embryonic hippocampus, aucubin significantly elevated the number of GAD65/67 immunoreactive cells and the expression of GAD65/67 proteins was upregulated dramatically by more than three-fold at relatively low concentrations of aucubin (0.01 µM to 10 µM). The expression of both NeuN and vGluT1 of NPCs, the markers for neurons and glutamatergic cells, respectively, and the number of vGluT1 immunoreactive cells also increased with higher concentrations of aucubin (1 µM and 10 µM), but the ratio of the increases was largely lower than GAD expression and GAD immunoreactive cells. The GABAergic differentiation of pax6-expressing late NPCs into GABA-producing cells was further supported in cortical NPCs primarily cultured from transgenic mouse brains, which express recombinant GFP under the control of pax6 promoter. The results suggest that aucubin can be developed as a therapeutic candidate for neurodegenerative disorders caused by the loss of inhibitory GABAergic neurons.

Intracellular delivery of a native functional protein using cell-penetrating peptide functionalized cubic MSNs with ultra-large mesopores.

The intracellular delivery of functional proteins in their native forms into cells is a theme of paramount importance in research owing to their diverse biological applications. Porous inorganic nanoparticles are emerging as efficient nanocarriers for the delivery of small molecules and drugs. To expand the range of cargos from small molecules to large native functional proteins, cubic mesoporous silica nanoparticles (cMSNs) with a Pm3n pore symmetry with an average particle dimension of 180 nm were prepared. The as-prepared cMSNs were subsequently etched with a methanolic solution of Ca(NO3)2 to expand their mesopores and simultaneously remove the template. The original mesopores with a pore dimension of 2.41 nm partially collapsed and combined into ultra-large mesopores with an average pore diameter of 13.89 nm without perturbing the original cubic symmetry of the remaining mesopores. The maximum pore dimension was around 60 nm. Various techniques including powder X-ray diffraction, transmission electron microscopy, and electron tomography identified the unique three-dimensional structure of pore-enlarged cMSNs (Ca-cMSNs). Moreover, their surfaces were functionalized with a guanidinium-rich cell-penetrating R8-azido-peptide (p-azidophenylalanine-GSGSGGRRRRRRRR) through the click reaction. The intracellular delivery of functional proteins such as Cre recombinase into human TE671(LoxP-LacZ) cells was realized by using R8-Ca-cMSNs as native protein delivery synthetic nanocarriers. The delivery efficiency when using the R8-Ca-cMSNs significantly enhanced compared to that when using Ca-cMSNs without surface-bound cell-penetrating peptides.

Upregulation of amyloid precursor protein by platelet-derived growth factor in hippocampal precursor cells.

Amyloid precursor protein generates the secreted amyloid precursor protein alpha, which protects hippocampal neurons from ischemic injury and facilitates neuronal survival and synaptogenesis in the developing nervous system. Here, we examined whether platelet-derived growth factor regulates the generation of secreted amyloid precursor protein alpha during the neuronal differentiation of hippocampal precursor cells, HiB5. We showed that platelet-derived growth factor promoted amyloid precursor protein production and secreted amyloid precursor protein alpha secretion. These effects of platelet-derived growth factor were diminished by the PI3K-specific inhibitor wortmannin and the protein kinase C-specific inhibitor GF109203X, suggesting the involvement of the PI3K and protein kinase C-signaling pathway. Furthermore, the conditioned media enriched with secreted amyloid precursor protein alpha promoted the survival of HiB5 cells during neuronal differentiation. These results suggest that the neurotrophic effect of platelet-derived growth factor is mediated in part via upregulation of the expression and release of secreted amyloid precursor protein alpha.

Association of BAF53 with mitotic chromosomes.

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.

Shp2 is involved in neuronal differentiation of hippocampal precursor cells.

HiB5 is a multipotent hippocampal stem cell line whose differentiation into cells of a neuronal phenotype is promoted by neurotrophic factors such as PDGF and brain-derived neurotrophic factor (BDNF). We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatase (Shp2) in this differentiation process. We found that Shp2 became tyrosine phosphorylated following PDGF treatment. Wild-type Shp2 enhanced the expression of neurofilament, synapsin I and PSD95 by PDGF and BDNF, whereas their expression was attenuated by the catalytically inactive mutants Shp2C/S and Shp2DeltaP. Formation of dendritic spine-like structures increased with wild-type Shp2, but diminished with Shp2C/S and Shp2DeltaP. PSD95, localized in the post-synaptic density region of dendritic spines, PDGFRbeta and TrkB were co-immunoprecipitated with Shp2 antibodies. These results suggest that Shp2 plays a positive role in mediating PDGF- and BDNF-activated signaling which promotes the formation of dendritic spines.

Effects of Ser2 and Tyr6 mutants of BAF53 on cell growth and p53-dependent transcription.

BAF53 is an actin-related protein that shuttles between nucleus and cytoplasm. In the nucleus, it constitutes an integral component of many chromatin-modifying complexes such as the SWI/SNF, TIP60, TRRAP, and TIP48/49 complexes. BAF53 is essential for growth, but its function remains elusive. BAF53 homologues from yeast to humans have a conserved N-terminal motif, MS_(G/A)(G/A)_(V/L)YGG, which is unique to these proteins. Previously we showed that over-expression of an N-terminal deletion mutant of BAF53 (BAF53_deltaN) reduced the viability of HEK293 and HeLa cells. When we replaced the serine 2 and tyrosine 6 of this N-terminal motif with alanine, over-expression of the alanine-replaced BAF53 strongly impaired the growth of HEK293 cells whereas replacement with aspartate/glutamate had no effect. The alanine-replaced BAF53 mutants also stimulated p53-dependent transcription, in which the SWI/SNF and TRRAP complexes are involved. Our results demonstrate that serine 2 and tyrosine 6 play important roles in BAF53 activity.

The Actin-Related Protein BAF53 Is Essential for Chromosomal Subdomain Integrity.

A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

The effect of ER stress on activity-dependent induction of brain-derived neurotrophic factor exon III.

Neuronal activity promotes neuronal survival and increases synaptic strength. These effects are primarily mediated by influx of calcium through L-type calcium channels during membrane depolarization. This activates the transcription factor CREB by phosphorylating it on Ser-133 and induces several genes including BDNF exon III that play important roles in activity-dependent neuronal survival. Since ER stress is known to perturb intracellular calcium signaling, we examined whether it attenuated depolarization-induced CREB Ser-133 phosphorylation and BDNF exon III expression in PC12 cells. ER stress induced by tunicamycin, beta-mercaptoethanol, or thapsigargin did not reduce depolarization-induced CREB Ser-133 phosphorylation. However it did suppress the induction of BDNF exon III by membrane depolarization, indicating that it exerts this effect by blocking a pathway distinct from CREB Ser-133 phosphorylation.

Cytoplasmic localization and nucleo-cytoplasmic shuttling of BAF53, a component of chromatin-modifying complexes.

BAF53 (also known as ArpNbeta) is a nuclear arp that is a component of a number of chromatin-modifying complexes, such as the SWI/SNF and TRRAP complexes. These complexes play specific roles in chromatin remodeling and transcription. Previous studies have suggested that BAF53 is localized exclusively in the nucleus in HeLa and SW-13 cells. In this study, however, we provide evidence that it is also present in the cytoplasm of HeLa, NIH3T3, and WI-38 cells. Cytoplasmic localization was more prominent in NIH3T3 and WI-38 cells, and the cytoplasmic BAF53 staining in NIH3T3 cells appeared punctate. We also show by interspecies heterokaryon assays and in vitro export assays that BAF53 shuttles between the cytoplasm and nucleus in an energy-dependent manner. Shuttling was not blocked by leptomycin B, indicating that export of BAF53 is not mediated by CRM1. The nucleocytoplasmic shuttling and the punctate staining of the cytoplasm suggest that BAF53 can play a role distinct from its previously recognized function in transcriptional regulation.

Coumarin-malonitrile conjugate as a fluorescence turn-on probe for biothiols and its cellular expression.

A nonfluorescent coumarin-malonitrile conjugate (1) was transformed into a strongly fluorescent molecule through the Michael addition of a thiol group to the α,ß-unsaturated malonitrile of 1. The molecular probe has exhibited a highly selective fluorescence response toward biothiols (Cys, Hcy, GSH) with micromolar sensitivity both in vitro and in vivo.

Ratiometric fluorescence imaging of cellular glutathione.

A fluorescent probe (1) with a hydrogen bond was designed for the detection of GSH. The probe exhibited a rapid and ratiometric fluorescence response to GSH through a Michael reaction and allowed us to obtain clear cellular images for GSH.

The viral oncogene human papillomavirus E7 deregulates transcriptional silencing by Brm-related gene 1 via molecular interactions.

BRG-1, a component of the human SWI/SNF complex, either activates or represses cellular promoters by modulating chromatin structure via the formation of a multiple polypeptide complex. Human papillomavirus E7 binds and destabilizes pRb, resulting in the blockage of G(1) arrest in the cell cycle. We show here that the high-risk human papillomavirus E7 protein group binds BRG-1 and modulates repression of the c-fos promoter mediated by this protein. In addition, both wild-type and Rb binding-defective E7 proteins abolish flat cell formation by BRG-1 in SW13 cells, whereas E7 COOH-terminal mutants do not affect this process. BRG-1-triggered repression of the c-fos promoter is sensitive to trichostatin A. We further establish that BRG-1 contains an activation domain and a trichostatin A-sensitive repression domain. These results collectively suggest that the viral oncogene E7 targets both pRb and BRG-1 via protein-protein interactions, resulting in the deregulation of host cell cycle control.