F-box genes and proteins

Details F-box genes and proteins F-box genes and proteins

1997-10-16

2003-06-03

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C536S023100, C536S023500, C435S252300, C435S320100

Reexamination Certificate

active

06573094

ABSTRACT:

FIELD OF THE INVENTION
The present invention provides compositions and methods for identification of F-box proteins, as well as for drug discovery and assessment. In particular, the present invention provides components of an E3 complex involved in ubiquitination of cell cycle regulators and other proteins, as well as members of a class of proteins that directly function in recognition of ubiquitination targets.
BACKGROUND OF THE INVENTION
The proper development and maintenance of a multicellular organism is a complex process that requires precise spatial and temporal control of cell proliferation. Cell proliferation is controlled via an intricate network of extracellular and intracellular signaling pathways that process growth regulatory signals. This signaling network is superimposed upon the basic cell cycle regulatory machinery that controls particular cell cycle transitions. In eukaryotes, the cell cycle is comprised of an ordered series of discrete events. In contrast to the periodicity of eukaryotic DNA replication and mitosis, cellular growth requires that most metabolic reactions occur continuously. The cell cycle regulatory machinery coordinates the events that occur during the cell cycle, as well as cell growth. Protein degradation is an important aspect of the development and maintenance of multicellular organisms, as it provides direction, order, and the appropriate timing for the key events that occur during the cell cycle.
The problem of how cell division is controlled has long been a topic of intense research. Early models suggested the existence of an initiator that would accumulate during the cell cycle, and induce DNA replication or mitosis when it reached a critical concentration. The mitotic process would then inactivate the initiator, thereby “resetting” the cell cycle. Subsequent research showed that mitotic cyclins accumulate during interphase to drive entry of cells into mitosis. These cyclins are then degraded at the end of mitosis, in order to reset the cycle. Protein degradation has been shown to have a pervasive role in the regulation of cell cycle progression. For example, proteolysis is required for multiple mitotic processes, and for initiating DNA replication (See, King et aL, Science 274:1652-1659 [1996]). Nonetheless, much remains unknown regarding the proteins and the interactions that are involved in the proteolytic regulation of the cell cycle and other processes. Indeed, many proteins are likely to be involved in proteolysis and cellular maintenance (as well as other processes). Such information is needed for the development of compounds to regulate the cell cycle and prevent or treat diseases associated with abnormal cell proliferation.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for gene identification (e.g., F-box genes), as well as drug discovery and assessment. The present invention provides components of an E3 complex involved in ubiquitination of cell cycle regulators and other proteins, as well as members of a class of proteins that directly function in recognition of ubiquitination targets.
Thus, the present invention provides the function of a class of proteins referred to as F-box proteins in targeted ubiquitination. The present invention finds utility in methods for developing compounds that affect ubiquitination. The present invention also provides numerous novel F-box containing mammalian genes whose encoded proteins are contemplated to function in processes including, but not limited, to targeted ubiquitination of cellular proteins.
The present invention also provides amino acid and DNA sequence information for eighteen novel F-box-containing human or mouse genes. As with Cdc4, Grr1, Skp2, and cyclin F, these novel F-box proteins have the capacity to associate with Skp1 and to simultaneously interact with other proteins through other protein-protein interaction motifs encoded by regions of their genes other than the F-box. Thus, the present invention provides compositions and methods for determining the interaction of these proteins with other proteins.
In one embodiment, the present invention provides an isolated polypeptide comprising at least one functionally active fragment of an F-box protein. In a preferred alternative embodiment, the F-box protein is mammalian, while in a particularly preferred embodiment, the F-box protein is human or murine.
In another embodiment, the functionally active fragment comprises the amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS:1, 3, 5, 9, 13, 17, 19, 25, 27, 41, 45, 47, 51, 53, 55, and 57, while in alternative embodiment, the functionally active fragment comprises the amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS:7, 11, 15, 21, 23, 29, 31, 33, 35, 37, 39, 43, and 49.
The present invention also provides a purified antibody which binds specifically to the isolated polypeptide encoding an F-box protein. In one embodiment, the antibody is monoclonal, while in another embodiment, the antibody is polyclonal. In another embodiment, the present invention provides a purified antibody which specifically binds to a complex comprised of an F-box protein and an F-box protein target. In yet another embodiment, the present invention provides an antibody which specifically binds to a complex comprised of an F-box protein and Skp1; it is contemplated that the Skp1 in the complex may be bound to another protein, but such binding is not required.
The present invention also provides an isolated nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In a preferred embodiment, the F-box protein is mammalian, while in particularly preferred embodiments, the F-box protein is human or murine. In one embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58. In another embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides a vector comprising a nucleotide sequence, wherein the nucleotide sequence comprises the nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In one preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides a host cell transformed with at least one vector comprising a nucleotide sequence, wherein the nucleotide sequence comprises the nucleotide sequence encoding at least one functionally active fragment of an F-box protein, wherein the nucleotide sequence encodes at least a portion of an F-box protein. In one preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another preferred embodiment, the isolated nucleotide sequence comprises at least a portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.
The present invention also provides an isolated nucleotide sequence encoding the amino acid sequence selected from group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In one embodiment, the present invention provides a vector comprising an isolated nucleotide sequence encoding the amino acid sequence selected from group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11

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