Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Utility Patent
1998-01-27
2001-01-02
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C530S324000, C530S350000, C530S826000
Utility Patent
active
06168929
ABSTRACT:
BACKGROUND OF THE INVENTION
Simian virus 40 (SV40) encodes two proteins involved in tumorigenesis, the large and small tumor antigens. Large tumor antigen (T antigen) orchestrates many aspects of productive viral infection and is necessary, and in many cases sufficient, for tumorigenesis. T antigen is a 708 amino acid multifunctional protein that elicits cellular transformation by acting on multiple targets, including members of the retinoblastoma tumor suppressor family (pRb, p107, and p130), members of the CBP-family of transcriptional coactivators (CBP, p300, and p400), and the tumor suppressor, p53. T antigen sequences important for transformation have been mapped to two different regions of the molecule: the amino-terminal domain, which encompasses the first 125 amino acids, and a region located within the carboxy-terminal half of the molecule (FIG.
1
).
Evidence that one or more independent transforming functions reside in the carboxy-terminal half of T antigen stems from the observation that carboxy-terminal fragments of T antigen have the ability to immortalize primary C57B1/6 mouse embryo fibroblasts (Cavender et al. (1995)
J. Virol.
69:923). This activity maps to a bipartite region including amino acids 351-450 and 533-626 that correspond to sequences required for T antigen association with p53 (Kierstead et al. (1993)
J. Virol.
67:1817). This same region has been shown to be necessary for T antigen association with CBP, p300, and p400 (Eckner et al. (1996)
Mol. Cell. Biol.
12:3288). It is not clear whether the CBP-family proteins bind directly to T antigen, or indirectly by associating with T antigen-bound p53.
The amino-terminal region of T antigen also carries independently acting transforming functions since it is capable of immortalizing primary cells, transforming established cell lines in culture, and inducing tumors in transgenic mice (Chen et al. (1992)
Oncogene
7:1167; Clayton et al. (1982)
Nature
299:59; Colby et al. (1982)
Proc. Nat'l. Acad. Sci. USA
79:515189; Srinivasan et al. (1989)
J. Virol.
63:5459). Three separate sequence motifs within the amino-terminal domain contribute to transformation. First, the sequence extending from amino acids 101-118 is similar to the cr2 motif found in adenovirus E1A proteins, and papillomavirus E7 proteins (Chen et al. (1990)
J. Virol.
64:3350). This sequence is required for the association of T antigen with the three members of the retinoblastoma tumor suppressor family of proteins: pRb, p107, and p130 (DeCaprio et al. (1988)
Cell
54:275). Second, a region near the amino-terminus of T antigen (amino acids 17-32) is similar to the cr1 motif found in adenovirus E1A proteins. Deletion of this sequence alters the transforming properties of T antigen, but a cellular target for this region has not been identified (Pipas et al. (1983)
Mol. Cell. Biol.
3:203; Symonds et al. (1993)
Mol. Cell. Biol.
13:3255). Finally, mutations within or near a hexapeptide (HPDKGG; amino acids 42-47 of SEQ ID NO: 1) conserved among the T antigens in all known polyomaviruses render T antigen transformation-defective (Peden et al. (1992)
Virus Genes
6:107).
The amino-terminal 82 amino acids of T antigen are similar to the J domain of the DnaJ family of molecular chaperones (Kelley et al. (1994)
TIBS
19:277). This similarity includes the HPDKGG motif (amino acids 42-47 of SEQ ID NO: 1) as well as the cr1-like sequence (FIG.
1
). Since the 82 amino-terminal amino acids of large T antigen and small t antigen are identical, the J-domain homology region is included in small t as well. One function of J-domain containing chaperones is to interact with, and modulate the activity of, a specific member of the DnaK family (reviewed by Caplan et al. (1993)
Mol. Biol. Cell
4:555; Hartl, (1996)
Nature
381:571). The cytosolic mammalian DnaK homologue, hsc70, associates with T antigen through its amino-terminal domain (Sawai et al. (1989)
J. Virol.
63:3691; Campbell et al. (1997)
Genes
&
Devel.
11:1098-1110; Sawai et al. (1994)
Virus Res.
31:367-378).
Mutations within the J-domain region of T antigen affect diverse viral functions including DNA replication, transcriptional regulation, virion assembly, and tumorigenesis (Pipas et al. (1983); Peden, (1992); Srinivasan et al. (1989)
J. Virol.
63:5459; Spence et al. (1994)
Virology
204:200). Other reports suggest a role for the T antigen/hsc70 association in stimulating the degradation of the tumor suppressors p107 and p130 (Stubdal et al. (1996)
J. Virol.
70:2781; Harris et al. (1996)
J. Virol.
70:2378; Stubdal et al. (1997)
Mol. Cell. Biol.
17:4979-4990).
A collection of T antigen mutants with altered amino acids within each of the known sequence motifs important for transformation have been studied in an attempt to characterize the roles of T antigen in transformation. Mutant d11135 contains a deletion of amino acids 17 through 27 and synthesizes a T antigen lacking the cr1-like motif, and fails to transform established cell lines and to immortalize primary cells (Pipas et al. (1983); Michalovitz et al. (1987)
J. Virol.
61:2648). On the other hand, d11135 induces T cell lymphomas in transgenic mice at the same efficiency as wild-type T antigen (Symonds et al. (1993)
Mol. Cell. Biol.
13:3255). Even though d11135 is defective for viral DNA replication in vivo, the purified mutant protein supports the replication of viral DNA in vitro nearly as well as wild-type (Collins et al. (1995)
J. Biol. Chem.
270:15377). Mutant 5110 carries a single amino acid substitution of D44N within the conserved HPDKGG motif (amino acids 42-47 of SEQ ID NO: 1), and has a reduced ability to transform established cell lines. Mutant 3213 containing two amino acid substitutions (E107K, E108K) within the cr2-like sequences, is defective for T antigen association with pRb.
The foregoing studies with these mutants suggest that T antigen's contribution to cellular transformation is complex. An elucidation of the role of the J-domain homology region of T antigen in transformation and chaperone action is necessary to devise strategies to interfere with viral and nonviral tumorigenicity.
SUMMARY OF THE INVENTION
The present invention provides a method of identifying agents that interfere with the interaction of a J-domain and a DnaK protein. In one embodiment, agents are identified by measuring their ability to block the stimulation of the ATPase activity of a DnaK protein by a polypeptide comprising a J-domain. In another embodiment, agents are identified by measuring their ability to block the J-domain-catalyzed release of a denatured polypeptide substrate from a DnaK protein. In a preferred embodiment, the polypeptide comprising a J-domain comprises amino acids 1-82 or amino acids 1-136 of the SV40 large T antigen, and the DnaK protein is the yeast Ssa1p protein or the human hsc70 protein. In another embodiment, agents are identified by measuring their ability to block the J-domain dependent growth of the
S. cervisiae
mutant ydj1-151 at elevated temperatures.
The present invention further provides a compartmentalized kit comprising a first container containing a polypeptide comprising a J-domain and a second container containing a DnaK protein. In another embodiment, the kit further comprises a third container containing a denatured polypeptide substrate for a DnaK protein.
REFERENCES:
Cheetham et al. (1992)Biochem. J.284:469.
Campbell et al. (1997)Genes&Development11:1098.
Karzai et al. (1'996)J. Biol. Chem.271:11236.
Minami et al. (1996)J. Biol. Chem.271:19617.
Sawai et al. (1994)Virus Research31:367.
Sheng et al. (1997)J. Virol.71:9410.
Srinivasan et al. (1997)Mol. Cell. Biol.17:4761.
Stubal et al. (1997)Mol. Cell Biol.17:4979.
Stubdal et al. (1996)J. Virol.70:2781.
Wall et al. (1994) J. Biol. Chem., vol. 269, pp. 5446-5451.
Kelley et al. (1994) TIBS, vol. 19, pp. 277-278.
Tsai et al. (Apr. 19, 1996) J. Biol. Chem., vol. 271, pp. 9347-9354.*
Brodsky Jeffrey L.
Pipas James M.
Baker & Botts LLP
Slobodyansky Elizabeth
University of Pittsburgh
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