Delivery of Superoxide Dismutase Using Cys2-His2 Zinc-Finger Proteins.

Therapeutic proteins have shown great potential in treating life-threatening diseases, but the hydrophilicity and high molecular weight hamper their passing through the cell membrane. Cell-penetrating peptide (CPP)-assisted protein delivery is a simple and efficacious strategy to promote the cellular uptake of therapeutic proteins. We recently demonstrated that the engineered Cys2-His2 zinc-finger domains possess intrinsic cell permeability, which could be leveraged for intracellular protein delivery. Here we applied this method to deliver superoxide dismutase (SOD), a therapeutic protein widely used in preclinical and clinical studies. We present a protocol for the production and delivery of zinc-finger domain-fused SOD. This protocol can be extended for delivering other therapeutic proteins.

PI4KIIα regulates insulin secretion and glucose homeostasis via a PKD-dependent pathway.

Insulin release by pancreatic ß cells plays a key role in regulating blood glucose levels in humans, and to understand the mechanism for insulin secretion may reveal therapeutic strategies for diabetes. We found that PI4KIIα transgenic (TG) mice have abnormal glucose tolerance and higher serum glucose levels than wild-type mice. Glucose-stimulated insulin secretion was significantly reduced in both PI4KIIα TG mice and PI4KIIα-overexpressing pancreatic ß cell lines. A proximity-based biotin labeling technique, BioID, was used to identify proteins that interact with PI4KIIα, and the results revealed that PI4KIIα interacts with PKD and negatively regulates its activity. The effect of PI4KIIα on insulin secretion was completely rescued by altering PKD activity. PI4KIIα overexpression also worsened glucose tolerance in streptozotocin/high-fat diet-induced diabetic mice by impairing insulin secretion. Our study has shed new light on PI4KIIα function and mechanism in diabetes and identified PI4KIIα as an important regulator of insulin secretion.

PI-273, a Substrate-Competitive, Specific Small-Molecule Inhibitor of PI4KIIα, Inhibits the Growth of Breast Cancer Cells.

While phosphatidylinositol 4-kinase (PI4KIIα) has been identified as a potential target for antitumor therapy, the clinical applications of PI4KIIα are limited by a lack of specific inhibitors. Here we report the first small-molecule inhibitor (SMI) of human PI4KIIα. Docking-based and ligand-based virtual screening strategies were first employed to identify promising hits, followed by two rounds of kinase activity inhibition validation. 2-(3-(4-Chlorobenzoyl)thioureido)-4-ethyl-5-methylthiophene-3-carboxamide (PI-273) exhibited the greatest inhibitory effect on PI4KIIα kinase activity (IC50 = 0.47 µmol/L) and suppressed cell proliferation. Surface plasmon resonance and thermal shift assays indicated that PI-273 interacted directly with PI4KIIα. Kinetic analysis identified PI-273 as a reversible competitive inhibitor with respect to the substrate phosphatidylinositol (PI), which contrasted with most other PI kinase inhibitors that bind the ATP binding site. PI-273 reduced PI4P content, cell viability, and AKT signaling in wild-type MCF-7 cells, but not in PI4KIIα knockout MCF-7 cells, indicating that PI-273 is highly selective for PI4KIIα. Mutant analysis revealed a role of palmitoylation insertion in the selectivity of PI-273 for PI4KIIα. In addition, PI-273 treatment retarded cell proliferation by blocking cells in G2-M, inducing cell apoptosis and suppressing colony-forming ability. Importantly, PI-273 significantly inhibited MCF-7 cell-induced breast tumor growth without toxicity. PI-273 is the first substrate-competitive, subtype-specific inhibitor of PI4KIIα, the use of which will facilitate evaluations of PI4KIIα as a cancer therapeutic target. Cancer Res; 77(22); 6253-66. ©2017 AACR.

Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα as anti-tumor strategy.

Our previous studies indicate that phosphatidylinositol 4-kinase IIα can promote the growth of multi-malignant tumors via HER-2/PI3K and MAPK pathways. However, the molecular mechanisms of this pathway and its potential for clinical application remain unknown. In this study, we found that PI4KIIα could be an ideal combinatorial target for EGFR treatment via regulating EGFR degradation. Results showed that PI4KIIα knockdown reduced EGFR protein level, and the expression of PI4KIIα shows a strong correlation with EGFR in human breast cancer tissues (r = 0.77, P < 0.01). PI4KIIα knockdown greatly prolonged the effects and decreased the effective dosage of AG-1478, a specific inhibitor of EGFR. In addition, it significantly enhanced AG1478-induced inhibition of tumor cell survival and strengthened the effect of the EGFR-targeting anti-cancer drug Iressa in xenograft tumor models. Mechanistically, we found that PI4KIIα suppression increased EGFR ligand-independent degradation. Quantitative proteomic analysis by stable isotope labeling with amino acids in cell culture (SILAC) and LC-MS/MS suggested that HSP90 mediated the effect of PI4KIIα on EGFR. Furthermore, we found that combined inhibition of PI4KIIα and EGFR suppressed both PI3K/AKT and MAPK/ERK pathways, and resulted in downregulation of multiple oncogenes like PRDX2, FASN, MTA2, ultimately leading to suppression of tumor growth. Therefore, we conclude that combined inhibition of PI4KIIα and EGFR exerts a multiple anti-tumor effect. Dual inhibition of EGFR at protein and activity level via combinatorial blocking of PI4KIIα presents a novel strategy to combat EGFR-dependent tumors.

Molecular insights into the membrane-associated phosphatidylinositol 4-kinase IIα.

Phosphatidylinositol 4-kinase IIα (PI4KIIα), a membrane-associated PI kinase, plays a central role in cell signalling and trafficking. Its kinase activity critically depends on palmitoylation of its cysteine-rich motif (-CCPCC-) and is modulated by the membrane environment. Lack of atomic structure impairs our understanding of the mechanism regulating kinase activity. Here we present the crystal structure of human PI4KIIα in ADP-bound form. The structure identifies the nucleotide-binding pocket that differs notably from that found in PI3Ks. Two structural insertions, a palmitoylation insertion and an RK-rich insertion, endow PI4KIIα with the 'integral' membrane-binding feature. Molecular dynamics simulations, biochemical and mutagenesis studies reveal that the palmitoylation insertion, containing an amphipathic helix, contributes to the PI-binding pocket and anchors PI4KIIα to the membrane, suggesting that fluctuation of the palmitoylation insertion affects PI4KIIα's activity. We conclude from our results that PI4KIIα's activity is regulated indirectly through changes in the membrane environment.

Molecularly imprinted solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of safranine T in wolfberry.

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001-1.0 µg mL(-1) (r = 0.9999) with a detection limit (S/N = 3) of 0.4 ng g(-1). Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry.

Preparation and evaluation of a novel molecularly imprinted SPE monolithic capillary column for the determination of auramine O in shrimp.

A novel method combining molecular imprinting and SPE was developed in a capillary column for the determination of auramine O in shrimp. The capillary monolithic column was prepared by UV-initiated in situ polymerization, using auramine O as template and methacrylic acid and ethylene dimethacrylate as functional monomer and cross-linker, respectively. The properties of the prepared capillary monolithic column were investigated under the optimized conditions coupled with HPLC, and then the morphologies of the inner polymers were characterized by SEM. The calibration curve was expressed as A = 103C + 19.8 (r = 0.9992) with a linear range of 0.25-25.0 µg/mL, and the recoveries of auramine O at different concentrations in shrimp ranged from 90.5 to 92.4% with RSDs ranging from 2.1 to 4.4%. The capacities of the molecularly imprinted polymer and nonimprinted polymer columns were 0.722 and 0.147 µg/mg, respectively, and the LOD (S/N = 3) of auramine O in shrimp was 17.85 µg/kg. Under the selected conditions, the enrichment factors obtained were higher than 70-fold. The results indicate that the prepared molecularly imprinted capillary monolithic column was reliable and applicable to the analysis of auramine O in shrimp.

ESNOQ, proteomic quantification of endogenous S-nitrosation.

S-nitrosation is a post-translational protein modification and is one of the most important mechanisms of NO signaling. Endogenous S-nitrosothiol (SNO) quantification is a challenge for detailed functional studies. Here we developed an ESNOQ (Endogenous SNO Quantification) method which combines the stable isotope labeling by amino acids in cell culture (SILAC) technique with the detergent-free biotin-switch assay and LC-MS/MS. After confirming the accuracy of quantification in this method, we obtained an endogenous S-nitrosation proteome for LPS/IFN-gamma induced RAW264.7 cells. 27 S-nitrosated protein targets were confirmed and using our method we were able to obtain quantitative information on the level of S-nitrosation on each modified Cys. With this quantitative information, over 15 more S-nitrosated targets were identified than in previous studies. Based on the quantification results, we found that the S-nitrosation levels of different cysteines varied within one protein, providing direct evidence for differences in the sensitivity of cysteine residues to reactive nitrosative stress and that S-nitrosation is a site-specific modification. Gene ontology clustering shows that S-nitrosation targets in the LPS/IFN-gamma induced RAW264.7 cell model were functionally enriched in protein translation and glycolysis, suggesting that S-nitrosation may function by regulating multiple pathways. The ESNOQ method described here thus provides a solution for quantification of multiple endogenous S-nitrosation events, and makes it possible to elucidate the network of relationships between endogenous S-nitrosation targets involved in different cellular processes.

Nitric oxide destabilizes Pias3 and regulates sumoylation.

Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes.