Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
1999-09-10
2003-05-06
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S007100, C435S455000, C435S320100, C435S325000, C530S350000, C536S023500, C536S023400
Reexamination Certificate
active
06558950
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Phase application of International Application No. PCT/IB98/00706, filed Mar. 2, 1998.
BACKGROUND OF THE INVENTION
This invention relates to apoptosis.
Apoptosis, which is also referred to as programmed cell death, is a form of cell death characterized by membrane blebbing and nuclear DNA fragmentation. Apoptotic cell death is morphologically distinct from necrotic cell death and is important in the normal development and maintenance of multicellular organisms. Dysregulation of apoptosis has been implicated in a number of human diseases. An inappropriate suppression of apoptosis in a cell may lead to the uncontrolled propagation of that cell. Such an event would favor, for example, the development of cancer. In contrast, a failure to control the extent of apoptotic cell death may lead to degeneration of specific tissues and cell-types. For example, an inappropriately high level of apoptosis in leukocytes may result in acquired immunodeficiency. Likewise, certain neurodegenerative disorders may result from an inappropriately high level of apoptosis in neuronal cells and tissues.
Although apoptotic cell death is initially triggered by a specific death signal received, for example, by ligation of the Fas cell surface molecule, execution of the apoptotic pathway occurs only upon the activation of members of the Ced-3/ICE (caspase) family of cysteine proteases. There are at least 10 known members of the caspase family whose activities lead to site-specific cleavage and consequent activation/inactivation of various target molecules. FLICE and related caspases may initiate apoptosis by activating a downstream caspase cascade, including CPP32 (caspase-3).
The decision to engage the apoptotic execution pathway in response to specific death signals depends on the status of various cellular regulators of apoptosis, including p53 and the Bcl-2/Bax set point. The latter set point arises through heterodimerization between the Bcl-2/Bcl-X
L
family of suppressors and promoters, respectively, in which the ratio of the heterodimerizing partners determines the outcome—cell death or cell survival—in response to various death signals. Bad, a more distantly related family member, is a direct regulator of the set point, by a mechanism that is governed by phosphorylation. The phosphorylation may, in turn, be affected by Bcl-2-dependent recruitment of Raf-1 kinase.
Although it is now known that Bcl-2/Bcl-X
L
controls the apoptotic execution pathway at a point that is either at or upstream of pro-enzyme activation of the caspases, how this is achieved remains to be elucidated. There thus remains a need to identify Bcl-2 binding proteins that are functionally linked to apoptosis. There also remains a need to identify factors that interact with Bcl-2 and modulate the apoptotic signalling pathway. Further, it would be useful to identify factors which enable a signalling of the apoptotic pathway from Bcl-2 and interacting factors to the caspases involved therein.
SUMMARY OF THE INVENTION
We have discovered that p28 Bap31 polypeptides, nucleic acids, and antibodies may be used for the detection and treatment of conditions involving apoptosis and for the identification of therapeutic molecules.
In a first aspect, the invention features a substantially pure p28 Bap31 polypeptide fragment that modulates apoptosis.
In a second aspect, the invention features a substantially purified nucleic acid molecule encoding a substantially pure p28 Bap31 polypeptide fragment that modulates apoptosis.
In various embodiments of the first two aspects of the invention, the fragment includes a domain that is required for an association of p28 Bap31 with pro-FLICE, or a domain that is required for an association of p28 Bap31 with a Bcl-2 protein (e.g., Bcl-2 or Bcl-X
L
). In other preferred embodiments, the fragment either increases apoptosis or inhibits apoptosis. In yet another embodiment of the first and second aspects of the invention, the fragment is from a mammal (e.g., a human or a mouse).
In an third aspect, the invention features a substantially pure polypeptide that modulates apoptosis, the polypeptide having 50% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In preferred embodiments, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or has 80% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.
In a fourth aspect, the invention features a method for modulating apoptosis in a cell that includes administering to the cell a compound that alters p28 Bap31 biological activity, the compound being administered at a dosage which is sufficient to modulate the p28 Bap31 biological activity. In various preferred embodiments of this aspect of the invention, the p28 Bap31 biological activity may be cleavage of the p28 Bap31 polypeptide to produce a p20 product, formation of a complex of the p28 Bap31 polypeptide with pro-FLICE, formation of a complex of the p28 Bap31 polypeptide with a Bcl-2 protein (e.g., Bcl-2 or Bcl-X
L
), binding of the p28 Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or expression of the p28 Bap31 polypeptide in the cell. In various other embodiments, the cell is from a mammal (e.g., a human or a rodent).
In another embodiment of the fourth aspect of the invention, the modulating is inhibiting. In another embodiment, where the modulating is inhibiting, the cell is in a mammal with a degenerative disease (e.g., a neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome, an ischemic injury, an infection with HIV, or a bone degenerative disease). In yet another embodiment, where the-modulating is inhibiting, the compound may be a p28 Bap31 antisense nucleic acid molecule, an antibody that specifically binds to p28 Bap31 (e.g., a p28 Bap31 neutralizing antibody), a p20 antisense nucleic acid molecule, an antibody that specifically binds to p20 (e.g., a p20 neutralizing antibody), a p20-inhibiting amount of a Bcl-2 protein, or a p20-inhibiting amount of a nucleic acid molecule encoding a Bcl-2 polypeptide, where the nucleic acid molecules are positioned for expression in said cell.
In another embodiment of the fourth aspect of the invention, the modulating is increasing. In another embodiment, where the modulating is increasing, the cell is in a mammal with a neoplasia. In another embodiment, where the modulating is increasing, the compound may be a p28 Bap31 polypeptide, a p20 product that is a cleavage product of p28 Bap31, or a nucleic acid molecule encoding a p28 Bap31 polypeptide, where the nucleic acid molecule is positioned for expression in the cell.
In a fifth aspect, the invention features a method for detecting a compound that modulates apoptosis that includes the steps of: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, the first fusion protein including a polypeptide fragment of p28 Bap31 covalently bonded to a binding moiety, the binding moiety capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein, the second fusion protein including a polypeptide fragment of a second protein covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, a change in the reporter gene expression identifying a compound that modulates apoptosis. In one embodiment of this aspect, the cell is a yeast cell.
In a sixth aspect, the invention features a method for detecting a compound that modulates apoptosis that includes the steps of: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, the first fusion protein including a polypeptide fragment of a se
Branton Philip E.
Ng Florence W. H.
Nguyen Mai
Shore Gordon C.
Bieker-Brady Kristina
Clark & Elbing LLP
McGill University
Nguyen Dave T.
Shukla Ram R.
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