Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-01-26
2004-12-07
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S005000, C435S006120, C435S007200, C435S007800, C435S069700, C530S380000, C530S387300, C530S387100, C530S320000, C530S385000, C530S350000, C536S023500, C514S418000, C514S486000
Reexamination Certificate
active
06828106
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detecting or monitoring the activity of an enzyme. In particular, the invention relates to the use of a polypeptide multimer capable of generating a multimerisation-dependent signal and whose multimerisation properties are modulated by the activity of a protease in such a method.
BACKGROUND
Proteolysis has long been recognised as an important intra- and extracellular modification of proteins. Endopeptidase enzymes recognise particular primary sequence signals (and sometimes also secondary or tertiary structural cues) within a substrate protein and cleave the peptide bond following a particular amino acid. Exopeptidases, on the other hand, digest polypeptides from the N or C terminus. Exopeptidases are generally not sequence-specific. These enzymes play a role in, for example, digestion, the coagulation of blood, the complement cascade and the destruction of inactive, mutated or foreign forms of proteins in the cell. Proteolysis is also important as a method of recycling amino acids within the cell for the synthesis of new proteins or for utilisation as a fuel source. More recently, the role of proteolysis in signalling and in specific intracellular processes has been recognised.
It is clear that aberrant proteolysis plays a significant role in a number of disease processes. Examples include the processing of &bgr;-amyloid precursor protein (inappropriate processing of this protein is thought to play a role in Alzheimer's Disease), the inappropriate activation of proteolytic enzymes of digestion leading to pancreatitis and a loss of proteolysis of the insulin receptor precursor leading to diabetes. Proteolysis is now understood to play important roles both within the cell and in processes important in homeostasis in multi-cellular organisms. These include:
Production of bioactive molecules from inactive precursors. A hallmark of proteolytic enzymes is their production in many cases as inactive proenzymes and their subsequent rapid activation by a proteolytic event. This may be an autocatalytic process or part of a cascade. This is exemplified by the blood clotting cascade and also the cleavage of digestive proproteases to their active form. One of the central events in acute pancreatitis is the premature proteolysis and activation of pancreatic enzymes (especially trypsin) leading to autodigestion of pancreatic tissue amongst other effects (Acute pancreatitis, Mergener, K. & Baillie, J. British Medical Journal (1998) 316 44-48). Proteases are also known to activate other proenzymes and to play a role in the generation of other bioactive molecules. An important clinical example of this is the generation of angiotensin II by the enzyme angiotensin converting enzyme (ACE). ACE cleaves the C-terminal two residues from the inactive angiotensin I to produce the active form, angiotensin II. Angiotensin II has potent vasoconstrictive and salt-retentive properties, the control of ACE activity by ACE inhibitors has an important clinical role in the treatment of hypertension, heart failure, myocardial infarction and diabetic nephropathy (Angiotensin converting enzyme inhibitors, Brown N J. & Vaughan, D E., Circulation (1998) 97 1411-1420).
Destruction of bioactive molecules. An important aspect of a regulatory process is the presence not only of an ‘on switch’ but also the potential to switch it off again. This is an area in which proteolysis is particularly important as it is an irreversible modification. The only way in which the process can be restarted is by a resynthesis of the destroyed component. This affords a high level of control over timing. An important clinical example of this is the degradation of bradykinin by ACE. Bradykinin has a number of effects in the body including inducing smooth muscle contraction, increasing vascular permeability and promoting vasodilation and natriuresis. This, together with the example above, indicates that ACE is important in the regulation of the balance between the antagonistic effects of angiotensin II and bradykinin (Angiotensin converting enzyme inhibitors, Brown N J. & Vaughan, D E., Circulation (1998) 97 1411-1420).
Protein turnover. The ability of the cell to degrade unwanted, damaged or foreign proteins is of great importance in the maintenance of the cell. Limited proteolysis of foreign proteins is also important in the antigen presentation process and therefore in an appropriate immune response to pathogens.
Post-translational modification. The proteolysis of certain proteins is key in their ability to perform their function in the cell. For example, the biosynthesis of the insulin receptor involves the cleavage of a large precursor to produce the subunits of the receptor complex (Biosynthesis and glycosylation of the insulin receptor, Hedo, J. A., Kahn, C. R., Hayashi, M., Yamada, K. M., Kasuga, M., Journal of Biological Chemistry (1983) 258 10020-10026). The assembly of the plant lectin concanavalin A (con A) also involves the proteolysis of a precursor protein and the religation of fragments in an altered order to generate the mature protein (Traffic and assembly of concanavalin A, Bowles, D. J. & Pappin, D. J., Trends in Biochemical Science (1988) 13 60-64).
A process coincident with other forms of post-translational modification. Proteolysis is an important feature of the processes leading to the addition of glycosylphosphatidylinositol (GPI) anchors to proteins and also in some fatty acylation reactions (such as farnesylation or geranylgeranylation).
Thus, proteolysis is an important post-translational modification of proteins and peptides which occurs both within and outside of the cell and can be an essential part of other forms of post-translational modification such as addition of a GPI anchor or some fatty acids. The ability to measure the cleavage of a protein or peptide at a specific site where that protein or peptide is also accessible for the addition of a prenyl moiety or a GPI anchor will allow the in vitro and in vivo study of processes for which the methods currently available are limited.
However, methods presently available for monitoring or detecting protease activity are not sufficiently sophisticated to be useful. Reporters are currently available to follow proteolysis where a peptide containing the cut site of the protease of interest has fluorophores at either end. Modification is followed by a change in the fluorescent output on cleavage of the peptide (causing physical separation of the fluorophores). Methods are available for monitoring both in vivo and in vitro proteolysis given the availability of various chemical fluorophores and quenchers and also a number of GFP variants which can be expressed in the cell (Compositions for the detection of proteases in biological samples and methods of use thereof, Komoriya, A. & Packard, B. S., WO96/13607; Tandem fluorescent protein constructs, Tsien, R. Y., Heim, R. & Cubitt, A., WO97/28261). All such methods, however, rely on the use of a synthetic reporter which is typically not the natural substrate for the enzyme being assayed. Moreover, the flexibility of the prior art systems is limited.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention, there is provided a polypeptide multimer comprising a first polypeptide and a second polypeptide, wherein
a) at least one of the polypeptides is susceptible to protease digestion;
b) association of the polypeptides to form a multimer is detectable via a signal; and
c) digestion of at least one polypeptide results in modulation of the association state of the multimer and modulation of the signal.
The invention accordingly provides a polypeptide multimer, or a constituent polypeptide thereof, which is susceptible to protease digestion such that digestion leads to dissociation of the constituent polypeptides, or a part thereof, from the multimer. The dissociation and association of the polypeptides in the multimer is in turn detectable, for example via a label (further described below), or by monitoring of molecular weight, suc
Colyer John
Lightowler Joanne
Cheu Changhwa J
Cyclacel Limited
Le Long V.
Palmer & Dodge LLP
Williams Kathleen M.
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