Session VIIB

Wednesday, May 18th

Session VIIB


Investigation of Changes in Lipid Composition During Regulated Necrosis

Laura Parisi and G. Ekin Atilla-Gokcumen

University at Buffalo, Department of Chemistry

Necroptosis is a type of highly regulated necrotic cell death.  As a type of necrotic cell death, necroptosis is characterized by plasma membrane rupture, release of intracellular contents, and an associated inflammatory response.  Accumulating evidence has linked necroptosis to the pathogenesis of many disease states including ischaemia-reperfusion injury, inflammatory bowel disease, neurodegeneration, and pathogen infection.  Although much research has been devoted to understanding the protein signaling pathways involved in necroptosis, surprisingly little research has studied the role of lipids in necroptosis until very recently.  Given the changes in plasma membrane permeability and other drastic morphological changes that occur during necroptosis, we suspect that lipids may play crucial roles in this process.  In order to study the role of lipids in necroptosis, an untargeted LC-MS-based approach was employed to compare the global lipid composition of control cells with that of cells undergoing necroptosis.  This comparison provides an unbiased, comprehensive analysis of the lipid species which change in abundance during necroptosis and thus may be involved in the execution of cell death.  Our analysis has revealed significant changes in lipid composition during necroptosis in a colorectal adenocarcinoma cell line.   Among our findings, we observe that ceramides accumulate during necroptosis.  Quantitative PCR reveals that this accumulation of ceramides is due, at least in part, to increased expression of ceramide synthase genes.  Further studies to determine the mechanisms by which these and other lipids change during necroptosis and to understand their functional role are currently underway.


Stereoselective Synthesis of Isoxazolidines Via Copper (II)-catalyzed Alkene Diamination

Zainab Khoder and Sherry R. Chemler

University at Buffalo, Department of Chemistry

Isoxazolidine motifs are a basic scaffold in many molecules with biological activities such as antibacterial, antiviral, anti-inflammatory, central nervous system (CNS), antidiabetics, blood pressure regulation and anticancer. Because of the significant value of isoxazolidine scaffold, many scientists have shown wide efforts in the field of isoxazolidine synthesis. Alkene difunctionalization is the addition of two functional groups across a double bond. It is a class of reaction that shows great potential for the synthesis of a large number of useful organic molecules. This strategy has been extensively used in the synthesis of the isoxazolidine moiety.The first intra/intermolecular diamination reaction of the unactivated alkenes for the stereoselective synthesis of isoxazolidine rings was achieved using catalytic copper(II) ethylhexanoate in the presence of MnO2. This diamination reaction tolerated different classes of substrates (aliphatic and aromatic) as well as different external amines (aliphatic amines, sulfonamides, benzamide, secondary amines and anilines). The yields varied from good to excellent.


Towards Zn (II) Complexes for Selective Binding of i-motif and G-quadruplex Structures in the Human c-MYC Promoter

Naomi Bryner and Janet R. Morrow

University at Buffalo, Department of Chemistry

The nuclear hypersensitivity element III (NHEIII) is a GC-rich sequence of DNA located in the c-MYC proto-oncogene. This sequence has been reported to form i-motif and G-quadruplex structures from complementary stretches of cytosines and guanines, respectively. Both structures have thymine-containing loops connecting the stretches of consecutive nucleotides. The presence of thymine provides a viable target for the macrocyclic complex, Zn (cyclen), which is known to coordinate the deprotonated N3 site of thymine (cyclen = 1, 4, 7, 10-tetraazacyclododecane). Functionalization of Zn (cyclen) with an aromatic pendant can enhance selectivity and binding strength by providing another point of interaction with the other structural features of the DNA. Interactions of the DNA with Zn (II) complexes can be characterized via circular dichroism, optical thermal melting, isothermal titration calorimetry, and, when applicable, direct fluorescence studies. Preliminary results are promising for the binding of Zn (cyclen) derivatives to the targeted c-MYC structures.


Searching for Metallothionein 2a

Devika Jayawardena1 and Martin Stillman1,2

(1)   University of Western Ontario, Department of Biology (2)   University of Western Ontario, Department of Chemistry

Acute metal toxicity is easily diagnosed by a patient’s symptoms. However, chronic low-level exposure to toxic metals cannot be easily identified until later in life, when the damage has already been done. Most toxic metals such as mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As) are sulfhydryl-reactive metals, which bind to the sulfhydryl groups of the proteins and disrupt their function. One such metal binding protein that has been shown to bind to metals is human liver metallothionein (MT), which forms metal-thiolate complexes. MT protects other proteins from dysfunction as well as bind to essential metals like zinc (Zn) and copper (Cu), and in acts as a metal reservoir for other metalloproteins. Human MT2a (the predominant isoform in livers) is a very difficult protein to express in E. coli. My research is being carried out to determine the optimal growth conditions to make E. coli express the maximum yield of MT2a and to investigate how MT2a is involved in transfer of Zn to metal free carbonic anhydrase, which has a zinc atom in its active state, in vitro.

Acknowledgements: Professor Ilka Heinemann, department of Biochemistry, University of Western Ontario for technical support. NSERC for funding.