Spring 2012 Biomedical Seminar Series
Friday, April 6, 2012
Abstract: The ability of a cell to appropriately respond to stresses and maintain its redox potential is essential for life. Stressors that can alter the redox potential in the cell include UV light, reactive oxygen intermediates (ROI), heat shock, ionizing radiation, and DNA damage. The transcription factor p53 is a tetramer protein, containing two identical dimer subunits that together form a fully functional protein that can bind to DNA. Adverse changes in redox potential can alter the tertiary structure of p53, and affect its DNA binding capabilities. It has been shown that the tumor suppressor protein p53 is subjected to a reversible modification called S-glutathionylation within the DNA binding domain. It has been shown that Cysteines 124, 141, and 182 are sites for S-glutathionylation, with Cysteine 141 being the most reactive. Recent studies have revealed that major interface contacts between dimers occur between L2 loop of a single subunit and the S2Â/S3 loop from another subunit. The S2Â/S3 loop is immediately adjacent to Cys141. It is possible that Cys141 S-glutathionylation prevents dimerization of subunits and formation into a functional tetramer. To test this hypothesis, p53 DNA binding domain will be purified and glutathionylated at Cys141. The changes in protein structure will be analyzed by NMR and compared to the unmodified p53 DNA binding domain.
Abstract: The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers, p53 is inactivated by abnormally high levels of MDM2Âan E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3,244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present a subset of FDA-approved drugs predicted to inhibit p53/MDM2 interaction.
Abstract: Our defense mechanisms against infectious agents include epithelial cells lining mucosal surfaces and their secretions that are rich in antimicrobial proteins. Lipids are also present in mucosal secretions and we have discovered that host derived-lipids are essential antimicrobial effector molecules in the airways. However, their native carrier molecule is unknown. Palate Lung Nasal epithelium Clone (PLUNC) is a highly hydrophobic protein abundantly expressed in the airways with putative lipid binding pockets. Hypothesizing that PLUNC is a carrier for antimicrobial lipids, we assessed whether PLUNC is associated with lipids in airway secretions we utilized two-systems: (1) in vitro cultures of Calu3 cells transfected to secrete PLUNC; and (2) bronchial alveolar lavages (BAL) collected from pigs which express PLUNC naturally and who have lipid profiles similar to that of humans. We quantified PLUNC in apical secretions of Calu3 cells by Western immunoblotting and assessed the corresponding lipid profiles by thin layer chromatography. Pig BAL was subjected to P60 size exclusion gel column chromatography and PLUNC containing fractions identified by western immunoblotting were analyzed for their lipid content. Preliminary data indicate that PLUNC-expressing Calu3 cell secretions contain more polar lipids compared to secretions from vector transfected control cells correlating with the concentration of PLUNC and that in pig BAL cholesteryl esters co-elute with PLUNC. Infectious diseases continue to be among the top ten causes of death in the U.S. Identification of carrier proteins of antimicrobial lipids may lead to the development of suitable lipid carriers that will facilitate the delivery of novel lipid antimicrobials. Acknowledgements: We thank Dr. Paul McCray, Jr. and Dr. Jennifer Bartlett at the University of Iowa for providing us with cell culture supernatants and pig bronchial alveolar lavages. NSF-HRD-1026102-518741; NIH 1SC1GM096916-01; NIH PO1 HL091842, and Roy J. Carver Trust.