Identification and comparison of protein-protein...

Details Identification and comparison of protein-protein... Identification and comparison of protein-protein...

1999-01-12

2002-05-28

Zitomer, Stephanie W. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S069700, C435S070100, C435S071100, C435S254110, C436S501000

Reexamination Certificate

active

06395478

ABSTRACT:

1. INTRODUCTION
The present method relates to the identification of protein-protein interactions and inhibitors of these interactions that, preferably, are specific to a cell type, tissue type, stage of development, or disease state or stage.
2. BACKGROUND OF THE INVENTION
Proteins and protein-protein interactions play a central role in the various essential biochemical processes. For example, these interactions are evident in the interaction of hormones with their respective receptors, in the intracellular and extracellular signalling events mediated by proteins, in enzyme substrate interactions, in intracellular protein trafficking, in the formation of complex structures like ribosomes, viral coat proteins, and filaments, and in antigen-antibody interactions. These interactions are usually facilitated by the interaction of small regions within the proteins that can fold independently of the rest of the protein. These independent units are called protein domains. Abnormal or disease states can be the direct result of aberrant protein-protein interactions. For example, oncoproteins can cause cancer by interacting with and activating proteins responsible for cell division. Protein-protein interactions are also central to the mechanism of a virus recognizing its receptor on the cell surface as a prelude to infection. Identification of domains that interact with each other not only leads to a broader understanding of protein-protein interactions, but also aids in the design of inhibitors of these interactions.
Protein-protein interactions have been studied by both biochemical and genetic methods. The biochemical methods are laborious and slow, often involving painstaking isolation, purification, sequencing and further biochemical characterization of the proteins being tested for interaction. As an alternative to the biochemical approaches, genetic approaches to detect protein-protein interactions have gained in popularity as these methods allow the rapid detection of the domains involved in protein-protein interactions.
An example of a genetic system to detect protein-protein interactions is the “Two-Hybrid” system to detect protein-protein interactions in the yeast
Saccharomyces cerevisiae
(Fields and Song, 1989, Nature 340:245-246; U.S. Pat. No. 5,283,173 by Fields and Song). This assay utilizes the reconstitution of a transcriptional activator like GAL4 (Johnston, 1987, Microbiol. Rev. 51:458-476) through the interaction of two protein domains that have been fused to the two functional units of the transcriptional activator: the DNA-binding domain and the activation domain. This is possible due to the bipartite nature of certain transcription factors like GAL4. Being characterized as bipartite signifies that the DNA-binding and activation functions reside in separate domains and can function in trans (Keegan et al., 1986, Science 231:699-704). The reconstitution of the transcriptional activator is monitored by the activation of a reporter gene like the lacZ gene that is under the influence of a promoter that contains a binding site (Upstream Activating Sequence or UAS) for the DNA-binding domain of the transcriptional activator. This method is most commonly used either to detect an interaction between two known proteins (Fields and Song, 1989, Nature 340:245-246) or to identify interacting proteins from a population that would bind to a known protein (Durfee et al., 1993, Genes Dev. 7:555-569; Gyuris et al., 1993, Cell 75:791-803; Harper et al., 1993, Cell 75:805-816; Vojtek et al., 1993, Cell 74:205-214).
Another system that is similar to the Two-Hybrid system is the “Interaction-Trap system” devised by Brent and colleagues (Gyuris et al., 1993, Cell 75:791-803). This system is similar to the Two-Hybrid system except that it uses a LEU2 reporter gene and a lacz reporter gene. Thus protein-protein interactions leading to the reconstitution of the transcriptional activator also allow cells to grow in media lacking leucine and enable them to express &bgr;-galactosidase. The DNA-binding domain used in this system is the LexA DNA-binding domain, while the activator sequence is obtained from the B42 transcriptional activation domain (Ma and Ptashne, 1987, Cell 51:113-119). The promoters of the reporter genes contain LexA binding sequences and hence will be activated by the reconstitution of the transcriptional activator. Another feature of this system is that the gene encoding the DNA-binding domain fusion protein is under the influence of an inducible GAL promoter so that confirmatory tests can be performed under inducing and non-inducing conditions.
In yet another version of this system developed by Elledge and colleagues, the reporter genes HIS3 and lacz (Durfee et al., 1993, Genes Dev. 7:555-569) are used. The transcriptional activator that is reconstituted in this case is GAL4 and protein-protein interactions allow cells to grow in media lacking histidine and containing 3-aminotriazole (3-AT) and to express &bgr;-galactosidase. 3-AT inhibits the growth of his3 auxotrophs in media lacking histidine (Kishore and Shah, 1988, Annu. Rev. Biochem. 57:627-663).
In a different two-hybrid assay, a URA3 reporter gene under the control of Estrogen Response Elements (ERE) has been used to monitor protein-protein interactions. Here, the DNA-binding domain is derived from the human estrogen receptor. The authors of the ERE assay propose that inhibition of the protein-protein interactions can be identified by negative selection on 5-FOA medium (Le Douarin et al., 1995, Nucleic Acids Res. 23:876-878), but do not provide any details.
A version of the two-hybrid approach called the “Contingent Replication Assay” that is applicable in mammalian cells has also been reported (Nallur et al., 1993, Nucleic Acids Res. 21:3867-3873; Vasavada et al., 1991, Proc. Natl. Acad. Sci. USA 88:10686-10690). In this case, the reconstitution of the transcription factor in mammalian cells due to the interaction of the two fusion proteins leads to the activation of the SV40 T antigen. This antigen allows the replication of the activation domain fusion plasmids. Another modification of the two-hybrid approach using mammalian cells is the “Karyoplasmic Interaction Selection Strategy” that also uses the reconstitution of a transcriptional activator (Fearon et al., 1992, Proc. Natl. Acad. Sci. USA 89:7958-7962). Reporter genes used in this case have included the gene encoding the bacterial chloramphenicol acetyl transferase, the gene for cell-surface antigen CD4, and the gene encoding resistance to Hygromycin B. In both of the mammalian systems, the transcription factor that is reconstituted is a hybrid transcriptional activator in which the DNA-binding domain is from GAL4 and the activation domain is from VP16.
In all of the assays described above, the identity of one (or both) of the proteins being tested for interaction is known. All of the assays mentioned above can be used to identify novel proteins that interact with a known protein of interest. In a variation of the “Interaction Trap” system, a “mating-grid” strategy has been used to characterize interactions between proteins that are thought to be involved in the Drosophila cell cycle (Finley and Brent, 1994, Proc. Natl. Acad. Sci. USA 91:12980-12984). This strategy is based on a technique first established by Rothstein and colleagues (Bendixen et al., 1994, Nucleic Acids Res. 22:1778-1779) who used a yeast-mating assay to detect protein-protein interactions. Here, the DNA-binding and activation domain fusion proteins were expressed in two different haploid yeast strains, a and &agr;, and the two were brought together by mating. Thus, interactions between proteins can be studied in this method. However, even in this method, the identities of at least one of the proteins in the interacting pairs of proteins was known prior to analyzing the interactions between pairs of proteins.
Stanley Fields and coworkers have recently performed an analysis of all possible protein-protein interactions that can take place in the
E. coli
bacteriophage T7 (Bartel et al., 19

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