Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Reexamination Certificate
1998-10-05
2001-06-12
Bansal, Geetha P. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
C530S300000, C530S324000
Reexamination Certificate
active
06245885
ABSTRACT:
BACKGROUND OF THE INVENTION
The response of metazoan cells to apoptotic death signals depends on the status of various regulatory checkpoints in the cell. Prominent among these is the BCL-2 family of proteins whose members include dominant suppressors of cell death (Ced-9, BCL-2, BCL-XL, BCL-w, A1, and MCL-1) and proapoptotic inducers of cell death (BAX, BAK, and BCL-Xs), as well as proapoptotic inhibitors of BCL-2/BCL-X
L
function (BAD, BID) (Yang and Korsmeyer, Blood 88:386-401, 1996). The relationship between these family members is complex and, in the case of the BCL-2 suppressor and BAX inducer, is further complicated by their apparent ability to function autonomously in regulating cell death, while at the same time influencing one another's activities via heterodimeric interactions.
BCL-2 suppressors function upstream of caspase death effectors, such as caspase-3, to inhibit cell death. This inhibition of cell death may be accomplished in several ways, including recruitment and regulation of Ced-4-like molecules and Ced-4-like adaptors that are required for activation of initiator caspases, and recruitment of kinases and phosphatases that may regulate the activity of BCL-2-associated complexes. Moreover, regulation of BCL-2 complexes may influence the formation of ion conducting pores or the channel activities of membranes in which BCL-2 resides.
While BAX may affect all of these BCL-2 mediated events via heterodimeric modulation, BAX is also capable of autonomous pore formation in lipid bilayers. The ability of elevated levels of BAX or BAK to initiate cell death in the absence of any additional signal in vivo correlates with severe intracellular membrane dysfunction that includes redistribution of mitochondrial cytochrome c to the cytosol and induced mitochondria permeability transition.
Most BCL-2 and BAX family proteins contain at their extreme COOH-terminus a single predicted transmembrane segment (TM). In the case of BCL-2, the TM functions as a signal-anchor that targets and inserts the protein in a N
cyto
-C
in
orientation into the two main membrane locations for this protein, the mitochondrial outer membrane and the endoplasmic reticulum
uclear envelope. Strikingly, however, the ability of BAX to translocate to membrane sites, including mitochondria, is regulated in certain contexts and depends upon the cell receiving a death signal.
SUMMARY OF THE INVENTION
We have discovered that BAX targeting to mitochondria can be regulated by zVAD-sensitive caspase(s), and we have mapped two regions of the BAX protein, the NH
2
-terminal ART domain and the COOH-terminal TM, as the regions controlling the prevention of BAX targeting to the mitochondria in the absence of a death signal. Our results demonstrate a regulated mechanism by which a death signal can cause translocation of BAX to mitochondria. Based on our results, in general, the invention features methods and reagents for modulating BAX-mediated apoptosis.
Accordingly, in a first aspect, the invention features a substantially pure polypeptide fragment that includes a BAX protein lacking an ART domain. In one embodiment, the polypeptide fragment increases apoptosis of a cell when administered to the cell (e.g., a cancer cell).
In a second aspect, the invention features a substantially pure polypeptide fragment that includes a BAX ART domain. In one embodiment, the polypeptide fragment decreases apoptosis of a cell when administered to the cell (e.g., a degenerative cell).
In preferred embodiments of the first and second aspects of the invention, polypeptide fragment is administered with a pharmaceutically acceptable carrier, and the cell is in a mammal that has or is likely to develop a disease involving altered apoptosis.
In a third aspect and a fourth aspect, the invention features two methods for diagnosing a mammal for the presence of a disease involving altered apoptosis or for an increased likelihood of developing the disease. The method of the third aspect includes the steps of: (a) isolating a sample of nucleic acid from the mammal; and (b) determining whether the nucleic acid encodes a mutated BAX protein that has an alteration in the amino acid sequence of the ART domain or the amino acid sequence of the transmembrane domain, where the presence of the alteration indicates that the mammal has a disease involving altered apoptosis or that the mammal has an increased likelihood of developing the disease. The method of the fourth aspect includes the steps of: (a) isolating a cell sample from the mammal; and (b) determining whether the sample includes a mutated BAX protein that has an alteration in the amino acid sequence of the ART domain or the amino acid sequence of the transmembrane domain, where the presence of the alteration indicates that the mammal has a disease involving altered apoptosis or that the mammal has an increased likelihood of developing the disease.
In one embodiment of the third and fourth aspects of the invention, the alteration is in the amino acid sequence of the ART domain and the alteration inhibits the interaction of the ART domain with the transmembrane domain. In another embodiment, the alteration is a deletion of the ART domain. Preferably, the mammal has a disease involving increased apoptosis or the mammal has an increased likelihood of developing a disease involving increased apoptosis.
In another embodiment of the third and fourth aspects of the invention, the alteration is in the transmembrane domain and the alteration inhibits the ability of the transmembrane domain to insert into a mitochondrial membrane. In another embodiment, the mutation strengthens the interaction of the ART domain with the transmembrane domain. Preferably, the mammal has a disease involving decreased apoptosis or the mammal has an increased likelihood of developing a disease involving decreased apoptosis.
In a fifth aspect, the invention features a method for identifying a compound that is likely to increase apoptosis of a cell that includes the steps of: (a) providing a substantially pure polypeptide fragment that includes a BAX ART domain; (b) contacting the fragment with a candidate compound; and (c) determining binding of the candidate compound to the fragment, where binding of the candidate compound to the fragment indicates that the candidate compound is a compound likely to increase apoptosis of a cell (e.g., a cancer cell).
In a sixth aspect the invention features a method for identifying a compound that is likely to decrease apoptosis of a cell that includes the steps of: (a) providing a substantially pure polypeptide fragment that includes a BAX transmembrane domain; (b) contacting the fragment with a candidate compound; and (c) determining binding of the candidate compound to the fragment, where binding of the candidate compound to the fragment indicates that the candidate compound is a compound likely to decrease apoptosis of a cell (e.g., a degenerative cell).
In various preferred aspect of the fifth and sixth aspects of the invention, the polypeptide fragment is immobilized or is part of a fusion protein. In other embodiments, the candidate compound is a protein or is a chemical (e.g., an inorganic chemical).
In a seventh aspect, the invention features a method for increasing apoptosis in a cell (e.g., a cancer cell) that includes administering to the cell an apoptosis-increasing amount of a BAX polypeptide that lacks a functional ART domain. Preferably, the ART domain is deleted.
In an eighth aspect, the invention features a method for increasing apoptosis in a cell (e.g., a cancer cell) that includes administering to the cell an apoptosis-increasing amount of a compound that inhibits the interaction of the ART domain of a BAX polypeptide with the transmembrane domain of the BAX polypeptide. In one embodiment, the compound is an antibody that specifically binds to a BAX polypeptide.
In a ninth aspect, the invention features a method for increasing apoptosis in a cell (e.g., a cancer cell) that includes administering to the cell an apoptosis-increasing amount of a compound that s
Goping Ing Swie
Shore Gordon C.
Bansal Geetha P.
Bieker-Brady Kristina
Clark & Elbing LLP
McGill University
LandOfFree
Bax-mediated apoptosis modulating reagents and methods does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Bax-mediated apoptosis modulating reagents and methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Bax-mediated apoptosis modulating reagents and methods will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2511558