Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1998-11-03
2002-05-21
Achutamurthy, Ponnathapu (Department: 1659)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S219000, C435S252300, C435S320100, C435S325000, C435S471000, C435S006120, C536S023200
Reexamination Certificate
active
06391574
ABSTRACT:
BACKGROUND OF THE INVENTION
A cell possesses thousands of unique proteins that serve structural, enzymatic or signaling functions. The intracellular environment is composed of a myriad of structures and membrane-enclosed compartments. The correct subcellular localization is critical for the proper functioning of many proteins. Proteins situated at the lipid bilayer membrane are classified as peripheral proteins, whereas proteins situated within the lipid bilayer membrane are classified as integral membrane proteins. Integral membrane proteins possess intrinsic hydrophobic regions which are inserted into the lipid bilayer as they are synthesized. Typically, peripheral membrane proteins are less tightly associated with membranes and are localized to the lipid bilayer by protein-protein interactions, by intrinsic hydrophobic properties or by the post-translational addition of a lipid group.
A major class of peripheral membrane proteins, known as prenylated proteins, are modified by isoprenoids on a so-called CAaa
1
Aaa
2
Xaa (CAAX) motif, wherein C is cysteine, Aaa
1
and Aaa
2
are aliphatic amino acids and Xaa is any amino acid. This tetra-peptide sequence is located at the proteins' carboxyl termini and triggers a series of modification reactions. Of the approximately 30 known CAAX-containing proteins, the Ras family of small GTP-binding proteins are major constituents. Ras proteins localize at the inner surface of the plasma membrane where they function as key components of various signal transduction pathways or participate in cytoskeletal organization and establishment of cell polarity. The critical role of the Ras proto-oncogene in controlling cell division is exemplified by the participation of mutated forms of the Ras protein in a variety of human tumors, including colorectal carcinoma, exocrine pancreatic carcinoma and myeloid leukemias. Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
The presence of the CAaa
1
Aaa
2
Xaa motif sequence targets the protein for at least 3 post-translational modifications. Generally, such modifications include prenylation of the cysteine amino acid, proteolytic removal of the terminal three amino acids (i.e., the Aaa
1
Aaa
2
Xaa tripeptide) and methylesterification of the prenylated cysteine, i.e., the C-terminus. More particularly, in the first step, a 15 carbon farnesyl or a 20 carbon geranylgeranyl isoprenyl lipid is added to the cysteine residue. The lipid which is added depends upon the amino acid at the “X” position. Following prenylation, the terminal tripeptide, i.e., the Aaa
1
Aaa
2
Xaa tripeptide, is removed by a membrane-bound endoprotease. Thereafter, the resulting C-terminal isoprenylated cysteine is methylesterified.
It has been determined that prenylation of the CAAX motif is essential for the proper functioning of every prenylated protein that has been tested to date. However, the functional requirement of CAAX proteolysis has not been rigorously evaluated because the gene encoding the protease has been elusive. This is true despite the fact that the entire yeast genome has been sequenced and the sequences deposited in GenBank. Unfortunately, elucidation of the complete yeast genome in the absence of functional information for each yeast gene is insufficient for identification of any particular gene. Although many open reading frames (ORFs) have been identified, it is not known whether these ORFs encode functional mRNAs.
Kato, et al. (
Proc. Natl. Acad. Sci. USA,
89:9554-9558 (1992)) monitored foci formation of NIH3T3 cells transformed with activated forms of Ras with altered CAAX sequences. They found that one sequence, CVYS, when substituted for the normal Ras CAAX sequence appeared not to have undergone proteolysis and resulted in approximately 50% reduction in foci formation. Unfortunately, the design of this experiment was not ideal because it relied on the heterologous expression of Ras from an SV40 promoter, which resulted in a considerably higher expression level than the physiological Ras promoter. Moreover, the CAAX sequence CVYS displayed a prenylation defect.
In view of the foregoing, there remains a need in the art for the identification of the genes encoding the polypeptides that participate in the post-prenylation modification reactions so that the functional importance of such enzymes can be elucidated.
SUMMARY OF THE INVENTION
The present invention includes the discovery of two families of genes which encode polypeptides that mediate the proteolytic removal of an AAX tripeptide from a prenylated CAAX protein in a cell. In yeast, the families of genes are represented by the genes AFC1 and RCE1 which encode the polypeptides Afc1p and Rce1p, respectively.
Accordingly, the invention provides vectors that includes a nucleic acid sequence which encodes an Afc1p or Rce1p polypeptide (or both polypeptides), or conservatively modified variations of Afc1p or Rce1p. Exemplar nucleic acids which encode Afc1p, or Rce1p include those set forth in SEQ ID NO:1 and SEQ ID NO:2. Recombinant cells, including recombinant yeast cells, which comprise a vector nucleic acid of the invention are also provided.
In one class of embodiments, the vector of the invention provides a nucleic acid sequence which hybridizes under stringent conditions to a nucleic acid selected from the group consisting of the AFC1, and RCE1 genes. Exemplar nucleic acids with the desired hybridization properties include those represented by the sequences of SEQ ID NO:1 and SEQ ID NO:3.
The invention provides isolated polypeptides, such as Afc1p and Rce1p, encoded by the vectors of the invention. Exemplar polypeptides include those represented by SEQ ID NO:2 and SEQ ID NO:4. Antibodies which specifically bind to the polypeptides of the invention are also provided.
In addition to nucleic acids, cells, polypeptides and antibodies, a variety of useful methods and assays are provided by the present invention. In one embodiment, the invention provides methods for inhibiting the proteolytic removal of an AAX tripeptide from a prenylated CAAX protein in a cell. Exemplar prenylated CAAX proteins include the Ras protein, a-factor, and the &ggr;-subunit of the heterotrimeric G-protein. In these methods, a mutation is introduced into an AFC1 and/or RCE1 gene.
In one class of embodiments, the invention provides methods for inhibiting the proteolytic removal of an AAX tripeptide from a prenylated CAAX protein in a cell. In this class of embodiments, the activity of the Afc1p or Rce1p protein is blocked using an inhibitor. Exemplar inhibitors include 1,10-phenanthroline and NME 181.
The invention provides assays for testing the inhibitory activity of a potential inhibitor of the Afc1p or Rce1p proteases, which are responsible for the proteolytic removal of an AAX tripeptide of a CAAX protein in a cell. In the assay method, a test compound to be tested for inhibitory activity is provided. The test compound is contacted to a cell expressing either the AFC1 or RCE1 genes, or both. The transcriptional or translational activity of the genes or, alternatively, the activity of the encoded proteins, is measured, and typically compared to a reference, such as a control assay which establishes the activity of the measured activity in the absence of the test compound. One convenient activity which is mediated by the AFC1 and RCE1 genes is heat shock sensitivity of cells. Accordingly, in one embodiment, the measured activity is heat shock sensitivity. In a second convenient assay, the level of Afc1p or Rce1p protein in a population of cells is measured in a standard immunological assay, such as an ELISA.
In addition, the present invention provides an improved method for monitoring heat shock sensitivity, particularly in yeast, is provided. In this method, a plurality of aliquoted yeast strains in liquid are provided. Each strain is separated into a test population of cells and a control population of cells. The test population of cells is heated to a heat shock temperature of between about 40° C. and about 60° C. for a time per
Ashby Matthew N.
Boyartchuk Victor L.
Rine Jasper D.
Achutamurthy Ponnathapu
Osman Richard Aron
The Regents of the University of California
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