Structure and Function of the Bax Apoptosis Regulator

Institution: The Burnham Institute for Medical Research
Investigator(s): Francesca Marassi, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8WB-0110 Award: $285,141
Award Type: STEP Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2002)
The molecular structures of cancer-related molecules bring insight to their functions, and provide targets for the design of drugs that offer potential approaches to treating tumors. The Bcl2 genes encode a family of proteins that regulate cell survival and cell death in many tissues, including mammary tissue and breast cancer. They are believed to exert their pro- or anti- cell death activities through the transport of salts and biological materials across the membranes of mitochondria. These transport processes are essential for maintaining cell viability, while their de-regulation can lead to cancer.

Our objective is to understand the relationship between the molecular structures of the membrane-associated Bcl2 proteins, and their activities as transporters in artificial membranes and in cells. The research is designed to test the hypothesis that the transport activity of these proteins is important in regulating cell death in breast cancer. The structures of Bcl2 proteins are essential for understanding their modes of action, and they are the first step towards in the development of structure-based therapeutic drugs to breast cancer. The two principal specific aims are: (1) to determine the structure of the cell death-inducing membrane-associated Bcl2 protein Bax; and (2) to understand its role as a membrane transporter in breast cancer cells.

The molecular structure of Bax will be determined using magnetic resonance spectroscopy of the protein in artificial membranes. We have already obtained preliminary data that demonstrate experimental feasibility and that give an initial view of the protein architecture. The transport activity of Bax will be characterized in artificial membranes - transport across membranes leads to electrical currents that can be measured and characterized in the laboratory - and in cells - using assays developed by in our laboratory.

One of the most important research goals for the treatment of cancers is the identification and characterization of new therapeutic targets, and proteins that regulate cell death and membrane transport, like those of the Bcl2 family, represent a very attractive approach. The structure-activity study that we describe is an important step for understanding the biology of both normal breast and breast cancer cells, and the structures of Bcl2 proteins in membranes may provide specific targets for breast cancer drugs: the structures guide the design and screening of drug molecules.

Final Report (2004)
The proteins of the Bcl-2 family play a major regulatory role in programmed cell death. Imbalances in their relative amounts and activities are associated with major human diseases, characterized by either insufficient (cancer, autoimmunity) or excessive (AIDS, Alzheimer's disease) cell death. Many Bcl-2 proteins are expressed in normal breast and in breast cancer, where they are involved in tissue development and maintenance. The structures of Bcl-2 proteins in solution provided the first clues to their activity, however these studies focused on truncated, soluble forms of the proteins because of the difficulties associated with recombinant membrane protein expression and structure determination. Nevertheless, the Bcl-2 family proteins exercise some of their most important functions at mitochondrial membranes suggesting that their membrane-associated structures are key to understanding their functions.

The goal of this research is to determine the structures of membrane-associated Bcl-2 family proteins, and to correlate the structures with their biological activities assayed in lipid bilayers and in cells. We focus first on BclxL and Bax, because they represent two distinct protective and killer subsets of the Bcl-2 family, and because their solution structures have been determined. The information derived from these studies will bring important insights to the physiology of both normal breast and breast cancer, and will be useful for developing structure-based approaches to breast cancer therapy.

In the past year we continued our efforts to express milligram quantities of Bax and of other Bcl-2 family proteins, through the use of different expression vectors, including pET with Histidine and various other fusion protein tags. Recently, we described the conformations of the cell-death inducing protein Bid (Gong et al., 2004) and of the cell-death protective protein in membranes, and have published these results in the Journal of Biological Chemistry and in a Special Issue of Magnetic Resonance in Chemistry (Franzin et al., 2004), respectively. These results give the first insights to the mechanisms of action of Bcl-2 family proteins at membranes, and we are focusing our efforts on obtaining high-resolution structural data that will pave the way for the design of drug molecules.

Symposium Abstract (2003)
The Bcl-2 family includes proteins that cause programmed cell death (apoptosis) and those that inhibit it. Their structures in solution have been determined by NMR spectroscopy, and are similar to those of bacterial channel-forming toxins. Indeed, just like these toxins, the Bcl-2 proteins cycle between soluble and membrane-associated forms and form channels in mitochondrial membranes. We are determining the structures of Bcl-2 family proteins in membrane environments using NMR spectroscopy. They appear to associate with membranes through two central hydrophobic helices that are also required for both channel formation and cytoprotective activity. The solution and solid-state NMR spectra of these proteins, in lipid micelles and lipid bilayers, provide the first view of their membrane-associated architectures.

Using Pisa pies to resolve ambiguities in angular constraints from PISEMA spectra of aligned proteins.
Periodical:Journal of Biomolecular NMR
Index Medicus: J. Biomol. NMR
Authors: Marassi FM, Opella SJ
Yr: 2002 Vol: 23 Nbr: Abs: Pg:239-242

Dipolar waves as NMR maps of protein structure
Periodical:Journal of the American Chemistry Society
Index Medicus: J Am Chem Soc
Authors: Mesleh MF, Veglia G, DeSilva TM, Marassi FM and Opella SJ
Yr: 2002 Vol: 124 Nbr: Abs: Pg:4206-4207

Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints
Periodical:Protein Science
Index Medicus: Protein Sci
Authors: Marassi FM, Opella SJ
Yr: 2003 Vol: 12 Nbr: Abs: Pg:403-411

Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers.
Periodical:Biochemica et Biophysica Acta
Index Medicus: Biochem Biophys Acta
Authors: Crowell KC, Franzin CM, Lee S, Lucchese AM, Snyder BS, Marassi FM
Yr: 2003 Vol: 1645 Nbr: Abs: Pg:15-21

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins
Periodical:Journal of Magnetic Resonance Imaging
Index Medicus: J Magn Reson Imaging
Authors: Marassi FM, Crowell KC
Yr: 2003 Vol: 161 Nbr: Abs: Pg:64-69

Structure determination of membrane proteins by NMR spectroscopy: Using pisa pies to resolve ambiguities in angular constraints from PISEMA spectra of aligned proteins.
Periodical:Biochem Cell Biol
Index Medicus: Biochem Cell Biol
Authors: Opella SJ, Nevzorov A, Mesleh MF, Marassi FM
Yr: 2002 Vol: 80 Nbr: Abs: Pg:597-604