Novel genes and methods that modulate apoptosis

Abstract

This invention generally relates to the nucleic acid sequences of a novel gene FAIM that encodes an apoptosis inhibiting protein. Furthermore, this invention relates to methods of identifying and testing antagonists of FAIM activity and screening for inter- and intra-specific homologs and mutants of FAIM.

Description

[0002] This invention generally relates to the nucleic acid sequences of a novel gene called the Fas Apoptosis Inhibitory Molecule (faim) that encodes an apoptosis inhibiting protein. Furthermore, this invention relates to methods of identifying and testing antagonists of FAIM activity and to methods to assay for FAIM expression.[0003] Programmed cell death (PCD) is mediated by a process called apoptosis. Although the investigation of cell death is a relatively new field of study, it has become readily apparent that many disease states are manifested due to the aberrant control of programmed cell death. Recent evidence suggest that the failure of cells to undergo apoptotic cell death might be involved in the pathogenesis of a variety of human diseases including cancer, autoimmune diseases and viral infections. The understanding of survival pathways would be critical in disease states where excessive cell numbers, such as in various cancers, are the result of cell death rather than c...

AI Technical Summary

Benefits of technology

[0061] Programmed cell death, or apoptosis, is the genetically controlled, systematic dismantling of a cell. Apoptosis typically happens during embryogenesis when much tissue remodeling is taking place, but continues to happen throughout the life of an organism. For example, the elimination of senescent cells, the involution of tissues and the elimination of diseased cells happens by apoptosis. The hallmarks of the apoptotic process are morphological changes consisting of chromatin condensation, membrane blebbing, loss of membrane integrity and, ultimately, the disintegration of the cell into apoptotic bodies that are engulfed by phagocytic cells. On the molecular scale, DNA is cleaved into 180-200 kb nucleosomal fragments resulting in a laddering appearance when run on an agarose gel. Apoptosis prevents the release of cellular constituents into the extracellular space thereby preventing an inflammatory response and allows for the orderly remodeling of tissues. (In contrast, necrotic or accidental cell death is exemplified by membrane rupture and the release of cellular constituents into the extracellular space resulting in an inflammatory response by the body).

[0075] One embodiment of this invention would be to produce antibodies from peptides generated from the invention. This would allow for immunological blotting assays to test for expression of naturally occurring mutant FAIM in various cell or tissue types, the ability to isolate homologs via immunoprecipitation assays and the ability to purify large quantities of protein from expression systems.

[0077] One embodiment of this invention would be to produce from the invention faim lof and gof RNA and cDNA. This would make it possible to perform a wild range of standard molecular biological assays including, but not limited to, Northern and Southern blotting, PCR, cloning and various screening assays for the detection of intraspecific and interspecific homologs.

[0084] There are several different approaches contemplated by the present invention to confirm the ability of small molecules to specifically inhibit or enhance FAIM activation. One approach is to transfect expression constructs specific for the invention into cells and measure changes in the rate of apoptosis as compared to controls transfected with wild type FAIM after the cells have been exposed to the compound suspected of modulating FAIM activity. Cells may be transiently transfected or stably transfected with the construct under control of an inducible promoter. Furthermore, transgenic animal could be produced allowing for in vivo assays to be conducted.

[0088] In another preferred embodiment stably transfected cells lines would be developed. The use of an inducible promoter could be utilized in these systems. Screening assays for compounds suspected of modulating FAIM activity would be conducted in the same manner as with the transient transfection assays. Using stably transfected cell lines would allow for greater consistency between experiments and allow for inter-experimental comparisons.

[0101] The yeast two-hybrid system that identifies the interaction between two proteins by reconstructing active transcription factor dimers. The dimers are formed between two fusion proteins, one of which contains a DNA-binding domain (DB) fused to the first protein of interest (DB-X) and the other, an activation domain (AD) fused to the second protein of interest (AD-Y). The DB-X:AD-Y interaction reconstitutes a functional transcription factor that activates chromosomally-integrated reporter genes driven by promoters containing the relevant DB binding sites. In the present invention FAIM would be the first protein of interest (protein X) and proteins generated from the cDNA libraries would constitute the second protein of interest (protein Y). Large cDNA libraries can be easily screened with the yeast-two hybrid system. Yeast cDNA libraries are commercially available. Standard molecular biological techniques can be employed to isolate and characterize the interacting protein.

Problems solved by technology

The screening of potential therapeutics has been hindered by both a lack of understanding of the physiological basis of cell death and by a dearth of reagents specific for critical points in the cell death signaling pathways.

It may, in effect, function as a diminisher of natural gene function if the natural gene is present and functional in the host into which the lof oligonucleotide was transfected, or may negatively interfere with processes in the host if the natural gene is not present or is non-functional.

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