Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-26
2002-10-15
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100
Reexamination Certificate
active
06465199
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to monitoring of the post-translational modification of a protein.
BACKGROUND OF THE INVENTION
The post-translational modification of proteins have been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such modifications include, but are not limited to, the addition of a carbohydrate (glycosylation), ADP-ribosyl (ADP ribosylation), fatty acid (prenylation, which includes but is not limited to: myrisoylation and palmitylation), ubiquitin (ubiquitination) and sentrin (sentrinization; a ubiquitination-like protein modification). Additional examples of post-translational modification include methylation, actylation, hydroxylation, iodination and flavin linkage. Many of the identified modifications have important consequences for the activity of those polypeptides so modified.
Currently there are several approaches to analyzing the state of modification of target proteins in vivo:
1. In vivo labelling of cellular substrate pools with radioactive substrate or substrate precursor molecules to result in incorporation of labeled (for example, radiolabeled) moieties (e.g., fatty acyl (including, but not limited to, myristoyl and palmityl) sentrin, methyl, actyl, hydroxyl, iodine, flavin, ubiquitin or ADP-ribosyls), which are added to target proteins. Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis.
2. Back-labeling. The enzymatic incorporation of a labeled (including, but not limited to, with a radioactive and fluorescent label) moiety into a protein in vitro to estimate the state of modification in vivo.
3. Detection of alteration in electrophoretic mobility of modified protein compared with unmodified (e.g., glycosylated or ubiquitinated) protein.
4. Thin-layer chromatography of radiolabelled fatty acids extracted from the protein of interest.
5. Partitioning of protein into detergent-rich or detergent-poor layer by phase separation, and the effects of enzyme treatment of the protein of interest on the partitioning between aqueous and detergent-rich environments.
6. The use of cell-membrane-permeable protein-modifying enzyme inhibitors (e.g., Wortmannin, staurosporine) to block modification of target proteins and comparable inhibitors of the enzymes involved in other forms of protein modification (above).
7. Antibody recognition of the modified form of the protein (e.g., using an antibody directed at ubiquitin or carbohydrate epitopes), e.g., by Western blotting, of either 1- or 2-dimensional gels bearing test protein samples.
8. Lectin-protein interaction in Western blot format as an assay of the presence of particular carbohydrate groups (defined by the specificity of the lectin in use).
9. The exploitation of eukaryotic microbial systems to identify mutations in protein-modifying enzymes.
These strategies have certain limitations. Monitoring states of modification by pulse or steady-state labelling is merely a descriptive strategy to show which proteins are modified when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are modified. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions.
The use of enzyme inhibitors to block activity is also problematic. For example, very few enzyme inhibitors have adequate specificity to allow for the unequivocal correlation of a given enzyme with a specific modification reaction. Indeed, many inhibitors have a broad inhibitory range. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
Finally, yeast (
Saccharomyces cervisiae
and
Schizosaccharomyces pombe
) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein-modifying enzymes have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
A non-limiting example of post-translational modification is provided by the Ras proteins, which are a conserved group of polypeptides located at the plasma membrane which exist in either a GTP-bound active state or in a GDP-bound inactive state. This family of proteins operates in signal transduction pathways that regulate cell growth and differentiation. In higher eukaryotes, Ras is a key regulator that mediates signal transduction from cell surface tyrosine kinase receptors to the nucleus via activation of the MAP kinase cascade. Recent studies have demonstrated that Ras directly binds a serine/threonine kinase, Raf-1, a product of the c-raf-1 proto-oncogene, and that this association leads to stimulation of the activity of Raf-1 to phosphorylate MAP kinase kinase (MEK).
An important post-translational modification is the addition of ubiquitin to selected polypeptides. This provides a key mechanism by which to control the abundance of important regulatory proteins, for example, G1 and mitotic cyclins and the p53 tumor suppressor protein. Ubiquitin is a highly conserved 76-amino-acid cellular polypeptide. The role of ubiquitin in targeting proteins for degradation involves the specific ligation of ubiquitin to the &egr; group of lysine residues in proteins that are to be degraded or internalized from the plasma membrane. The ubiquitin tag determines the fate of the protein and results in its selective proteolysis. Recently a number of factors have been isolated and shown to be involved in the ubiquitination process.
The initial step in the addition of ubiquitin to a protein is the activation of ubiquitin by the ubiquitin activating enzyme, E1. This is an ATP-dependent step resulting in the formation of a thioester bond between the carboxyl terminal glycine of ubiquitin and the active site cysteine residue of E1. Activated ubiquitin then interacts with a second factor, the E2 protein. A thioester bond forms between the activated glycine residue of ubiquitin and a cysteine residue in a specific E2 protein. The E2 proteins represent a family of closely-related proteins encoded by different genes that confer specificity in the proteolytic process. The ligation of ubiquitin to target proteins is effected by the involvement of a further factor, a ubiquitin ligase, E3, of which a number are known (in yeast, reviewed by Haas and Siepmann, 1997,
FASEB J.,
11: 1257-1268; in humans, see Honda et al., 1997,
FEBS Lett.,
420: 25-27). E3 completes the final step of ubiquitination by attaching ubiquitin via the &egr; amino group on lysine residues in proteins to be targeted for degradation. Moreover, E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins, thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi-subunit proteasome.
An example of heterodimer association is described in patent application number W092/00388. It describes an adenosine 3:5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R
2
C
2
. In the absence of cAMP
Colyer John
Craig Roger K.
Cyclacel Ltd.
Palmer & Dodge LLP
Weber Jon P.
Williams Kathleen M.
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