Faculty Bibliography

Determining arterial macrophage expression is an important goal in the molecular imaging of atherosclerosis. Here, we compare the efficacy of two synthetic, high density lipoprotein (HDL) based contrast agents for magnetic resonance imaging (MRI) of macrophage burden. Each form of HDL was labeled with gadolinium and rhodamine to allow MRI and fluorescence microscopy. Either the 37 or 18 amino acid peptide replaced the apolipoprotein A-I in these agents, which were termed 37pA-Gd or 18A-Gd. The diameters of 37pA-Gd and 18A-Gd are 7.6 and 8.0 nm, respectively, while the longitudinal relaxivities are 9.8 and 10.0 (mM s)(-1). 37pA has better lipid binding properties. In vitro tests with J774A.1 macrophages proved the particles possessed the functionality of HDL by eliciting cholesterol efflux and were taken up in a receptor-like fashion by the cells. Both agents produced enhancements in atherosclerotic plaques of apolipoprotein E knockout mice of approximately 90% (n = 7 per agent) and are macrophage specific as evidenced by confocal microscopy on aortic sections. The half-lives of 37pA-Gd and 18A-Gd are 2.6 and 2.1 h, respectively. Despite the more favorable lipid interactions of 37pA, both agents gave similar, excellent contrast for the detection of atherosclerotic macrophages using MRI.

Spherical supported bilayer membranes (SS-BLMs) are very attractive candidates in modern bioanalytics and biorecognition studies. A uniform, facile method of preparing different SS-BLMs on silica beads is reported. Confocal fluorescence microscopy and cryo-TEM imaging have been used to characterize these SS-BLMs. Thermal analysis data and FRAP experiments show that the bilayer properties of the SS-BLM are consistent with those of lipid vesicles from which they are formed. The possibility of modulating the size, lipid type and functionality, and mechanical stability makes these rigid liposomes very attractive candidates in biosensors, drug screening, and gene delivery-related applications. This is especially true in work with native vesicle membranes derived from living cells because the existing methods can only accommodate anionic membranes to a limited extent.

We have determined the structure of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in an E2.P(i)-like form stabilized as a complex with MgF(4)(2-), an ATP analog, adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5A resolution leads to a significantly revised model of CPA binding when compared with earlier reports. It shows that a divalent metal ion is required for CPA binding through coordination of the tetramic acid moiety at a characteristic kink of the M1 helix found in all P-type ATPase structures, which is expected to be part of the cytoplasmic cation access pathway. Our model is consistent with the biochemical data on CPA function and provides new measures in structure-based drug design targeting Ca(2+)-ATPases, e.g. from pathogens. We also present an extended structural basis of ATP modulation pinpointing key residues at or near the ATP binding site. A structural comparison to the Na(+),K(+)-ATPase reveals that the Phe(93) side chain occupies the equivalent binding pocket of the CPA site in SERCA, suggesting an important role of this residue in stabilization of the potassium-occluded E2 state of Na(+),K(+)-ATPase.

Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for Cul3 Rbx1-mediated degradation of Nrf2. A majority of the INrf2/Cul3 Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that Cul3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-alpha both bind to the DGR region of INrf2. However, it is prothymosin-alpha and not Nrf2 that mediates nuclear import of INrf2/Cul3 Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/Cul3 Rbx1 complex. The INrf2/Cul3 Rbx1 complex inside the nucleus exchanges prothymosin-alpha with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-alpha-mediated nuclear import of INrf2/Cul3 Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.