WO2002066982A2 - ANALYSIS OF MODIFICATIONS AND DEMODIFICATIONS OF PROTEINS WITH UBIQUINTIN-RELATED PROTEINS BY FRET (FLUORESCENCE RESONANCE ENERGY TRANSFER) - Google Patents

Abstract

The present invention provides methods for assaying protein modifications with ubiquitin-related proteins and protein demodifications related to ubiquitin-related proteins. The invention further provides methods for determining substances that influence such modifications or demodifications, as well as methods for detecting a defect in such modifications or demodifications. The invention further relates to a method for identifying target proteins for modification with ubiquitin-related proteins, as well as methods for producing a pharmaceutical composition. Furthermore, the invention relates to a kit, a fusion protein, a cell and an isopeptidase substrate, and their use in the analysis and / or diagnosis of protein modifications or demodifications with proteins related to ubiquitin.

Description

 Analysis of Modifications and Demodifications of Proteins with U-biquitin-Related Proteins Using FRET (Fluorescence Resonance Energy Transfer)

The present invention relates to methods for analyzing modifications of proteins with ubiquitin-related proteins and of demodifications of proteins linked to ubiquitin-related proteins. Furthermore, the present invention relates to methods for determining substances which influence these modifications or demodifications, as well as methods for detecting a defect in these modifications or demodifications. Finally, the present invention relates to a method of identifying target proteins for modification with ubiquitin-related proteins, as well as methods of making a pharmaceutical composition. Moreover, the invention relates to a kit, a fusion protein, a cell and an isopeptidase substrate and their uses in the analysis and / or diagnosis of ubiquitin-related modifications or demodifications of proteins.

A variety of proteins are affected in their activity, localization and stability by covalent linkage with ubiquitin or ubiquitin-related proteins such as SUMO1 or Νeddδ (Hershko and Ciechanover, 1998, Hochstrasser, 1998, Jentsch and Pyrowolakis, 2000, Melchior, 2000; Yeh et al, 2000). In a cascade of enzymatic steps, an isopeptide bond is attached between the carboxy-terminal end of ubiquitin (or the ubiquitin-related protein) and the ε-amino group in lysine side chains of the target protein. Elucidating enzymes, E2 conjugating enzymes, and (in part) E3 ligases are involved in this linkage. The activity of isopeptidases, which can cleave this binding, reverses these posttranslational modifications (Chung and Baek, 1999, Wilkinson, 1997). The Ubiquitination (and its subsequent degradation) and modification with ubiquitin-related proteins appear to be useful for a variety of cellular processes such as cell cycle control, signal transduction, intracellular transport or apoptosis, but also for many pathological scenarios (viral infections, tumorigenesis, neurodegenerative diseases, etc.) (Bonifacino and Weissman, 1998; Pagano, 1997; Schwartz and Ciechanover, 1999). Both the modification and the demodification of many target proteins are subject to regulation specific for the target protein (eg by target-protein-specific E3 ligases and isopeptidases and / or by cell-cycle or stress-dependent phosphorylation or dephosphorylation). Therefore, a diagnosis of modification of demodification of specific target proteins, as well as the inhibition or activation of these reactions as a basis for a disease treatment and appropriate diagnostics on.

Modification of proteins with ubiquitin or ubiquitin-related proteins results in a significant size change of the target protein and can be analyzed both in vivo and in vitro. Previously, in vivo modification of the modification was usually by Western blot analysis of cell extracts with antibodies to the protein of interest or by immunoprecipitation of the modified protein and subsequent Western blot analysis with antibodies to the ubiquitin-related protein. This analysis often involves prior transfection of the cells with plasmids encoding either the ubiquitin-related protein or the protein to be modified.

An in vt ^{'tro-reconstitution} of ubiquitylations or modifications with the ubiquitin-related proteins is so far performed by purified or recombinant target proteins, purified or recombinant ubiquitin or ubiquitin-related proteins and the corresponding purified or rekombinan- ten enzymes (or cell extracts containing containing the necessary enzymes) in the presence of ATP for a defined time. The analysis of Modification is then performed by SDS-PAGE, followed by Western transfer and I rimun staining, or (using radiolabeled proteins) by SDS-PAGE followed by autoradiography (see, eg, Buschmann et al., 2000, Deng et al. 2000, Kaiser et al., 2000).

A method of detecting inhibitors of ubiquitin-dependent degradation of regulatory proteins of the cell cycle has been described by Kirschner et al. (US 5,726,025). This document describes, inter alia, a quantitative test procedure for the degree of ubiquitination. It is proposed to provide the ubiquitinierende protein or ubiquitin with a fusion moiety or mark with biotin or a fluorochrome and it after the reaction from the cells or the reaction mixture by means of so-called. "Pull downs" or ELISA-related techniques remove and prove the modification. A general procedure for the detection of posttranslational modifications (including the modification with ubiquitin or ubiquitin-related proteins) by means of the so-called "fluorescence resonance energy transfer" (FRET) has been described by Bastiaens and Parker (WO 00/43780). The authors suggest that the target protein be labeled fluorescently and that the modification be detected by means of a likewise fluorescently labeled probe which recognizes only the modified protein (eg an anti-ubiquitin antibody). It is also proposed herein to use such a Nerfahren in so-called. "Drag screenings". Similarly, Boisclair and co-workers suggest using FRET technology to search for inhibitors of ubiquitination by fluorescently labeling probes (streptavidin and anti-Gst antibodies) with donor and acceptor chromophores (Boisclair et al , 2000). Although these procedures represent a simplification compared to the commonly used methods, they still require a considerable amount of manipulation, and are very susceptible to interference by the indirect detection methods, are poorly practicable in vivo, and at most semiquantitative. It was therefore an object of the present invention to provide a quantitative, rapid and in particular versatile test system for the analysis or the diagnosis of post-translational ubiquitin-related modifications or demodifications, which can be used both in vitro and in vivo. Another object of the present invention is to provide and use suitable components of this test system.

It has now surprisingly been found that quantitative and rapid assays can be provided for post-translational modifications with both ubiquitin and ubiquitin-related proteins. For example, in contrast to the methods described above, these test methods and the universally applicable components proposed for them are not limited to the determination of inhibitors of ubiquitination alone. Rather, the proposed methods now allow both in vitro and in vivo so-called "high throughput drug screenings" (hereinafter HTS method) and the provision of diagnostic kits for detecting defects in the modification / demodification and in particular the discovery of both inhibitors and also activators involved in the modification and / or demodification of specific target proteins (eg E3 ligases, isopeptidases, kinases or other binding partners). In addition, it is also possible for the first time to discover new target proteins for the modification with U-biquitin-related proteins in the "high throughput" method, for which the corresponding inhibitors and / or activators can then be determined with the aid of the proposed methods which in turn makes it possible, by means of the proposed further methods, to carry out the analysis of the modifications and demodifications or the detection of a defect in the modification / demodification for this new target protein. This systematic and practical approach is particularly advantageous because it provides a complete chain from identification through characterization to analysis and diagnostics. The proposed test systems make use of the FRET technology (Periasamy and Day, 1999, Pollok and Heim, 1999) and, in the application examples described below, use the fluorescent proteins "Yellow Fluorescent Protein" (YFP) and "Cyan" described by Tsien and co-workers by way of example Fluorescent Protein "(CFP) (US 5,981,200; US 5,998,204; Tsien, 1998). However, none of the above documents describes the use of FRET for the direct and quantitative analysis of covalent modifications as proposed herein.

The term "quantitative" is used to express that, in contrast to other methods described so far, the signal to be measured is directly proportional to the measure of the linkage, since neither manipulations after the reaction has taken place nor probes for detecting the linkage have to be used , The ability of probes to recognize the reaction product is dependent on their affinity, as well as on the accessibility of the product, and thus is not linear in every concentration range. In addition, the previously described methods are susceptible to interference if additional binding partners for the reactants are present.

A very significant advantage over that of Kirschner et al. proposed method (see above) is beyond that reaction sequences can be followed kinetically. While the method of Kirschner allows the analysis of the degree of coupling of a reaction mixture by the required manipulations only at a given time (end point measurement), our method allows non-invasive "on-line" measurements over the entire period of the reaction (100 measurements of a single reaction in the Distance of seconds or minutes are readily possible).

Since FRET is dependent on the distance and orientation of the chromophores (with maximum energy transfer at distances below 5 nm), it was initially surprising to find that the size of the proteins to be conjugated FRET was sufficiently and reliably detectable. Thus, it is described herein that Reliable FRET detection occurs when proteins of 10 and 20 kDa, respectively, are inserted between exemplary fluorescent proteins, while in the methods proposed by Tsien and co-workers for the analysis of proteases, a linker peptide of only 5-50 amino acids, containing the desired protease Carries recognition sequence between the fluorescent proteins YFP and CFP was set (US 5,981,200). Furthermore, in the methods known in the prior art, only the probes, but not the actual reactants, are labeled with fluorescent groups. On the other hand, it has surprisingly been found that the direct linkage of the reactive reaction components of the test system, ie target protein and ubiquitin-related protein, with the respective chromophores does not lead to an inhibition thereof. The inventors made the decisive breakthrough in this technological concept by successfully establishing an in vitro test system with recombinant components, in which the quantitative modification of a target protein and the demodification of a modified target protein were achieved. Based on the findings of the inventors, it is now possible to develop analogous modification and demodification systems with all the desired ubiquitin-related proteins and target proteins. Since proteins of the ubiquitin family not only have the same three-dimensional folding, but are furthermore linked to target proteins via conserved mechanisms (Jentsch and Pyrowolakis, 2000), the person skilled in the art can now assume, for example, that fusion proteins between a fluorescent protein and a ubiquitin -related protein (including ubiquitin) can be used in the FRET technology. The linkage may preferably be such that the ubiquitin-related protein is fused to the C-terminus of the fluorescent protein (analogously to the application examples). While the target proteins to the corresponding ubiquitin-related proteins are more heterogeneous in fold and size, it will be readily apparent to those skilled in the art, in light of the present disclosure, to construct a fusion protein or otherwise fluorescently labeled target protein such that it can be used in the assay system. For example, N-terminal or C-terminal fusions with fluorescent proteins can be used. the integration of the fluorescent protein between two domains of a protein, or also the linking of chromophores to specific amino acids in the target protein (if appropriate after introduction of suitable side chains by mutagenesis of the target protein). It should be noted that the acceptor chromophore is possibly brought close to the modification site in the target protein in order to achieve efficient FRET. Here, "close" does not refer to the primary sequence but to the spatial proximity in the folded protein. Even if the structural data for a target protein should not be known, the person skilled in the art will recognize that a suitable target protein can be constructed according to the abovementioned specifications without great experimental effort.

In a first aspect, the present invention relates to a method for the analysis of modifications of proteins with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) at least one target protein, comprising a covalently linked to the first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET), (ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair, (iii) Enzymes which cause the formation of an isopeptide bond between the at least one target protein and the at least one ubiquitin-related protein; (iv) ATP and / or an ATP derivative, and contacting components (i) - (iv) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of conjugate of target protein and ubiquitin-related protein formed by determining FRET between the first and second chromophore of the donor-acceptor pair.

The principle of this FRET-based test system for the analysis of post-translational modifications of proteins with ubiquitin-related proteins

Proteins is as follows. A target protein linked to a donor chromophore becomes an acceptor chromophore-linked ubiquitin-related protein in the presence of the appropriate enzymes and energy in the form of ATP (or ATP derivatives such as AMP-PNP) incubated. The formation of the isopeptide bond brings donor and acceptor chromophores into close proximity, allowing FRET (see also FIG. 1). This is detected by means of a fluorescence meter (eg in microtiter plate fluorescence instruments) by exciting the donor chromophore by irradiation of the appropriate wavelength and measuring both donor and acceptor fluorescence as a function of reaction time. The change in the ratio of acceptor fluorescence to donor fluorescence correlates directly with the change in the amount of conjugation. Therefore, this method is suitable ubiquitin-related modifications without further manipulations in vt ^'tro test systems recombine nanten or purified components and / or cell extracts to be analyzed quickly and quantitatively.

In a further aspect, the invention relates to a method of analyzing demodifications of proteins associated with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) an isopeptidase substrate which a target protein covalently linked to a first chromophore of a donor-acceptor pair for FRET (fluorescence resonance energy transfer), and at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair , includes; these being linked together by an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein; and (ii) at least one enzyme which effects cleavage of the isopeptide bond of the isopeptidase substrate; and contacting components (i) and (ii) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of cleaved isopeptidase substrate, ie the conjugate of target protein and ubiquitin-related protein, by determining FRET, in particular the decrease of FRET, between the first and the second chromophore of the donor-acceptor pair. The ubiquitination is an enzymatically catalyzed formation of an isopeptide bond between the C-terminus of the 9-kDa polypeptide ubiquitin with the ε-amino groups in the lysines of acceptor or target proteins. This modification is reversible, ie a deubiquitination can take place in which the ubiquitin moiety can be cleaved off from the target protein by deubiquitinating enzymes (isopeptidases). The terms "ubiquitination" and "deubiquitination" as used herein, therefore, in a narrower sense refer to ubiquitin itself, while with respect to ubiquitin-related proteins in a broader sense, "modifications of proteins with ubiquitin-related proteins" or " Demodifications of proteins associated with ubiquitin-related proteins "is mentioned. By a "ubiquitin-related protein" is meant in a broader sense a protein structurally related to ubiquitin, and its C-terminal end linked by enzymatic reaction by means of an isopeptide bond to ε-amino groups in lysines of the target proteins can. In addition to eg SUMO1 and its homologues SUMO2 and SUMO3, Nedd8 / Rubl, Apgl2 or Ucrp, this also includes ubiquitin itself in a broader sense (for review see Hershko and Ciechanover, 1998, Hochstrasser, 1998, Jentsch and Pyrowolakis, 2000, Melchior, 2000; Yeh et al., 2000). In a narrower sense, this term means such ubiquitin-related proteins to the exclusion of ubiquitin itself.

By a "target protein" is meant herein a protein that can be linked by means of the appropriate enzymes by the formation of an isopeptide bond with ubiquitin or ubiquitin-related proteins. The target protein is usually covalently linked to a first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET), while the at least one ubiquitin-related protein is covalently linked to a second chromophore of the donor-acceptor pair. the Persons skilled in the art are aware of suitable methods for achieving a covalent linkage. By a "chromophore" herein is meant the "coloring" group of a molecule, that is, the portion of a molecule responsible for light absorption and emission due to its electronic transitions. Chromophores can be both low molecular weight substances, for example the indocyanine dyes Cy3, Cy3.5, Cy5, Cy7 (available from Amersham International plc, GB) or also fluorescent proteins, such as certain GFPs ("Green Fluorescent Protein") and mutant GFPs (available, for example, from Clontech Laboratories Ine, Palo Alto, Calif., USA). Preferably, the first chromophore is the FRET donor CFP ("Cyan Fluorescent Protein") and the second chormophore is the FRET acceptor YFP ("Yellow Fluorescent Protein"). In fluorescence resonance energy transfer (FRET), upon overlap of the emission spectrum of a chromophore (donor chromophore or FRET donor) with the absorption spectrum of the second chromophore (acceptor chromophore or FRET acceptor) after excitation of the donor Chromophors are excited by energy transfer and the acceptor chromophore, if both chromophores are in the immediate vicinity. FRET is detectable by the decrease / loss of emission of the donor chromophore with simultaneous emission of the acceptor chromophore. In the life sciences, a variety of chromophores are used as donor-acceptor pairs for the FRET technology, and thus they can also be used for the methods described below. By way of example, fluorescein and rhodamine are here to be mentioned, which are commercially available in activated form and can be coupled to proteins according to standard protocols; and CFP and YFP, which can be generated by standard techniques of molecular biology and biochemistry as fusions with other proteins (plasmids encoding YFP and CFP proteins are also commercially available). By "isopeptidase substrate" herein is meant a substrate for an enzyme which removes ubiquitin or a ubiquitin-related protein from a target protein linked to ubiquitin or a ubiquitin-related protein by cleavage of the isopeptide bond. Typically, this includes a target protein covalently linked to a first chromophore of a donor-acceptor pair for FRET and at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair in particular, these being linked to one another by an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein.

In providing the test system and contacting the ingredients under conditions which permit the enzymatic reaction to proceed, unless otherwise stated, it is not important that the ingredients be present as separate ingredients or in a particular order mixed or brought into contact with each other. Rather, it should be ensured that the respectively required constituents of the respective test system are present, and that the contacting of these constituents takes place in such a way that the enzymatic reaction can proceed and a FRET measurement can be carried out. If ATP and / or an ATP derivative are required for this purpose, the latter may be the derivatives known to the person skilled in the art, in particular AMP-PNP or ATPγS (for example from Roche Diagnostics). It will be apparent to those skilled in the art, in light of the present disclosure, how the conditions suitable for the reaction under study, e.g. in terms of temperature or pH.

In a preferred embodiment, the target protein and / or the ubiquitin-related protein can be used in the form of a fusion protein with a fluorescent protein. The at least one target protein is preferably mouse or human RanGAP1 (SWISS-PROT: P46061; SWISS-PROT: P46060) and the at least one ubiquitin-related protein for SUMO (small ubiquitin-related modifier), in particular SUMO1, SUMO2 and / or SUMO3. The accession numbers (SWISS-PROT) for the human SUMO proteins are as follows: Q93068 (SUMO1); P55854 (SUMO2) and P55855 (SUMO3). These proteins are identical for the mouse (for a review, see also Melchior, 2000). Advantageously, RanGAP1 and / or CFP may be used in the form of a fusion protein, in particular wherein the C-terminal domain of RanGAP1 is fused to the N- or C-terminal end of CFP. Similarly, SUMO and / or YFP can be used in the form of a fusion protein, particularly where amino acids 1-91 of SUMO1, amino acids of 1-92 of SUMO2, or amino acids 1-93 of SUMO3 are attached to the C-terminal end of YFP are merged.

Conveniently, the target protein, the ubiquitin-related protein and / or the respective enzymes can be used in the form of a purified protein (partial or to homogeneity) and / or a cell extract. This may in particular be an extract of mammalian, insect, yeast and / or bacterial cells, which the person skilled in the art can prepare by means of suitable techniques. This may in particular also be a derivative of the target protein, the ubiquitin-related protein and / or the enzymes. The term "derivative" is understood here to mean a protein or polypeptide which has a sequence homology, in particular a sequence identity to the abovementioned proteins of at least 50%, such as at least 60%, 65%, 70%, 75%, 80%, 85%. , 90% or even 95%. Furthermore, these include deletions of the respective proteins as well as substitutions and additions (eg K48R ubiquitin, which can still be linked to target proteins, but no longer forms the ubiquitin ubiquitin chains linked via lysine 48. This mutant thus offers a clear advantage over wild-type -Ubiquitin in the FRET analysis). Accordingly, the present disclosure also encompasses nucleic acids which are suitable for the above-mentioned encode proteins or polypeptides. Examples of such related nucleic acids are nucleic acids from different human cells or tissues or allelic variants, as well as nucleic acids which can originate from different human individuals. In a broader sense, a nucleic acid encoding such a derivative means a nucleic acid having a homology, in particular a sequence identity to a nucleic acid encoding a protein mentioned above of at least 50%, such as at least 60%, 65%, 70%, 75 %, 80%, 85%, 90% or even 95%. Suitable techniques and methods for the production and mutagenesis of nucleic acids as well as for gene expression and protein analysis are available to the person skilled in the art (see the following manuals: 1. Molecular Cloning: A Laboratory Manual (3rd Edition), Sambrook and Russell (2001) Spring Harbor Press, 2. Current Protocol in Protein Science, Coligan, JE, et al., (Quarterly updated), published by John Wiley & Sons, 3. Current Protocols in Molecular Biology, Ausubel FM, et al., Updated Quarterly. Publisher John Wiley & Sons.)

With regard to the enzymes contained in the particular test system, those of ordinary skill in the art will readily recognize which enzymes are each suitable for facilitating the completion of the corresponding enzymatic reaction (for review, see eg Hershko and A. Ciechanover, 1998; Yeh et al., 2000; Jentsch and Pyrowolakis, 2000.). By way of illustration, for example, in the analysis of modifications of proteins with ubiquitin-related proteins, these are the corresponding E1 and E2 enzymes, optionally in combination with at least one E3 enzyme. For the ubiquitin-related protein SUMO these are in particular the Aosl / Uba2 heterodimer as El enzyme and the Ubc9 protein as E2 enzyme. If an as yet unidentified E3 enzyme exists, this can be isolated and identified, for example, by means of the method described in this application, and then optionally used in the proposed test systems. In the case of deubiquitination analysis, the enzyme is one of the numerous ubiquitin isopeptidases (Review article by Chung and Baek, 1999). Proteins linked to the ubiquitin-related proteins SUMO are, for example, the previously known SUMO-specific isopeptidases Ulpl, Ulp2, Suspl and / or Senpl (original review of the cloning of SUMO-specific isopeptidases is reviewed in the review article Melchior Cited in 2000). If hitherto unidentified isopeptidases exist, they can be isolated and identified, for example, by means of the method described in this application, and then optionally used in the proposed test systems.

In a further aspect, the present invention relates to a method for determining substances that affect the modification of a target protein with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) at least one target protein, covalently linked to a first chromophore of a donor-acceptor pair for FRET (ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair (iii ) Enzymes which cause the formation of an isopeptide bond between the at least one target protein and the at least one ubiquitin-related protein, (iv) ATP and / or an ATP derivative, and (v) at least one substance to be tested; and contacting components (i) - (v) under conditions which allow the enzymatic reaction to proceed; and (b) detecting the formation of conjugate of target protein and ubiquitin-related protein and / or determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair. In a preferred embodiment, the enzymes of step (a) (iii) are the corresponding E1 and E2 enzymes, optionally in combination with at least one E3 enzyme. In the case of SUMO, the El enzyme is the Aosl / Uba2 heterodimer and the E2 enzyme is the Ubc9 protein. The at least one substance to be tested is a protein or polypeptide, in particular an E3 ligase, isopeptidase, kinase or phosphatase, or a derivative as defined above thereof, or a low-molecular substance, in particular an inhibitor or activator. The "low molecular weight substance" may be any inorganic or organic molecule or ion which is tested for its ability to influence the enzymatic reaction in the described test system, in particular to activate or inhibit this reaction. In particular, the low molecular weight substance has a molecular weight of at most 2500, in particular at most 2,250, 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,250, 1,200, 1,100, 1,000, 900, 800, 750, 700, 600, 500 , 450, 400, 350, 300, 250 and 200. For example, these may be molecules having a linear structure such as peptides, in particular oligopeptides, peptoids, linear oligosaccharides, nucleotides, in particular oligonucleotides, and their analogs, or, for example Monomers, such as heterocycles, in particular nitrogen heterocycles, or molecules with a nonlinear structure, such as branched oligosaccharides. Low molecular weight inorganic compounds may be, for example, arsenic and antimony compounds.

In view of the present description, it will be apparent to those skilled in the art that the protein or derivative thereof is from an expression library (Büssow et al., 2000) and / or the low molecular weight substance or a combinatorial library, especially a combinatorial peptide, non-peptide or "drug-like small molecule" bank (eg from Tecnogen SCpA Piana di Monte Verna, CE, Italy; ChemBridge Corporation, San Diego, USA; or Advanced ChemTech Europe Ltd, Cambridgeshire, UK) or from a cell extract can be present and / or in purified form, partially or to homogeneity. The person skilled in the art is familiar with suitable combinatorial strategies for screening, in particular "high throughput screening", of substances to be tested, in particular the use of combinatorial libraries which may comprise 100, 1000 or even 1,000,000 different compounds (see, for example, Meyers , Encyclopedia of Molecular Biology and Molecular Medicine, 1997). Another aspect relates to a method for determining substances that affect the demodification of a protein associated with at least one ubiquitin-related protein, the method comprising the steps of: (a) providing a test system comprising (i) a An isopeptidase substrate comprising a target protein covalently linked to a first chromophore of a donor acceptor pair for fluorescence energy transfer (FRET) and at least one ubiquitin-related protein conjugated to a second chromophore of the donor-acceptor pair covalently linked; these being linked to one another by an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein; (ii) at least one enzyme which effects cleavage of the isopeptide bond of the isopeptide substrate; and (iii) at least one substance to be tested; and contacting the components (i) - (iii) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of cleaved isopeptidase substrate, ie the conjugate of target protein and ubiquitin-related protein, by determining FRET, in particular the decrease of FRET, between the first and the second chromophore of the donor-acceptor pair. In a particular embodiment, the enzyme according to step (a) is an isopeptidase, in particular Ulpl, Ulp2, SUSP1 and / or Senpl (for a review see, for example, Melchior, 2000).

The at least one substance to be tested can be a protein or polypeptide, in particular a binding partner for the target protein, a kinase or phosphatase or a derivative thereof or a low molecular weight substance, in particular an inhibitor or activator, as defined above. Suitable quantitative and qualitative tests, for example ELISA methods, co-immunoprecipitation, microcalorimetry, etc., are available to the person skilled in the art in order to determine whether the protein or polypeptide is nonspecific or specific the target protein binds and thus represents a binding partner for the target protein (various techniques are described, for example, in Current Protocol in Protein Science, Coligan, JE et al., (updated quarterly), John Wiley & Sons.). In particular, herein binding is considered to be specific if the binding affinity is at least 10 ^-6 M, preferably 10 ^-7 , 10 ^-8 , 10 ^-9, or 10 ^-10 M. As described above, suitable methods and materials, eg combinatorial libraries, will be apparent to those skilled in the art , for screening, in particular HTS screening, of substances to be tested.

In a particular embodiment of the two aforementioned methods for determining substances which influence the modification / demodification, the first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein, in particular the chromophore is the FRET donor CFP ("Cyan Fluorescent Protein ") and the second chromophore of the FRET acceptor YFP (" Yellow Fluorescent Protein "). Conveniently, the target protein and / or ubiquitin-related protein can be used in the form of a fusion protein with a fluorescent protein. The person skilled in the art is familiar with suitable methods for producing such fusion proteins. In a specific embodiment, the at least one target protein is RanGAP1 and the at least one ubiquitin-related protein is SUMO (small ubiquitin-related modifier), in particular SUMO1, SUMO2 and / or SUMO3. Here, it may be preferable to use a fusion protein of RanGAP1 and CFP, in particular wherein the C-terminal domain of RanGAP1 is fused to the N- or C-terminal end of CFP. Similarly, a fusion protein of SUMO and YFP can be used, in particular amino acids 1-91 of SUMO1, amino acids of 1-92 of SUMO2 or amino acids 1-93 of SUMO3 fused to the C-terminal end of YFP. The target protein, the ubiquitin-related protein and / or the enzymes may be used in the form of a purified protein, partially or to homogeneity, or a cell extract. Such an extract can be prepared by means of those skilled in the art are made of mammalian, insect, yeast and / or bacterial cells. For example, if combinatorial libraries are used to test substances for effecting the enzymatic reaction, it may be desirable to verify and / or determine the identity of the substance or substance being tested in a further step. Molecular biology, biochemical and / or biophysical methods, such as protein and / or nucleic acid sequence analysis and eg the various methods of mass spectrometry, such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), are available for this purpose. In certain cases, it may also be desirable to use a derivative of the target protein, the ubiquitin-related protein, and / or the enzymes as defined above, for example, to study specific mutants of the target protein for their modification and / or demodification.

A particular embodiment is a so-called "cell based assay". Here, the contacting of the corresponding components in a cell, in particular a cell outside the human or animal body is effected, with mammalian or yeast cells are preferred. For example, for this purpose, the fusion protein of the target protein and the first fluorescent protein and / or the fusion protein of the ubiquitin-related protein and the second fluorescent protein can be expressed in the cell. The at least one substance to be tested can likewise be expressed in the cell, preferably by transfecting the cell with a cDNA coding for this substance, and / or the cell is incubated with the at least one substance to be tested.

In a further aspect, the present invention relates to a method for detecting a defect in the modification of protein with ubiquitin-related protein or in the demodification of proteins associated with ubiquitin-related proteins. It is probable that defects in the Modification and demodulation of specific target proteins are involved in a variety of diseases, as among the target proteins for this post-translational modification are growth factors, tumor suppressors and proto-oncogenes. Defects in the ubiquitination of target proteins have already been implicated in a number of diseases such as von Hippel Lindau syndrome, Angelman syndrome, or cervical cancer (reviewed in: Schwartz and Ciechanover, 1999, Vu and Sakamoto, 2000) For example, mutations in Parkin , an E3 ubiquitin ligase, contribute to the development of a form of Parkinson's disease.

The method for detecting a defect in the modification of proteins with ubiquitin-related proteins comprises the following steps: (a) providing a test system comprising (i) at least one target protein conjugated to a first chromophore of a donor-acceptor pair for FRET ( Flurescence Resonance Energy Transfer) and (ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair; (iii) at least one cell extract to be tested; and contacting components (i) - (iii) under conditions permitting formation of conjugate of target protein and ubiquitin-related protein; and (b) determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair.

The method for detecting a defect in the demodification of proteins linked to ubiquitin-related proteins comprises the following steps: (a) providing a test system comprising (i) an isopeptidase substrate containing a target protein which is linked to covalently linked to a first chromophore of a donor-acceptor pair for FRET, and at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair; in particular, this being due to an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group the at least one ubiquitin-related protein are linked together; and (ii) at least one cell extract to be tested; and contacting components (i) - (ii) under conditions permitting cleavage of conjugate of target protein and ubiquitin-related protein; and (b) determining the amount of cleaved conjugate by determining FRET, in particular the decrease in FRET, between the first and second chromophore of the donor-acceptor pair.

The cell extract to be tested can each originate from cells of tissue of an individual to be examined and / or cells from cell culture. The person skilled in the art is familiar with suitable methods for producing such cell extracts.

In yet another aspect, a novel method for identifying target proteins for modification with ubiquitin-related proteins is proposed, which comprises the following steps: (a) providing a test system comprising (i) at least one target protein to be tested which is linked to a first (Ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair, (iii) enzymes, the cause the formation of an isopeptide bond between the at least one target protein and the at least one ubiquitin-related protein; (iv) ATP and / or an ATP derivative; and contacting components (i) - (iv) under conditions which allow the enzymatic reaction to proceed; and (b) detecting the formation of conjugate of target protein and ubiquitin-related protein and / or determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair.

This method allows screening for target proteins for certain ubiquitin-related proteins. In this case, the contacting of the preceding constituents of the test system in a cell, in particular in a cell outside of the human or animal body, with a mammalian or yeast cell being preferred. The at least one target protein to be tested can be used in the form of a fusion protein with a first fluorescent protein, preferably a fusion protein with the FRET donor CFP. In a particularly practical embodiment, such a fusion protein originates from an expression library and can be introduced, for example, by means of a cDNA coding for this fusion protein into a cell and expressed therein. In this way, it is readily possible for a person skilled in the art, based on the combinatorial techniques and methods known in the art, to screen a very large number of potential target proteins. It is particularly advantageous if the second chromophore is a second fluorescent protein, preferably the FRET acceptor YFP, and, for example, the at least one ubiquitin-related protein is expressed in the form of a fusion protein with the second fluorescent protein in a cell. An example of a ubiquitin-related protein is SUMO (small ubiquitin-related modifier), in particular SUMO1, SUMO2 and / or SUMO3. Advantageously, a fusion protein comprising SUMO and YFP may be used, wherein in particular amino acids 1-91 of SUMO1, amino acids 1-92 of SUMO2 or amino acids 1-93 of SUMO3 are fused to the C-terminal end of YFP.

The person skilled in the art knows the corresponding enzymes according to step (a) (ii) of the aforementioned process (see also Hershko and Ciechanover, 1998, Hochstrasser, 1998, Jentsch and Pyrowolakis, 2000, Melchior, 2000, Yeh et al., 2000). So he will use appropriate El and E2 enzymes, optionally in combination with at least one E3 enzyme. In the case of SUMO, the El - enzyme can be the Aosl / Uba2 heterodimer and the E2 enzyme the Ubc9 protein.

Conveniently, the target protein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein, either partially or purified to homogeneity, or a cell extract, with an extract of mammalian, insect, yeast and / or bacterial cells being preferred.

The at least one target protein to be tested may also be a derivative, as defined above, of a known target protein, for example certain mutants. Alternatively or alternatively, an appropriately defined derivative of the ubiquitin-related protein and / or the enzymes may also be used. Should it be desired to verify the identity of the target protein (for example, when screening a combinatorial library of mutant target proteins obtained by so-called "DNA shuffling") or to determine the identity of the target protein (eg in the screening of an expression library with potential target proteins), this can be done by means of molecular biological, biochemical and / or biophysical methods known to the person skilled in the art as described above.

In a further aspect, the inventors propose a kit for the detection of modifications of proteins with ubiquitin-related proteins, which comprises the following components: (i) at least one target protein and / or derivative thereof, which is linked to a first chromophore of a donor-acceptor protein. Couple for FRET (Flurescence Resonance Energy Transfer) is covalently linked, and / or a nucleic acid encoding it; and (ii) at least one ubiquitin-related protein and / or derivative thereof covalently linked to a second chromophore of the donor-acceptor pair, and / or a nucleic acid encoding the same; and optionally (iii) enzymes which cause the formation of an isopep tide bond between the at least one target protein and the at least one ubiquitin-related protein; and / or at least one nucleic acid coding for this purpose; and / or (iv) ATP and / or an ATP derivative. Advantageous embodiments of said components are apparent from the foregoing description. In a further aspect, there is provided a fusion protein comprising SUMO and a fluorescent protein, preferably YFP, wherein in particular amino acids 1-91 of SUMO1, amino acids 1-91 of SUMO2 or amino acids 1-93 of SUMO3 with the C- terminal end of YFP are fused. Furthermore, a fusion protein is also proposed which comprises RanGAP1 and a fluorescent protein, preferably CFP, whereby, for example, the C-terminal domain of RanGAP1 may be fused to the N- or C-terminal end of CFP. Nucleic acids encoding the fusion proteins are also provided. Furthermore, a cell is provided, in particular a mammalian cell outside the human or animal body or a yeast cell which expresses one or both of the aforementioned fusion proteins.

In another aspect, there is provided an isopeptidase substrate comprising a targeting protein covalently linked to a first chromophore of a donor-acceptor pair for FRET (Fluorescent Resonance Energy Transfer), and a ubiquitin-related protein that modifies the targeting protein and covalently linked to a second chromophore of the donor-acceptor pair, wherein the target protein and the ubiquitin-related protein are linked via at least one isopeptide bond. The latter two proteins may also be derivatives as defined above. In particular, the at least one isopeptide bond is between at least one ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the ubiquitin-related protein. In a preferred embodiment, the ubiquitin-related protein is SUMO, in particular SUMO1, SUMO2 or SUMO3. The target protein for SUMO may be one of the following proteins, or still to be identified Protein: RanGAPl, RanBP2, PML, SplOO, I _κ B _α, D. melanogaster Dorsal, ρ53, c-Jun, HIPK2, S. cere- visiae CDC3 , S. cerevisiae Cdcll, S. cerevisiae Sep7 / Shsl, hCMV BEI, hCMV IE2, D. melanogaster Tramtrack, topoisomerase I, Glutl, Glut4 and Mdm2 (see Melchior, 2000, Table I). In a particular embodiment, the first one The first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein, preferably wherein the first chromophore is the FRET donor CFP ("cyan fluorescent protein") and the second chromophore is the FRET acceptor YFP ("yellow fluorescent protein"). In particular, the target protein and / or ubiquitin-related protein is used in the form of a fusion protein with a fluorescent protein, eg in the form of CFP-RanGAP1 and / or YFP-SUMO.

In yet another aspect of the invention, the inventors propose the use of a kit, a fusion protein, and / or a cell, each as described above, for the analysis of modifications of proteins with ubiquitin-related proteins, for the determination of materials which inhibit the proteins Modification of a target protein with ubiquitin-related proteins, for the identification of target proteins for the modification with ubiquitin-related proteins and / or for the diagnosis, in particular for the detection of a defect in the modification of proteins with ubiquitin-related proteins before. Similarly, the use of the described isopeptidase substrate for the analysis of demodifications of proteins with ubiquitin-related proteins, for determining substances which influence the demodification of a target protein with ubiquitin-related proteins, and / or for the diagnosis, in particular for the detection of a Defect in the demodification of proteins with ubiquitin-related proteins provided.

Finally, methods for preparing a pharmaceutical composition are provided. The first method comprises, in a first step, the steps described above for determining substances that (i) affect the modification of a target protein with ubiquitin-related proteins, or (ii) the demodification of a protein that associates with at least one ubiquitin-related protein and, in a second step, mixing the tested and / or identified substance with a pharmaceutically acceptable carrier known to those skilled in the art (see, for example, Sucker et al. 1991). The second method comprises in a first step the steps described above for identifying a target protein for ubiquitin-related proteins, and in a second step mixing the identified target protein or a derivative thereof with a pharmaceutically acceptable carrier. The present disclosure thus provides effective methods of identifying agents, compounds, or so-called "lead compounds" for drugs that directly or indirectly affect the modification or demodification with ubiquitin-related proteins. The identified substances and / or target proteins may be used by automated and cost-effective HTS methods to screen combinatorial libraries for providing effective components of drugs. The latter are then optimized by conventional methods for their activity and minimal toxicity. Clinical applications include the treatment of viral diseases, neurodegenerative diseases, tumors, inflammation and immune system diseases.

The embodiments described above may be used either alone or in combination with other embodiments.

The following figures and examples are intended to explain the invention in more detail, without limiting it thereto. The following claims are hereby incorporated by reference into the text of this specification.

Description of the figures

1 shows the principle and possible applications of the test methods for the determination of post-translational modifications or demodifications with ubiquitin-related proteins.

Ubiquitin-related proteins are covalently attached to a variety of cellular proteins in enzyme-mediated reactions. At least one isopeptide bond between the ubiquitin formed related protein and the target protein. Isopeptidases can cleave this bond again. Utilizing "Fluorescence Resonance Energy Transfer" (FRET), both the linkage (modification) and the cleavage (demodification) in solution can be studied in solution. These test methods are suitable in principle for the analysis of all known pairs of target protein and ubiquitin-related proteins, and can be used, for example, for: 1. The identification of modified and demodified enzymes, as well as other factors that influence this modification / demodification (binding partner , Kinases, phosphatases, etc). 2. The identification of pharmaceutically relevant substances that inhibit or activate the modification / demodification of specific target proteins. 3. The identification and analysis of mutants of the target proteins, ubiquitin-related proteins and the required enzymes. 4. Identification of new target proteins for a known ubiquitin-related protein

Protein. 5. Diagnosis.

Fig. 2 shows the reconstitution of SUMO 1 modification with fluorescent fusion proteins and recombinant enzymes.

Standard: the El enzyme heterodimer His-Aosl / Uba2, the E2 enzyme Ubc9, CFP-RanGAP (amino acids (As.) 400-589), and YFP-SUMO1 (As. 1-97) were prepared in the presence of ATP Incubated for 30 min at 30 ° C, then mixed with sample application buffer, separated by SDS-PAGE and by immunoblot staining with anti-RanGAPl-

Antibodies (upper part of Fig. 1), or analyzed with anti-GFP antibodies (lower part).

ATP: as standard, but without ATP. + SUMO: as standard, but with excess of nonfluorescent SUMO1.

- Ubc9: as standard, but without Ubc9 addition.

- El: as standard, but without the addition of Aosl / Uba2. ** indicates the isopeptide bond.

Fig. 3 shows the detection of SUMO 1 modification of fluorescent fusion proteins by FRET.

The El enzyme heterodimer His-Aosl / Uba2, the E2 enzyme Ubc9, CFP-

RanGAP (aa 400-589) and YFP-SUMO1 (aa 1-97) were mixed in microtiter plates (reaction volume 25 μl) and the modification reaction started by addition of 1 mM ATP. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm versus time. It is the primary data, i. Emissions at 485 and 527 nm (Figure 3A), and the processed data, i. the quotient of emissions at 527 nm and 485 nm (Figure 3B).

Fig. 4 shows the analysis of inhibitors of modification in the FRET test system.

His-Aosl / Uba2, Ubc9, CFP-RanGAP (Aa 400-589), and YFP-SUMO1 (Aa 1-97) were mixed in microtiter plates (reaction volume 25 μl). In addition to the samples in FIG. 4A, increasing amounts of nonfluorescent RanGAP1 have been added as a competitor; to

Samples in Fig. 4B were given increasing amounts of Gst-RanBP2 (aa 7634-8806) which binds to the enzyme Ubc9 and this inhibited by. By addition of 1 mM ATP, the modification reactions were started. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm versus time. The addition of the competitor (see Fig. 4A) changes the rate and maximum magnitude of the resulting signal. The addition of the non-competitive inhibitor (see Fig. 4B) alters the rate but not the maximum magnitude of the resulting signal.

Fig. 5 shows the analysis of isopeptidase activity in the FRET test system.

In each case 25 μl of isopeptidase substrate, consisting of the conjugate of CFP-RanGAP (aa 400-589) and YFP-SUMO1 (aa 1-97), were incubated with recombinant isopeptidase (Gst-Ulpl) at 37 ° C. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm versus time. The cleavage of the isopeptide bond leads to a decrease in the FRET signal.

Examples

1. Preparation of Recombinant YFP-SUMO CFP-RanGAP. SUMO1

Activating enzyme Aosl / Uba2, SUMO 1 -conjugating enzyme Ubc9 and isopeptidase Ulpl

The 20 kDa C-terminal domain of the SUMO 1 target protein RanGAP1 (mouse, As, 400-589; GenBank: U08111; Mahajan et al., 1997) was inserted into the pE vector. CFP (Clontech), expressed as cyan fluorescent protein (CFP) fusion protein in E. coli and partially purified via DEAE-Sepharose and gel filtration.

SUMO1 (human, As. 1-97; GenBank: U67122; Mahajan et al., 1997) was cloned into the vector pE-YFP (Clontech), expressed as Yellow Fluorescent Protein (YFP) fusion protein in E. coli and transduced therefrom DEAE sepharose and gel filtration partially purified.

The coding region of Ubc9 (mouse, GenBank: U94402) was cloned into the Ndel and BamHI sites of the vector pET23a (Novagen, Madison, USA), expressed in E. coli and purified by S-Sepharose and gel filtration. The El heterodimer Aos1 / Uba2 (human Uba2, GenBank: AF090384, human Aos1, GenBank: NM_005500) was obtained by simultaneous expression of both subunits in E. coli from the plasmids pETI1d (Uba2) and pET28a (Aos1) (plasmids from Novagen, Madison , USA). Aosl was expressed with an N-terminal His6 tag. Purification was first by binding and elution of ProBond ™ Resin (Invitrogen, Groningen, The Netherlands), then by gel filtration followed by chromatography on Q-sepharose.

Ulpl was expressed as a Gst fusion protein in E. coli and purified by glutathione sepharose and gel filtration. The expression plasmid (Li and Hochstraser, 1999) was used by Dr. med. Mark Hochstrasser, Yale, USA.

in vttro modification reaction

The recombinant proteins Aosl / Uba2, Ubc9, YFP-SUMO1 and CFP-RanGAP described under 1. were combined in 386-well microtiter plates, ATP was added to initiate the reaction, and the fluorescence meter Fluoroskan Ascent FL from the company Labsystems GmbH (Frankfurt, Germany) at 38 ° C for up to 120 minutes every 30 or 60 seconds measured. In this case, light of wavelength 430 nm (optimum excitation wavelength for CFP) was inserted. radiates and the fluorescence at the wavelengths 485 nm (maximum of the emission of CFP) and 527 nm (maximum of the emission of YFP) measured. Alternatively, the samples were incubated for 30 min, taken in SDS-PAGE sample application buffer, and by Western blot analysis with anti-RanGAPl (Mahajan et al., 1997) or anti-GFP antibodies (available from Santa Cruz Biotechnology, Santa Cruz , USA).

3. in v / tro-demodification reaction (isopeptidase test)

Isopeptidase substrate consisting of the conjugate of CFP-RanGAP (As. 400-589) and YFP-SUMO1 (or YFP-SUMO2) was generated by first recombining the recombinant proteins Aosl / Uba2, Ubc9, YFP- described in 1. SUMO1 (or YFP-SUMO2) and CFP-RanGAP were incubated in the presence of ImM ATP at 30 ° C for 30 min, and the reaction mixture after linking YFP-SUMO and CFP-RanGAP via gel filtration of remaining ATP was freed. [In view of this disclosure, one skilled in the art will be able to prepare other isopeptidase substrates from the appropriate ingredients in a similar manner.]

In each case 25 .mu.l of this isopeptidase substrate were mixed in 386-well microtiter plates with increasing concentrations of the isopeptidase Ulpl, and in the fluorescence meter Fluoroskan Ascent FL from Labsystems (company, town, state) at 37 ° C for up to 120 minutes all Measure 60 seconds. In this case, light of wavelength 430 nm (optimal excitation wavelength for CFP) was irradiated and the fluorescence at the wavelengths 485 nm (maximum of the emission of CFP) and 527 nm (maximum of the emission of YFP) measured. Alternatively, the samples were incubated for 30 min, incubated in SDS-PAGE sample application buffer, and by Western blot analysis with anti-RanGAPI (Mahajan et al, 1997) or anti-GFP antibodies (available from Santa Cruz Biotechnology, Santa Cruz, USA). Quantitative detection of the modification / demodulation of a ubiquitin-related protein and the influence of inhibitors on this reaction

The general applicability of the FRET-based assay is demonstrated below with a specific pair of "target protein" and "ubiquitin-related protein."

RanGAP1, a protein involved in the transport of nuclear proteins, is covalently linked to the ubiquitin-related protein SUMO1 (Mahajan et al., 1997; Mahajan et al., 1998). This modification alters the cytoplasmic localization of the unmodified RanGAP1 by allowing a stable association of the RanGAP1 with the nuclear pore complex protein RanBP2. Modification of RanGAP1 with SUMOI can be reconstituted in vitro by using either mammalian cell extracts or purified (recombinant) enzymes as the enzyme source and starting and measuring the modification reaction as shown in Example 2. As demonstrated by Western blot analysis (Figure 2), the CFP-RanGAP fusion protein was quantitatively linked to the YFP-SUMO1 fusion protein. This reaction was dependent on energy (ATP was used here), Ubc9, and the heterodimer Aosl / Uba2. The addition of an excess of unfused SUMO (As. 1-97) prevents the association with YFP-SUMOl by competition.

The appearance of FRET is characterized by loss of acceptor fluorescence with concomitant increase in donor fluorescence, as can be observed after combining all of the components described in Example 1 and ATP, whereas FRET does not occur in the absence of ATP. FIG. 3A shows the actually measured data, and FIG. 3B shows the quotients of the emis- Increases in the quotient correlated strictly with the appearance of conjugation under all circumstances tested, and is therefore dependent on the presence and concentration of the appropriate enzymes (either recombinant or in the form of cell extracts), and energy ( in the form of ATP or other useful nucleotides).

For example, a FRET signal could be relieved by an inhibitor of the enzymes (e.g., N-ethylmaleimide) or nonfluorescent competitors (e.g., recombinant RanGAPl or SUMOI). An already generated FRET signal could be destroyed if, after removal of ATP by the hexokinase and glucose, cell extracts with isopeptidase activity were added. 4 shows by way of example the signal changes as a function of a competitive inhibitor (RanGAP1, FIG. 4A) and of a noncompetitive inhibitor (Gst-RanBP2, FIG. 4B), which very stably binds to Ubc9 and thereby deprives it of the reaction). In addition, this method was used to screen mutants of SUMO1 and RanGAP1 for their conjugation ability by adding the mutants as nonfluorescent competitors. Conjugating mutants reduced / prevented FRET, whereas certain non-conjugating mutants did not affect FRET.

The reverse reaction, that is, the cleavage of the isopeptide bond by isopeptidases, leads to the decrease of the FRET signal. Fig. 5 exemplifies the demodification by the yeast isopeptidase Ulpl. Furthermore, the isopidotidase activity in total extracts and in cell fractions of HeLA cells could be quantitatively determined by the FRET method (data not shown).

Taken together, these data demonstrate the suitability of the FRET test methods for the kinetic analysis of the modification and / or the demodification of RanGAPl with SUMOl and SUMO2. In principle, therefore, can be build up such assays for each pair of target protein and ubiquitin-related protein. Care should be taken to bring the donor and acceptor chromophores into sufficient proximity for FRET by covalent linkage through appropriate choice of chromophores, protein domains, and chromophore linkage. In view of the present disclosure, those skilled in the art will readily recognize the appropriate materials and methods.

5. Determine proteins that influence the modification of a known target protein

The above-demonstrated in vitro assays, but in principle also methods in vivo, can be used to systematically identify proteins that inhibit or activate the modification of a defined ubiquitin-related protein with a defined target protein. In addition to putative E3 ligases and isopeptidases, these include kinases, phosphatases and other binding partners.

In Vitro Procedure: This can be done, for example, by presenting all components to be provided for in vitro modification, including the acceptor-labeled ubiquitin-related protein and the corresponding donor-labeled target protein in microtiter plates, and unknown recombinant proteins, eg, from expression libraries in "High Throughput Screening" (HTS) methods (eg when using N-terminal His-tags on the fusion proteins), or added to cell extract fractions or purified proteins. Changes in the reaction kinetics indicate an influence of the unknown protein on the modification. In vtvo methods: For example, the identification of such proteins in vivo can be carried out by transfecting a cell line which expresses the acceptor-labeled ubiquitin-related protein and the donor-labeled target protein simultaneously or transiently with cDNAs which have been transfected Lead expression of unknown proteins. The decrease or enhancement of the (steady state) FRET signal indicates an influence of the protein to be tested on the modification.

6. Identification of low molecular weight substances that influence the modification of a known target protein

The in vivo and in vitro test methods described in Example 5 can equally be used to systematically identify substances that inhibit or activate the modification of a defined ubiquitin-related protein with a defined target protein.

In vitro methods: this can be done, for example, by providing all components to be provided for in vitro modification, including the acceptor-labeled ubiquitin-related protein and the corresponding donor-labeled target protein in microtiter plates, and unknown substances, e.g. from a so-called combinatorial "Small Molecule Library", be tested in the HTS process for an effect on the reaction kinetics.

In vivo methods: The identification of such substances in vivo can be carried out, for example, by incubating a cell line which expresses the acceptor-labeled ubiquitin-related protein and the donor-labeled target protein simultaneously or transiently with these substances. The decrease or enhancement of the FRET signal indicates an influence of the substance to be tested on the modification. 7. Analysis and / or identification of mutants

The in vivo and in vitro test methods described above may equally be used to analyze and / or identify mutants of known ubiquitin-related proteins, known target proteins, and / or the modifying / demodifying enzymes.

8. Use of the test procedures in diagnostics

Variants of the test methods described above can be used in diagnostics. This is e.g. desirable when defects in the modification of a particular (target) protein contribute to certain diseases or are associated with certain diseases and / or disorders. For example, the ability of various cell extracts (eg made from tissue or from cell cultures) to modify a particular target protein can be quantified by incubating the corresponding pair of FRET donor and FRET acceptor-labeled proteins with defined amounts of an extract to be tested and the occurrence of the FRET signal is determined time-dependently.

9. Identification of Target Proteins for Modification with Ubiquitin-Related Proteins

The in vivo and in vitro assays described herein may be used to systematically identify novel target proteins for the modification of ubiquitin-related proteins by substituting a known unknown and / or unidentified donor chromophore-labeled proteins to be tested, for example from an expression library or a combinatorial bank, in combination with the acceptor CmOmophore-labeled ubiquitin-related protein.

Identification by means of in / tro methods: This may be e.g. all of the ingredients to be provided for in vitro modification, including a YFP-labeled ubiquitin-related protein in microtiter plates, and unknown recombinant CFP fusion proteins are added thereto. FRET signals should arise here only if the unknown protein is covalently linked to the YFF-labeled ubiquitin-related protein (in this case, FRET arises only as a function of energy and enzyme addition) or does not interact covalently with it (energy- and enzyme-independent) , For example, unknown recombinant CFP fusion proteins can be obtained from expression libraries in HTS procedures (e.g., using N-terminal His tags to the fusion proteins).

For example, this can be done by transfecting cells that stably or transiently express the YFP-ubiquitin-related protein with cDNAs that result in the expression of unknown proteins fused to CFP. The analysis then takes place, for example, in microtiter plate fluorescence measuring devices and / or with fluorescence microscopes which are equipped with the corresponding FRET filters. The appearance of FRET signals indicates the linkage of the unknown protein to the known ubiquitin-related protein. This can additionally be independently verified by appropriate controls (eg mobility change in the SDS gel and use of mutant proteins that can no longer be linked). For vtvo modification, all cells containing the enzymes suitable for the linkage can be considered (endogenous or due to manipulations). Ubiquitination or modification with SUMO and its Homologs can therefore be studied, for example, both in established cultures of mammalian cells and in the baker's or fission yeast.

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Claims

claims 1. A method of analyzing modifications of proteins with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) at least one target protein covalently linked to a first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET), (ii) at least one ubiquitin-related protein that binds with covalently linked to a second chromophore of the donor-acceptor pair; (iii) enzymes which cause the formation of an isopeptide bond between the at least one target protein and the at least one ubiquitin-related protein; (iv) ATP and / or an ATP Derivative, and contacting the components (i) - (iv) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of target protein-U-biquitin-related protein conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor protein; Pair. 2. A method of analyzing demodifications of proteins linked to U-biquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) an isopeptidase substrate which is a target protein covalently linked to a first chromophore of a donor-acceptor pair for FRET (Fluoescence Resonance Energy Transfer), and at least one ubiquitin-related one Protein bound to a second chromophore of the donor Acceptor pair is covalently linked; these are characterized by an isopeptide bond is linked between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein; and (ii) at least one enzyme which effects cleavage of the isopeptide bond of the isopeptidase substrate; and contacting the ingredients (i) and (ii) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of cleaved isopeptidase substrate by determining FRET between the first and second chromophore of the donor-acceptor pair. The method of claim 1 or 2, wherein the first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein, in particular wherein the first chromophore is the FRET donor CFP (cyan fluorescent protein) and the second chromophore is the FRET acceptor YFP (Yellow Fluorescent Protein) is. 4. The method according to any one of claims 1-3, wherein the target protein and / or the ubiquitin-related protein are used in the form of a fusion protein with a fluorescent protein. 5. The method according to any one of the preceding claims, wherein the at least one target protein RanGAPl and the at least one ubiquitin-related protein is SUMO (small ubiquitin-related modifier), in particular SUMO 1, SUMO2 and / or SUMO3. 6. The method according to any one of the preceding claims, wherein RanGAPl and CFP are used in the form of a fusion protein, in particular wherein the C-terminal domain of RanGAPl is fused to the N- or C-terminal end of CFP. 7. Method according to one of the preceding claims, wherein SUMO and YFP are used in the form of a fusion protein, in particular wherein the amino acids 1-91 of SUMO1, the amino acids 1-92 of SU-MO2 or the amino acids 1-93 of SUMO3 with the C terminal end of YFP are fused. 8. The method according to any one of the preceding claims, wherein the target protein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein and / or a cell extract, in particular an extract from mammalian, insect, yeast and / or or bacteria cells. 9. Method according to one of the preceding claims, wherein a derivative of the target protein, the ubiquitin-related protein and / or the enzymes is used. 10. The method according to any one of Ansprächen 1, 3-9, wherein the enzymes according to step (a) (iii) of claim 1 are the El and E2 enzymes, optionally in combination with at least one E3 enzyme, in particular wherein the El Enzyme is the Aos 1 / Uba2 heterodimer and the E2 enzyme Ubc9. 11. The method according to any one of claims 2-9, wherein the at least one enzyme according to step (a) of spoke 2 is an isopeptidase (which is with C-terminal hydrolases), in particular Ulpl, Ulp2, SUSP1 and / or Senpl. 12. A method for determining substances that affect the modification of a target protein with ubiquitin-related proteins, said method comprising the steps of: (a) providing a test system comprising (i) at least one target protein conjugated to a first chromophore of a donor acceptor pair covalently linked to FRET (Fluorescence Resonance Energy Transfer), (ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair, (iii) enzymes that see the formation of an isopeptide bond the at least one target protein and the at least one Cause ubiquitin-related protein, (iv) ATP and / or an ATP derivative, and (v) at least one substance to be tested; and contacting components (i) - (v) under conditions which allow the enzymatic reaction to proceed; and (b) detecting the formation of conjugate of target protein and ubiquitin-related protein and / or determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair. 13. The method of claim 12, wherein the enzymes of step (a) (iii) are of the E1 and E2 enzymes, optionally in combination with at least one E3 enzyme, in particular wherein the E1 enzyme is the Aos1 / Uba2 heterodimer and the E2 enzyme is Ubc9. 14. The method according to claim 12 or 13, wherein the at least one substance to be tested is a protein, in particular an E3 ligase, isopeptidase, kinase or phosphatase, or derivative thereof or a low molecular weight substance, in particular an inhibitor or activator. 15. A method of determining substances that affect the demodification of a protein linked to at least one ubiquitin-related protein, the method comprising the steps of: (a) providing a test system comprising (i) an isopeptidase substrate , which is a target protein that interacts with a first chromophore Donor-acceptor pair for FRET (Fluorescence Resonance Energy Transfer), and at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair; these being linked together by an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein; (ii) at least one enzyme which effects the cleavage of the isopeptide bond of the iso-peptide substrate; and (iii) at least one substance to be tested; and contacting components (i) - (iii) under conditions which allow the enzymatic reaction to proceed; and (b) determining the amount of cleaved isopeptidase substrate by determining FRET between the first and second chromophore of the donor-acceptor pair. 16. The method of claim 15, wherein the at least one enzyme according to step (a) is an isopeptidase, in particular Ulpl, Ulp2, SUSP1 and / or Senpl. 17. Method according to claim 15, wherein the at least one substance to be tested is a protein, in particular a binding partner for the target protein, a kinase or phosphatase, or a derivative thereof, or a low-molecular substance, in particular an inhibitor or activator. 18. The method according to any one of claims 12-17, wherein the protein or derivative thereof and / or the low molecular weight substance from an expression library or a combinatorial bank, in particular a combinatorial peptide, non-peptide or small molecule bank, or comes from a cell extract and / or is present in purified form. 19. A method according to any of claims 12-18, wherein the first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein fluorescent protein, in particular wherein the first chromophore is the FRET donor CFP (cyan fluorescent protein) and the second chromophore is the FRET acceptor YFP (yellow fluorescent protein). 20. The method according to any one of claims 12-19, wherein the target protein and or the ubiquitin-related protein are used in the form of a fusion protein with a fluorescent protein. 21. The method according to any one of claims 12-20, wherein the at least one target protein is RanGAP1 and the at least one ubiquitin-related protein is SUMO (small ubiquitin-related modifier), in particular SUMO1, SUMO2 and / or SUMO3. 22. The method according to any one of claims 12 - 21, wherein RanGAPl and CFP are used in the form of a fusion protein, in particular wherein the C-terminal domain of RanGAPl is fused to the N- or C-terminal end of CFP. 23. The method according to any one of claims 12-22, wherein SUMO and YFP are used in the form of a fusion protein, in particular wherein the Amino acids 1-91 of SUMO1, amino acids 1-92 of SUMO2 or amino acids 1-93 of SUMO3 are fused to the C-terminal end of YFP. 24. The method according to any one of claims 12 - 23, wherein the target protein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein or a cell extract, in particular an extract of mammalian, insect, yeast and / or bacteria Cells, are used. 25. The method according to any one of claims 12 - 24, wherein the substance to be tested in a further step by suitable methods, in particular molecular kularbiologische, biochemical and / or biophysical method, is identified. 26. The method according to any one of claims 12-25, wherein a derivative of the target protein, the ubiquitin-related protein and / or the enzymes is used. 27. The method according to any one of claims 12-14, and 18-26, wherein the bringing into contact of the components (i) - (iv) is effected in a cell outside the human or animal body, in particular in one Mammalian or yeast cell; in particular, wherein the fusion protein of the target protein and the first fluorescent protein and / or the fusion protein of the ubiquitin-related protein and the second fluorescent protein are expressed in the cell. 28. The method according to claim 27, wherein the at least one substance to be tested is expressed in the cell, preferably by transfecting the cell with a cDNA coding for this substance, and / or wherein the cell is incubated with the at least one substance to be tested. 29. A method of detecting a defect in the modification of proteins with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) at least one target protein covalently linked to a first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET), and (ii) at least one ubiquitin-related protein which is covalently linked to a second chromophore of the donor-acceptor pair; (iii) at least one cell extract to be tested; and contacting the components (i) - (iii) under conditions that the Allow formation of conjugate of target protein and ubiquitin-related protein; and (b) determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair. 30. A method for detecting a defect in the demodification of proteins associated with ubiquitin-related proteins, said method comprising the steps of: (a) providing a test system comprising (i) an isopeptidase A substrate comprising a target protein covalently linked to a first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET) and at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair is, includes; these being linked together by an isopeptide bond between the ε-amino group of a lysine of the target protein and the C-terminal carboxy group of the at least one ubiquitin-related protein; and (ii) at least one cell extract to be tested; and contacting the components (i) - (ii) under conditions which cause the cleavage of Allow conjugate of target protein and ubiquitin-related protein; and (b) determining the amount of cleaved conjugate by determining FRET between the first and second chromophore of the donor-acceptor pair. 31. The method according to claim 29 or 30, wherein the cell extract is derived from cells of tissue of an individual to be examined and / or cells from cell culture. 32. A method of identifying target proteins for modification with ubiquitin-related proteins, the method comprising the steps of: (a) providing a test system comprising (i) at least one target protein to be tested which is coupled to a first chromophore of a donor (Ii) at least one ubiquitin-related protein covalently linked to a second chromophore of the donor-acceptor pair, (iii) enzymes that promote the formation of an isopeptide - effect binding between the at least one target protein and the at least one ubiquitin-related protein; (iv) ATP and / or an ATP derivative; and contacting the components (i) - (iv) under conditions which allow the enzymatic reaction to proceed; and (b) detecting the formation of conjugate of target protein and ubiquitin-related protein and / or determining the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair. A method according to claim 32, wherein the contacting of components (i) - (iv) is effected in a cell external to the human or animal body, especially in a mammalian or yeast cell. 34. The method according to claim 32 or 33, wherein the at least one target protein to be tested in the form of a fusion protein with a first fluorescent protein, preferably as a fusion protein with the FRET donor CFP, is used. 35. The method according to claim 34, wherein the at least one fusion protein originates from an expression bank, in particular wherein one for the fusion Onsprotein coding cDNA is introduced into a cell and expressed therein. 36. The method according to any one of claims 32-35, wherein the second chromophore a second fluorescent protein, preferably the FRET Acceptor YFP is; in particular wherein the at least one ubiquitin-related protein is expressed in the form of a fusion protein with the second fluorescent protein in a cell. 37. The method according to any one of claims 32-36, wherein the at least one ubiquitin-related protein is SUMO (small ubiquitin-related modifier), in particular SUMOl, SUMO2 and / or SUMO3. 38. The method of claim 37, wherein a fusion protein comprising SUMO and YFP is used, in particular wherein amino acids 1-91 of SUMO1, amino acids 1-92 of SUMO2 or amino acids 1-93 of SUMO3 with the C-terminal end are fused by YFP. 39. The method according to any one of claims 32-38, wherein the enzymes according to step (a) (iii) of claim 24, the El and E2 enzymes, optionally in In particular wherein the El enzyme is the Aosl Uba2 heterodimer and the E2 enzyme Ubc9. 40. The method according to any one of claims 32-39, wherein the target protein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein or a cell extract, in particular an extract of mammalian, insect, yeast and / or bacteria Cells, are used. 41. The method according to any of claims 32-40, wherein the at least one target protein to be tested is a derivative of a known Target protein, and / or wherein a derivative of the ubiquitin-related protein and / or the enzymes is used. 42. The method according to any one of claims 32-41, wherein the target protein is identified in a further step by suitable methods, in particular molecular biological, biochemical and / or biophysical methods. 43. Kit for detecting modifications of proteins with ubiquitin-related proteins, comprising: (i) at least one target protein and / or derivative thereof covalently linked to a first chromophore of a donor-acceptor pair for fluorescence resonance energy transfer (FRET), and / or a nucleic acid encoding the same; and (ii) at least one ubiquitin-related protein and / or derivative thereof covalently linked to a second chromophore of the donor-acceptor pair, and / or a nucleic acid encoding the same; as well as optional (iii) enzymes which cause the formation of an isopeptide bond between the at least one target protein and the at least one ubiquitin-related protein; and / or at least one nucleic acid coding for this purpose; and or (iv) ATP and / or an ATP derivative. 44. A fusion protein comprising SUMO and a fluorescent protein, preferably YFP, in particular wherein amino acids 1-91 of SUMO1, amino acids 1-92 of SUMO2 or amino acids 1-93 of SUMO3 are fused to the C-terminal end of YFP are; or a nucleic acid encoding it. 45. A fusion protein comprising RanGAP1 and a fluorescent protein, preferably CFP, in particular wherein the C-terminal domain of RanGAP1 is fused to the N- or C-terminal end of CFP; or a nucleic acid encoding it. 46. Cell, in particular a mammalian cell outside the human or animal body or a yeast cell expressing a fusion protein according to claim 44 and / or 45. 47. An isopeptidase substrate comprising a target protein covalently linked to a first chromophore of a donor-acceptor pair for FRET (Fluorescence Resonance Energy Transfer), and a ubiquitin-related protein that modifies the target protein and a second chromophore of the protein Donor-acceptor pair is covalently linked, wherein the target protein and the ubiquitin-related protein are linked via at least one isopeptide bond. 48. Isopeptidase substrate according to spoke 47, wherein the at least one isopeptide bond between at least one ε-Aminograppe of a lysine of the target protein and the C-terminal carboxy group of the ubiquitin-related protein, in particular wherein the isopeptidase substrate is a derivative of the target protein and / or of the ubiquitin-related protein. 49. Isopeptidase substrate according to claim 47 or 48, wherein the ubiquitin-related protein is SUMO, in particular SUMOl, SUMO2 or SU-MO3. 50. isopeptidase substrate according spoke 49, wherein the target protein of SUMO RanGAPl, RanBP2, PML, SplOO, I _κ B _α, D. melanogaster Dorsal, p53, c- Jun, HIPK2, S. cerevisiae Cdc3, S. cerevisiae Cdcll, S. cerevisiae Sep7 / Shsl, hCMV El, hCMV IE2, D. melanogaster Tramtrack, topoisomerase I, Glutl, Glut4 or Mdm2. The isopeptidase substrate of any of claims 47-50, wherein the first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein, in particular wherein the first chromophore is the FRET donor CFP (cyan fluorescent protein) and the second chromophore the FRET acceptor is YFP (Yellow Fluorescent Protein); in particular wherein the target protein and / or the ubiquitin-related protein are used in the form of a fusion protein with a fluorescent protein, more preferably in the form of CFP-RanGAP 1 and / or YFP-SUMO. 52. Use of a kit according to claim 43, a fusion protein according to claim 44 and / or 45, and / or a cell according to claim 46 for analysis of modifications of proteins with ubiquitin-related proteins, for the determination of substances, the modification of a target protein with ubiquitin-related proteins, for identifying target proteins for modification with ubiquitin-related proteins and / or for diagnosis, in particular for detecting a defect in the modification of proteins with ubiquitin-related proteins. 53. Use of an isopeptidase substrate according to one of the claims 47 - 51 for the analysis of demodifications of proteins with ubiquitin-related proteins, for the determination of substances which influence the demodification of a target protein with ubiquitin-related proteins, and / or for the diagnosis, in particular for Evidence of a defect in the demodification of proteins with ubiquitin-related proteins. 54. A process for the preparation of a pharmaceutical composition comprising the process according to any one of claims 12 - 28, and the Mixing the tested and / or identified substance with a pharmaceutically acceptable carrier. 55. A process for the preparation of a pharmaceutical composition comprising the process of any of claims 32-42, and admixing the identified target protein or a derivative thereof with a pharmaceutically acceptable carrier.