Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration.

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end-tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150(Glued) and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.

A new strategy for intracellular delivery of enzyme using mesoporous silica nanoparticles: superoxide dismutase.

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus 1 (HIV) transactivator protein (TAT) to mesoporous silica nanoparticle is shown to be an effective way to enhance transmembrane delivery of nanoparticles for intracellular and molecular therapy. Cu,Zn-superoxide dismutase (SOD) is a key antioxidant enzyme that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.

Specifically and reversibly immobilizing proteins/enzymes to nitriolotriacetic-acid-modified mesoporous silicas through histidine tags for purification or catalysis.

Six nitriolotriacetic-acid-modified ordered mesoporous silicas (NTA-OMPSs) with different pore sizes and surface features for specific and reversible protein immobilization were fabricated and characterized. Specific immobilization of a genetically engineered undecaprenyl pyrophosphate synthase (UPPs) from cell lysate and a chemically modified His-tagged horseradish peroxidase (HRP) in these Ni-NTA-OMPSs through histidine coordination to the nickelated NTA was demonstrated and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Moreover, histidine tags with different lengths (His6, His4, His3, and His2) applied to HRP were evaluated to find the minimum length for effective complexation. Enzymatic assessment studies indicated that the pore size of the OMPSs has minimal influence on the enzymatic activity, whereas chemical entities such as unreacted mercapto groups tailored on the interior surfaces of the OMPSs played certain roles in inhibiting the enzymatic activity and stability. On MCF-S-NTA, SBA-S-NTA, and film-S-NTA, which contained unreacted mercaptopropyl groups on the interior surface, immobilized His-tagged HRP showed lower catalytic activity and stability than on MCF-NTA, film-NTA, and SBA-NTA. Selective hydroxylation of optically pure L-tyrosine to (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid (L-DOPA) by the immobilized HRP was also demonstrated.

En route to white-light generation utilizing nanocomposites composed of ultrasmall CdSe nanodots and excited-state intramolecular proton transfer dyes.

One single material that emits white light is of paramount interest for the development of white light-emitting diodes (WLEDs). Here we report a novel nanocomposite, in which a new type of excited-sate intramolecular proton transfer (ESIPT) molecule, namely 5-(1,2-dithiolan-3-yl)-N-(2-{[4-(3-hydroxy-4-oxo-4H-chromen-2-yl)phenyl](methyl)amino}ethyl)pentanamide (HF-N-LA), is anchored onto the surface of ultrasmall CdSe quantum dots through dithiol functionality. Authentic white light with a CIE coordinate of (0.33, 0.33) could then be generated by confluence of 440 nm emission from CdSe and 570 nm proton-transfer tautomer emission from HF-N-LA. Moreover, linear color tunability could be achieved simply by altering relative amount of the two species, i.e., number of HF-N-LA onto CdSe, in one single nanocomposite, thus opening an innovative route toward applying nanocrystals in the field of WLEDs.

Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement.

Embryonic cell movement is essential for morphogenesis and the establishment of body shapes, but little is known about its mechanism. Here we report that pregnenolone, which is produced from cholesterol by the steroidogenic enzyme Cyp11a1 (cholesterol side-chain cleavage enzyme, P450scc), functions in promoting cell migration during epiboly. Epiboly is a process in which embryonic cells spread from the animal pole to cover the underlying yolk. During epiboly, cyp11a1 is expressed in an extra-embryonic yolk syncytial layer. Reducing cyp11a1 expression in zebrafish using antisense morpholino oligonucleotides did not perturb cell fates, but caused epibolic delay. This epibolic defect was partially rescued by the injection of cyp11a1 RNA or the supplementation of pregnenolone. We show that the epibolic delay is accompanied by a decrease in the level of polymerized microtubules, and that pregnenolone can rescue this microtubule defect. Our results indicate that pregnenolone preserves microtubule abundance and promotes cell movement during epiboly.