Prevette Group Research

Research Overview:

The field of biomaterials has exploded recently due to applications from drug/gene delivery to tissue engineering to medical devices.  The interaction of these materials with the cell surface is important, because it can determine cellular uptake mechanisms, cell biochemical response, cell differentiation and adherence.  There are many other associations that take place at the cell surface, which play roles in cell-cell communication and signaling processes.  Our group uses isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy to investigate the thermodynamics and structure, respectively, of a variety of cell surface interactions of both biomaterial and biochemical importance.  We are also interested in the development of model membranes to enhance the biological relevance of these studies, through incorporation of cell surface receptors and selectively isotopically labeled constituents into a vesicle with a fluid lipid bilayer.

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Interactions Between Cell-penetrating Compounds and Glycosaminoglycans:

Cellular uptake remains a significant hurdle in the development of drug and gene delivery technologies. Cell-penetrating compounds (CPCs) have been used to enhance the internalization of such systems, but their mechanism at the cell surface is poorly understood. We hypothesize that cell membrane glycosaminoglycans (GAGs) play an integral role in the recognition and subsequent internalization of CPCs and their attached cargo. Therefore, our research group is studying the CPC-GAG interaction through binding thermodynamics, identification of chemical functional groups involved in binding and the conformational changes that occur. This information will lead to structure-function relationships that facilitate future design of drug and gene delivery agents.

2

 

Isothermal Titration Calorimetry (ITC) binding curves
Tat peptide injected into heparin at 37 °C, 3 trials shown. Thermodynamic parameters of the interaction obtained by fitting this data: K = 2.24 x 106 M-1,
ΔH = -3.49 kJ/mol, stoichiometry = 10.4 (Tat + charges/heparin – charges)

Understanding Self-assembly at the Cell Surface:

There is a critical lack of knowledge in the mechanisms of association driving self-assembly at the cell surface, which govern the cellular uptake of materials through endocytosis, cell signaling processes and cell-cell communication.  Our group studies these interactions, beginning with the example of carbohydrate ligands, called galectins, which have been hypothesized to cause clustering of glycoprotein receptors on the cell surface.  We want to know:

•The NMR structure of the complete series of galectins
•Which of the galectins bind glycoprotein receptor components lactose and N-acetyllactosamine
•The comparison between binding simple and complex glycans
•The thermodynamics of the interactions

  With these results, we can understand the ability of these ligands to recognize, cross-link and cluster glycoprotein receptors on cell membranes, as well as the detailed mechanism of their interaction.  This information will aid in the understanding of why galectins appear to enhance many cell signaling events and may lead to development of biomaterials that can capitalize on these processes.

5

Developing Model Cell Membranes:

Biological membranes are complex.  They contain approximately 20% cholesterol, many proteins and a large variety of lipid types, with different head groups, chain lengths and degree of unsaturation, and overall charge.  However, many of the functions of molecules at the cell surface depend upon this complexity. Model membranes help us to understand biomaterial and biomolecule interactions at the  cell surface by simplifying certain aspects of the structure.  Model membranes have been used with atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP) and sum frequency generation (SFG) vibrational spectroscopy to study everything from protein targeting to polymer-induced cytotoxicity to antimicrobial peptide orientation in the membrane.  However, popular versions such as supported lipid bilayers and lipid vesicles are lacking key components which have significant effects on the behavior of the membrane and hence, the usefulness of the model.

  The model membrane must not only retain the constituents of a cell membrane but also retain its fluidity. Supported bilayers, which are convenient for AFM, SFG and surface plasmon resonance spectroscopy, maintain lipid fluidity on glass or quartz due to a 1-2 nm water-filled gap between the solid support and the lower leaflet.  However, integral membrane proteins incorporated into these supported bilayers are usually immobilized as a result of noncovalent interactions with the solid support. Our group uses a bottom-up approach to the development of model membranes for the study of biomaterial and biomolecule interactions with the cell surface.  To avoid interactions between the solid support and the bilayer, cholesterol or integral proteins, a vesicle structure is preferred.  This approach allows for building in complexity to the system as needed, such as comparing the binding of free receptors in solution to receptors buried in a homogenous bilayer, or to those buried in a diverse mixture of lipids, or to those buried in a vesicle containing cholesterol.  These models will be useful for ITC, NMR and fluorescent microscopy experiments, through the selective incorporation of fluorescent- or isotopically-labeled constituents.

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Giant Unilamellar Vesicles (GUVs)
Fluorescence microscopy images of 9:1 dipalmitoyl phosphatidylcholine (DPPC): dipalmitoyl phosphatidylglycerol (DPPG) membranes formed by electroformation and stained with lipophilic dye nile red.

Prevette Group Research Students

Education:

•Postdoctoral Fellowship 2008-2010 – University of Michigan (Ann Arbor, MI)

  Advisor: Mark Banaszak Holl

  NMR Collaborators: A. Ramamoorthy, Hashim Al-Hashimi

 Michigan Nanotechnology Institute for Medicine and Biological Sciences (MNIMBS)

•Ph.D in Physical Chemistry 2008 – University of Cincinnati (Cincinnati, OH)

  Advisor: Theresa Reineke (now at Virginia Tech)

  Co-Advisor: Matthew Lynch (Principle Scientist at Procter & Gamble Co.)

 

•B.A. in Chemistry and Mathematics 2001 – Transylvania University (Lexington, KY)

 

Publications: (undergraduate students underlined)

•Lisa E. Prevette, Evgenia N. Nikolova, Hashim M. Al-Hashimi, Mark M. Banaszak Holl. “Intrinsic Dynamics of DNA-Polymer Complexes:  a Mechanism for DNA Release.” 2012 (in prep)

•Lisa E. Prevette, Mrinal Shah, Mary Jane Cunningham, Mark M. Banaszak Holl. “Cellular Stress Response Induced by Poly(amidoamine) Dendrimers.” 2012 (in prep)

•Sascha N. Goonewardena, Lisa E. Prevette, Ankur A. Desai. “Metabolomics and Atherosclerosis.” Curr. Atheroscler. Rep. 2010, 12, 267-272.

•Lisa E. Prevette, Douglas G. Mullen, Mark M. Banaszak Holl. “Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA.” Mol. Pharmaceutics 2010, 7, 870-883.

•Lisa E. Prevette, Matthew L. Lynch, Theresa M. Reineke. “Amide Spacing Influences pDNA Binding of Poly(amidoamine)s.” Biomacromolecules, 2010, 11, 326-332.

•Lisa E. Prevette, Karina Kizjakina, Matthew L. Lynch, Theresa M. Reineke. “Correlation of Amine Number and pDNA Binding Mechanism for Trehalose-based Polycations.” Langmuir 2008, 24, 8090-8101.

•Sathya Srinivasachari, Yemin Liu, Lisa E. Prevette, Theresa M. Reineke. “Effects of Trehalose Click Polymer Length on pDNA Complex Stability and Delivery Efficacy.” Biomaterials 2007, 28, 2885-2898.

•Lisa E. Prevette, Tom E. Kodger, Theresa M. Reineke, Matthew L. Lynch. “Deciphering the Role of Hydrogen Bonding in Enhancing pDNA-Polycation Interactions.” Langmuir 2007, 23, 9773-9784.

•Sathya Srinivasachari, Yemin Liu, Guo-dong Zhang, Lisa E. Prevette, Theresa M. Reineke. “Trehalose Click Polymers Inhibit Nanoparticle Aggregation and Promote pDNA Delivery in Serum.” J. Am. Chem. Soc. 2006, 128, 8176-8184.