Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.

Characterization and real-time imaging of the FTLD-related protein aggregation induced by amyloidogenic peptides.

We identify a new amyloidogenic peptide from the glutamine/asparagine-rich region of the FTLD-related protein (TDP-43), which can seed both the full-length and N-terminus-truncated TDP-43. Through the microinjection and real-time fluorescence imaging, we also found that this novel peptide could trigger cell apoptosis and initiate TDP-43 aggregation in the cytosol.

Identification of a two-layer regulatory network of proliferation-related microRNAs in hepatoma cells.

To elucidate how microRNA (miRNA)-regulated networks contribute to the uncontrolled growth of hepatoma cells (HCCs), we identified several proliferation-related miRNAs by comparing miRNA expression patterns in clinical HCC samples and growth-arrested HepG2 cells. To explore the molecular functions targeted by these miRNAs, we classified genes differentially expressed in clinical HCC samples into six functional clusters based on their functional similarity. Using target enrichment analysis, we discovered that targets of three proliferation-related miRNAs-miR-101, miR-199a-3p and miR-139-5p-were significantly enriched in the 'transcription regulation' functional cluster. An interactome network consisting of these three miRNAs and genes in the 'transcriptional control' cluster revealed that all three miRNAs were highly connected hubs in the network. All three miRNA-centered subnetworks displayed characteristics of a two-layer regulatory architecture, with transcription factors and epigenetic modulators as the first neighbors and genes involved in cell-cycle progression as second neighbors. The overexpression of miR-101 in HepG2 cells reduced the expression of transcription regulators and genes in cell-cycle progression and suppressed the proliferation and colony formation of HepG2 cells. This study not only provides direct experimental data to support the 'miRNA-centered two-layer regulatory network' model, but our results also suggest that such a combinatorial network model may be widely used by miRNAs to regulate critical biological processes.

Phosphorylation-dependent association of the G4-1/G5PR regulatory subunit with IKKß negatively modulates NF-κB activation through recruitment of protein phosphatase 5.

The transcription factor NF-κB (nuclear factor κB) co-ordinates various gene expressions in response to diverse signals and is a critical regulator of inflammation and innate immunity. Several negative regulators of NF-κB have been identified as downstream targets of NF-κB and function as a feedback control of NF-κB activation. A few protein phosphatases have also been shown to inactivate NF-κB activation. However, little is known about how protein phosphatases detect and respond to NF-κB activation. In the present study, we report a regulatory subunit of PP5 (protein phosphatase 5), G4-1, that physically interacts with IKKß [IκB (inhibitor of NF-κB) kinase ß] and negatively regulates NF-κB activation. The association of G4-1 with IKKß depends on the kinase activity of IKKß. Mapping of the G4-1-binding domain of IKKß reveals that the serine-rich domain in the C-terminus of IKKß is required for G4-1 binding. When seven autophosphorylated serine residues in this domain were mutated to alanine, the mutant form of IKKß lost its ability to bind G4-1 and was more potent than the wild-type kinase to activate NF-κB. Knockdown of G4-1 enhanced TNFα (tumour necrosis factor α)-induced NF-κB activity, and knockdown of PP5 totally abolished the inhibitory activity of G4-1 on NF-κB activation. The results of the present study suggest that G4-1 functions as an adaptor to recruit PP5 to the phosphorylated C-terminus of activated IKKß and to down-regulate the activation of IKKß.

PKCalpha mediated induction of miR-101 in human hepatoma HepG2 cells.

BACKGROUND: Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCalpha mediated cell growth arrest is still unknown. METHODS: We first surveyed 270 miRNA expression profiles in 20 pairs of human hepatoma tissues. We identified 11 up-regulated and 23 down-regulated miRNAs (FDR < = 0.01; fold-change > = 2) in human hepatoma tissue after Student's T-test and Mann-Whitney rank test. We then examined miRNAs expression profile in TPA treated HepG2 cells. Two miRNAs, miR-101, and miR-29c, were shown to be significantly down regulated in human hepatoma tissues and induced over 4-fold in HepG2 cells under TPA treatment. RESULTS: In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. We identified two miRNAs, 101 and 29c, were induced by TPA and down regulated in human hepatoma tissues suggest that they might play as tumor suppressor gene and in tumor formation of HCC. Since induction kinetics of miR-101 by TPA was much faster than miR-29c suggests that the induction of miR-101 may be the primary response of TPA treatment. We then further investigated how miR-101 was regulated by TPA. MiR-101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play as a tumor suppressor gene in human prostate, breast and liver cancers. The target sequence of miR-101 located in the 3' UTR of both EZH2 and EED's mRNA was identified by bioinformatic analysis and was validated by reporter luciferase activity assay. Then we showed that TPA not only up regulated miR-101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti-virus-mediated shRNA to knockdown endogenous PKCalpha expression, we observed that TPA induced growth arrest, elevation of miR-101 and reduction of EZH2, EED and H3K27me3 proteins were all PKCalpha dependent. Specific inhibitor of ERK completely blocked TPA induced miR-101 expression. CONCLUSIONS: Therefore, this is the first time to show that PKCalpha and ERK pathway play important role to activate miR-101 expression, reduce PRC2 complex and H3K27me3 level. This epigenetic regulatory pathway may represent a novel mechanism of carcinogenesis and deserve further investigation.