Mona Gupta

Receptor-mediated targeting of magnetic nanoparticles using insulin as a surface ligand to prevent endocytosis

Superparamagnetic iron oxide nanoparticles have been used for many years as magnetic resonance imaging contrast agents or in drug delivery applications. Tissue and cell-specific drug targeting by these nanoparticles can be achieved by employing nanoparticle coatings or carrier-drug conjugates that contain a ligand recognized by a receptor on the target cell. In this study, superparamagnetic iron oxide nanoparticles with specific shape and size have been prepared and coupled to insulin for their targeting to cell expressed surface receptors and thereby preventing the endocytosis. The influence of these nanoparticles on human fibroblasts is studied using various techniques to observe cell-nanoparticle interaction that includes light, scanning, and transmission electron microscopy studies. The derivatization of the nanoparticle surface with insulin-induced alterations in cell behavior that were distinct from the underivatized nanoparticles suggests that cell response can be directed via specifically engineered particle surfaces. The results from cell culture studies showed that the uncoated particles were internalized by the fibroblasts due to endocytosis, which resulted in disruption of the cell membrane. In contradiction, insulin-coated nanoparticles attached to the cell membrane, most likely to the cell-expressed surface receptors, and were not endocytosed. The presence of insulin on the surface of the nanoparticles caused an apparent increase in cell proliferation and viability. One major problem with uncoated nanoparticles has been the endocytosis of particles leading to irreversible entry. These results provide a route to prevent this problem. The derivatized nanoparticles show high affinity for cell membrane and opens up new opportunities for magnetic cell separation and recovery that may be of crucial interest for the development of cellular therapies.

Microtubule-Associated Protein 1B-Light Chain 1 Enhances Activation of Rap1 by Exchange Protein Activated by Cyclic AMP but Not Intracellular Targeting

We have previously demonstrated that EPAC1 interacts with light chain (LC) 2 of microtubule-associated protein (MAP) 1A. In the present study, we investigated whether the structurally related LC1 of MAP1B also interacts with EPAC1. We demonstrate that LC1 copurifies with EPAC1 from extracts of PC-12 cells, using cyclic AMP-agarose. Using recombinant LC1 and LC2 in pull-down and solid phase binding assays, we demonstrate direct interaction with a glutathione S-transferase-fusion of the cyclic AMP-binding (CAMP) domain of EPAC1. We also tested whether LC1 directed intracellular targeting of EPAC1 through its interaction with the CAMP domain. EPAC1 was found be in the soluble and particulate, nuclear/perinuclear fractions of cells. We found that the catalytic (CAT) domain of EPAC1, and not the CAMP domain, was responsible for recruitment to the nuclear/perinuclear fraction of cells. The targeting sequence responsible was located between amino acids 764 and 838 of EPAC1. Overexpresssion of an isolated CAT domain in COS1 cells was found to displace endogenous EPAC1 from the nuclear/perinuclear fraction, thereby inhibiting EPAC-activated Rap1 in this compartment. In contrast, LC1 was not able to compete for the binding of EPAC1 to this fraction. LC1, however, was able to enhance interaction of EPAC1 with cyclic AMP and heightened the ability of EPAC to activate Rap1. Antibody disruption of EPAC1/LC1 interaction in PC-12 cells ablated the ability of cyclic AMP to activate Rap1. LC1 is therefore not involved in intracellular targeting of EPAC1, but it is rather a molecular chaperone of EPAC activity toward Rap1.

Exchange protein directly activated by cAMP (EPAC) interacts with the light chain (LC) 2 of MAP1A

Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.