Pro-MMP-2 activation by the PPARgamma agonist, ciglitazone, induces cell invasion through the generation of ROS and the activation of ERK

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating a variety of biological functions including cancer cell proliferation and differentiation. However, the role of PPARgamma and its ligands in tumor invasion is unclear. To evaluate a possible role for PPARgamma ligands in tumor invasion, we examined whether PPARgamma agonists including pioglitazone, troglitazone, rosiglitazone, and ciglitazone could affect the activity of matrix metalloproteinases (MMPs) in the HT1080 cell line, a well-studied and well-characterized cell line for MMP research. The gelatin zymography assay showed that ciglitazone activated pro-MMP-2 significantly. In addition, ciglitazone increased the expression of MMP-2, which was accompanied by an increase of membrane type 1-MMP (MT1-MMP) expression. The PPARgamma antagonist, GW9662 attenuated the ciglitazone-induced PPARgamma activation but it did not affect the pro-MMP2 activation by ciglitazone, suggesting that the action of ciglitazone on the pro-MMP-2 activation bypassed the PPARgamma pathway. Antioxidants and various inhibitors of signal transduction were used to investigate the mechanism of ciglitazone-induced pro-MMP-2 activation. We found that the sustained production of reactive oxygen species (ROS) was required for pro-MMP-2 activation by ciglitazone. We also found that PB98059, an inhibitor of MEK-ERK, significantly blocked ciglitazone-induced pro-MMP-2 activation and that extracellular signal-regulated kinase (ERK) was hyperphosphorylated by ciglitazone. Moreover, cell invasion was significantly increased by ciglitazone in the HT1080 cell lines, whereas cell motility was not affected. This study suggests that ciglitazone-induced pro-MMP-2 activation increases PPARgamma-independent tumor cell invasion through ROS production and ERK activation in some types of cancer cells.     

Measuring triamcinolone acetonide in aqueous humor by gas chromatography-electron-capture negative-ion mass spectrometry

Intravitreal triamcinolone acetonide (IVTA) injection has been used in the treatment of various posterior segment diseases. One of the side effects of IVTA is raised intraocular pressure, which may be secondary to triamcinolone acetonide (TAA)'s effects on the trabecular meshwork that affects aqueous outflow. In order to study the biological effects of TAA on the trabecular meshwork, we firstly need to reliably and accurately detect the concentration of TAA in tissue cells or fluids. In this study we have described a technique of using gas chromatography-electron-capture-negative-ion mass spectrometry (GC-NCI-MS) to develop a simple, sensitive, selective and validated method to detect TAA in aqueous humor (AH) of rabbits following IVTA and subconjunctival TAA injections. We derivatized TAA from extracted aqueous sample by acetic anhydride and BSTFA, respectively, and analyzed by GC-NCI-MS. The detection limit was 0.3ng/ml, linearity over 0.995 from 0 to 300ng/ml. The reproducibility ranged from 10.4 to 3.9 for concentrations from 3 to 300ng/ml, and recovery was over 95% for the concentrations 10, 60, and 200ng/ml. No interference was found from 159 aqueous samples. There was no TAA residue carried to the next injection from previously high concentration injection, 10,000ng/ml. We have provided an alternative, rapid, and robust method other than LC-MS-MS for TAA detection in AH.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Active Transport of Membrane Components by Self-Organization of the Min Proteins

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane.

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1

The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified ‘β-clasp’ structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.