DE10108263A1 - Analysis of modifications and demodifications of proteins with ubiquitin-related proteins using FRET (Fluorescence Resonance Energy Transfer) - Google Patents

Abstract

The invention relates to a method for analysing modifications of proteins with ubiquitin-related proteins and demodification of proteins which are concatenated with ubiquitin-related proteins. The invention also relates to a method for determining substances which influence said modifications or demodifications, in addition to a method for detecting a defect in said modifications or demodifications. Furthermore, the invention relates to a method for identifying target proteins for modification with ubiquitin-related proteins and a method for producing a pharmaceutical composition. The invention further relates to a kit, a fusion protein, a cell and an isopeptidase substrate and the use thereof in the analysis and/or diagnosis of modification or demodification of proteins with ubiquitin-related proteins.

Description

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. The far The present invention relates to methods for determining substances which influence these modifications or demodifications, as well as processes to detect a defect in these modifications or demodifications. Finally, the present invention relates to an identification method of target proteins for modification with ubiquitin-related proteins as also methods of making a pharmaceutical composition. Dar the invention further relates to a kit, a fusion protein, a cell and a Isopeptidase substrate and its uses in analysis and / or diagnosis ics of ubiquitin-related modifications or demodifications of Proteins.

A variety of proteins are found in their activity, location and stability by covalent linkage with ubiquitin or ubiquitin-related proteins such as SUMO1 or Nedd8 (Hershko and Ciechanover, 1998; Hoch strasser, 1998; Jentsch and Pyrowolakis, 2000; Melchior, 2000; Yeh et al., 2000). An isopeptide bond is created in a cascade of enzymatic steps between the carboxy-terminal end of ubiquitin (or the ubiquitin related protein) and the ε-amino group in lysine side chains of the target pro teins knotted. So-called E1-activating enzymes are involved in the linkage, E2 conjugating enzymes and (partly) E3 ligases. Through the activity of  Isopeptidases that can break this bond again are these posttransla tional modifications reversible (Chung and Baek, 1999; Wilkinson, 1997). Ubiquitination (and the subsequent breakdown) and the corresponding Mo Differentiation with ubiquitin-related proteins seems to be cellular for a large number processes such as cell cycle control, signal transduction, intracellular trans port or apoptosis, but also for many pathological scenarios (viral infection NEN, tumor formation, neurodegenerative diseases, etc.) of great importance tung (Bonifacino and Weissman, 1998; Pagano, 1997; Schwartz and Ciechanover, 1999). Both the modification and demodification of many Target proteins are subject to regulation specific to the target protein (e.g. by target protein-specific E3 ligases and isopeptidases and / or by Cell cycle or stress dependent phosphorylation or dephosphorylation). Therefore, diagnosis of the modification / demodification is more specific Target proteins and the inhibition or activation of these reactions as Basis for disease treatment and appropriate diagnostics.

Modification of proteins with ubiquitin or ubiquitin-related pro Stone leads to a significant change in the size of the target protein and can can be analyzed both in vivo and in vitro. So far, the analysis of Modification in vivo usually by Western blot analysis of cell extract with antibodies to the protein under investigation or by immunoprecision pitation of the modified protein and subsequent Western blot analysis with Antibodies to the ubiquitin-related protein. This analysis includes often a previous transfection of the cells with plasmids, either encode the ubiquitin-related protein or the protein to be modified.

An in vitro reconstitution of ubiquitinations or modifications with Ubiquitin-related proteins have so far been carried out by purified or recombinant target proteins, purified or recombinant ubiquitin or ubi quitin-related proteins and the corresponding purified or recombinant  Enzymes (or cell extracts containing the necessary enzymes) in Presence of ATP can be incubated for a defined time. The analysis of the Modification is then carried out by SDS-PAGE, followed by Western Transfer and immunostaining, or (when using radioactively labeled Proteins) by SDS-PAGE followed by autoradiography (see, e.g., Busch mann et al., 2000; Deng et al., 2000; Kaiser et al., 2000).

A method for the detection of inhibitors of ubiquitin-dependent degradation of regulatory proteins of the cell cycle has been described by Kirschner et al. (US 5,726,025) described. This document describes u. a. a quantitative test procedure for the degree of ubiquitination. It is suggested that to provide ubiquitinating protein or ubiquitin with a fusion component or to mark with biotin or a fluorochrome and after the expiration of the Reaction from the cells or the reaction mixture by means of so-called "pull downs" or ELISA-related techniques to remove and the modification after point. A general procedure for the detection of post-translational modifi adornments (including modification with ubiquitin or ubiquitin-related Pro with the help of the so-called "Fluorescence Resonance Energy Transfer" (FRET) was described by Bastiaens and Parker (WO 00/43780). The authors conclude gene to fluorescent mark the target protein and the modification with also a fluorescent labeled probe, which only contains the modified protein recognizes (e.g. an anti-ubiquitin antibody). Here too proposed to use such a method in so-called "drug screenings". Similarly, Boisclair and co-workers suggest using FRET technology for the Search for inhibitors of ubiquitination to use by probes (Strep tavidin and anti-Gst antibodies) with donor and acceptor chromophores fluo be marked resentively (Boisclair et al., 2000). Although this procedure is a Represent simplification compared to the commonly used methods, they still require a significant amount of manipulation, and are  due to the indirect detection methods, very susceptible to faults, poorly in vivo feasible, and at most semi-quantitative.

Therefore, it was an object of the present invention to be quantitative, quick les and especially versatile test system for analytics and diagnostics of post-translational ubiquitin-related modifications or demodifi to provide ornaments that are used both in vitro and in vivo can. Another object underlying the present invention is Provision and use of suitable components of this test system.

It has now surprisingly been found that quantitative and rapid Test procedure for post-translational modifications with both ubiquitin and ubiquitin-related proteins can also be provided. This test process and the proposed universally applicable components for these In contrast to the methods described above, for example, are not al limited to the detection of inhibitors for ubiquitination. Rather, the proposed methods now allow both in vitro and also in vivo so-called "high throughput drug screenings" (hereinafter HTS- Method) and the provision of diagnostic kits for the detection of Defects in the modification / demodification and in particular the Entdec tion of both inhibitors and activators involved in the modification and / or demodification of specific target proteins are involved (e.g. E3- Ligases, isopeptidases, kinases or other binding partners). Beyond that it is also possible for the first time to use new target proteins for modification Discovering ubiquitin-related proteins in the "high throughput" process for which then the corresponding with the help of the proposed procedures appropriate inhibitors and / or activators can be determined what again in order to enable 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 beneficial since it is a seamless chain from identification to characterization which provides analytics and diagnostics.

The proposed test systems take advantage of FRET technology (Periasamy and Day, 1999; Pollok and Heim, 1999) and use in the below written examples of use by Tsien and co-workers described fluorescent proteins "Yellow Fluorescent Protein" (YFP) and "Cyan Fluorescent Protein" (CFP) (US 5,981,200; US 5,998,204; Tsien, 1998). However, none of the aforementioned documents describes the use of FRET for the direct and quantitative analysis of covalent modifications as described herein is proposed.

The term "quantitative" is used to express that in the opposite the signal to be measured directly to other methods described so far is proportional to the degree of the linkage, since neither manipulation after he following reaction, probes are still used to detect the link have to. The ability of probes to recognize the reaction product depends gig of their affinity, as well as of the accessibility of the product and is therefore not linear in every concentration range. In addition, the so far be described procedures susceptible to interference if additional binding partners for the Re actants are present.

A very significant advantage over that of Kirschner et al. proposed The process (see above) is furthermore that reaction sequences ver kinetically ver can be followed. While Kirschner 's analysis of the Degree of linkage of a reaction approach through the required manipulations only allowed at a given point in time (end point measurement) our procedure non-interfering "on-line" measurements over the entire time reaction space (100 measurements of a single reaction batch at a distance seconds or minutes are easily possible).  

Since FRET depends on the distance and orientation of the chromophores (with maximum energy transfer at distances below 5 nm), it was initially over It is surprising to note that, given the size of the proteins to be conjugated, FRET was sufficiently and reliably detectable. It is described herein that a reliable FRET detection takes place when proteins of 10 or 20 kDa inserted between exemplary fluorescent proteins, while at the methods proposed by Tsien and co-workers for the analysis of Proteases a linker peptide of only 5-50 amino acids that the desired Pro tease recognition sequence carries between the fluorescent proteins YFP and CFP was set (US 5,981,200). Furthermore, in the state of the Technique known only the probes, but not the actual reak marked with fluorescent groups. In contrast, it was surpassed schenderenden found that the direct link between the react reaction components of the test system, d. H. Target protein and ubiquitin related protein, with the respective chromophores not an inhibition the same leads. The decisive breakthrough to this technological con The inventors succeeded in accepting an in vitro test system with rekom could establish binary components in which the quantitative modification a target protein and the demodification of a modified target protein was aimed. On the basis of the inventors' findings, one can now make analog ones Modification and demodification systems with all desired ubiquitin develop related proteins and target proteins. Since ubiquitin proteins Family not only have the same three-dimensional fold, but above it linked to target proteins via conserved mechanisms (Jentsch and Pyrowolakis, 2000), the expert can now, for. B. assume that Fusi proteins between a fluorescent protein and an ubiquitin related protein (including ubiquitin) can be used in FRET technology are. The linkage can preferably be carried out in such a way that the ubiquitin related protein is fused to the C-terminus of the fluorescent protein (analogous to the application examples). The target proteins for the corresponding Ubi Quitin-related proteins are more heterogeneous in folding and size, but they are  is readily possible to those skilled in the art in light of the present disclosure be a fusion protein or otherwise fluorescently labeled target protein construct that it can be used for the test system. Come into question at for example, N-terminal or C-terminal fusions with fluorescent protein NEN, the integration of the fluorescent protein between two domains of one Proteins, or the linking of chromophores to specific amino acids ren in the target protein (if necessary after the introduction of suitable side chains by mutage target protein). It should be noted that the acceptor chromophore is possible brought as close as possible to the modification site in the target protein in order to be efficient To reach FRET. "Near" does not refer to the primary sequence son the spatial proximity in the folded protein. Even if the structural data should not be known for a target protein, those skilled in the art will recognize that yourself according to the above-mentioned specifications without much experimental effort can construct a suitable target protein.

In a first aspect, the present invention relates to a method for ana lysis of modifications of proteins with ubiquitin-related proteins, the method comprising the steps of: (a) providing a testsy stems comprising (i) at least one target protein associated with a first chromo phor of a donor-acceptor pair for FRET (Fluorescence Resonance Energy Transfer) is covalently linked, (ii) at least one ubiquitin-related protein, that covalently ver with a second chromophore of the donor-acceptor pair (iii) Enzymes that form an isopeptide bond between the at least one target protein and the at least one ubiquitin-related Effect protein; (iv) ATP and / or an ATP derivative, and contacting the Constituents (i) - (iv) under conditions that affect the course of the enzymatic Re allow action; and (b) determining the amount of conjugate formed from target protein and ubiquitin-related protein 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 is as follows. A target protein linked to a donor chromophore is incubated with 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). Due to the formation of the isopeptide bond, donor and acceptor chromophores come into close proximity, which enables FRET (see also FIG. 1). This is detected by means of a fluorescence measuring device (e.g. in microtiter plate fluorescence measuring devices), in that the donor chromophore is excited by irradiation with the appropriate wavelength, and both donor and acceptor fluorescence are measured as a function of the reaction time. The change in the ratio of acceptor fluorescence to donor fluorescence correlates directly with the change in the amount of conjugation. This method is therefore suitable for quickly and quantitatively analyzing ubiquitin-related modifications without further manipulations in in vitro test systems with recombinant or purified components and / or cell extracts.

In a further aspect, the invention relates to a method for analyzing Demodification of proteins ver with ubiquitin-related proteins are linked, the method comprising the following steps: (a) providing a test system comprising (i) an isopeptidase substrate containing a target protein, that with a first chromophore of a donor-acceptor pair for FRET (Fluoescence Resonance Energy Transfer) is covalently linked, and at least an ubiquitin-related protein that is linked to a second chromophore of the donor Acceptor pair is covalently linked, includes; whereby this is by an isopep tide 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 are linked together; and (ii) at least one enzyme that cleaves the Causes isopeptide binding of the isopeptidase substrate; and bringing the Constituents (i) and (ii) under conditions that affect the course of the enzymatic  Allow reaction; and (b) determining the amount of cleaved isopeptidase Substrate, d. H. the conjugate of target protein and ubiquitin-related protein, by determining FRET, in particular the acceptance of FRET, between the first and second chromophore of the donor-acceptor pair.

Ubiquitination is an enzymatically catalyzed formation an isopeptide bond between the C-terminus of the 9-kDa poly peptide ubiquitin with the ε-amino groups in the lysines of acceptor or Target proteins. This modification is reversible, i. H. it can be a deubiquitine line tion in which the ubiquitin content is caused by deubiquitinating enzymes (Isopeptidases) can be cleaved from the target protein. The verein here The terms "ubiquitination" and "deubiquitination" used here refer forth in a narrower sense on ubiquitin itself, while in terms of having Ubiquitin-related proteins in a broader sense of "modifications of Proteins with ubiquitin-related proteins "or" demodifications of Proteins Linked to Ubiquitin-Related Proteins ". A "ubiquitin-related protein" is used here in a broader sense understood a protein that is structurally related to ubiquitin, and its C- terminal end by enzymatic reaction using an isopeptide bond ε-amino groups in lysines of the target proteins is linked or can be. In a broader sense, this includes z. B. SUMO1 and its homolo gene SUMO2 and SUMO3, Nedd8 / Rub1, Apg12 or Ucrp also Ubiquitin itself (For an overview, see Hershko and Ciechanover, 1998; Hochstrasser, 1998; Jentsch and Pyrowolakis, 2000; Melchior, 2000; Yeh et al., 2000). In a closer sense ne this term means such ubiquitin-related proteins to the exclusion from ubiquitin itself.

A “target protein” is understood here to mean a protein that by means of the for suitable enzymes by forming an isopeptide bond with ubiquitin or ubiquitin-related proteins. The  The target protein is usually associated with a first chromophore of a donor Acceptor pair for FRET (Fluorescence Resonance Energy Transfer) covalent linked, while the at least one ubiquitin-related protein with a second chromophore of the donor-acceptor pair is covalently linked. the Suitable methods for achieving a covalent linkage are known to those skilled in the art known. A "chromophore" is used herein to refer to the "coloring" group of Understood molecule, d. H. the part of a molecule that due to its electroni transitions is responsible for light absorption and emission. Chro mophores can contain both low molecular weight substances, e.g. B. the indocyanine Dyes Cy3, Cy3.5, Cy5, Cy7 (available from Amersham International plc, GB) or fluorescent proteins, such as certain GFPs ("Green Fluo rescent protein ") and mutant GFPs (available e.g. from Clontech Laboratories Inc. Palo Alto, CA, USA). It is preferably the first chromo phor around the FRET donor CFP ("Cyan Fluorescent Protein") and the two ten chorophore around the FRET acceptor YFP ("Yellow Fluorescent Protein"). With the so-called "Fluorescence Resonance Energy Transfer" (FRET), the Overlap of the emission spectrum of a chromophore (donor chromophore or FRET donor) with the absorption spectrum of the second chromophore (Ak zeptor chromophore or FRET acceptor) after excitation of the donor Chromophors also stimulate the acceptor chromophore through energy transfer if both chromophores are in the immediate vicinity. FRET can be demonstrated by the decrease / loss in the emission of the donor Chromophore with simultaneous emission of the acceptor Chromophore. A variety of chromophores are used in life sciences used as donor-acceptor pairs for FRET technology, and these are can therefore also be used for the processes described below. exemplary fluorescein and rhodamine should be mentioned here, which are activated in the Are commercially available and linked to proteins according to standard protocols that can; and CFP and YFP, which are based on standard molecular biology and biochemistry can be generated as fusions with other proteins  (Plasmids encoding YFP and CFP proteins are also commercially available available).

An "substrate for an enzyme" is used herein as an "isopeptidase substrate" who had ubiquitin or an ubiquitin-related protein from one Ubiquitin or an ubiquitin-related protein linked target protein removed by cleavage of the isopeptide bond. Usually this includes one Target protein that matches a first chromophore of a donor-acceptor pair for FRET (Fluoescence Resonance Energy Transfer) is covalently linked, and min at least one ubiquitin-related protein, which with a second chromophore of Donor-acceptor pair is covalently linked, in particular 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 Proteins are linked together.

When providing the test system and contacting the components under conditions that allow the enzymatic reaction to proceed unless otherwise stated, it does not matter indicates that the components are present as separate components or in a be mixed order or brought into contact with each other. Rather, it should be ensured that the required components of the respective test systems exist, and that contacting this inventory parts in such a way that the enzymatic reaction can proceed and a FRET Measurement can be made. Unless ATP and / or an ATP derivative are necessary for this, it can be the latter to the expert known derivatives, in particular AMP-PNP or ATPγS (e.g. from Roche Diagnostics). The person skilled in the art will be able to do so in the light of the present tion can be seen how the suitable for the reaction to be examined conditions, e.g. B. in terms of temperature or pH.  

In a preferred embodiment, the target protein and / or the Ubi quitin-related protein in the form of a fusion protein with a fluorescent the protein can be used. The at least one target protein is involved is preferably mouse or human RanGAP1 (SWISS-PROT: P46061; SWISS-PROT: P46060) and at least one ubiquitin-related pro complexion around SUMO (Small Ubiquitin-related Modifier), especially around SUMO1, SUMO2 and / or SUMO3. The access numbers (SWISS-PROT) for huma SUMO proteins are as follows: Q93068 (SUMO1); P55854 (SUMO2) and P55855 (SUMO3). These proteins are identical for the mouse (for an overview they see also Melchior, 2000). RanGAPI and / or CFP can advantageously be used in Form of a fusion protein can be used, in particular where the C- Terminal domain of RanGAP1 with the N- or C-terminal end of CFP is merged. Correspondingly, SUMO and / or YFP can be in the form of a Fusion protein can be used, especially where amino acids 1-91 from SUMO1, the amino acids from 1-92 from SUMO2 or the amino acids 1-93 of SUMO3 are fused to the C-terminal end of YFP.

Conveniently, the target protein, the ubiquitin-related protein and / or the respective enzymes in the form of a purified protein (partially or up to Homogeneity) and / or a cell extract can be used. It can be in particular an extract from mammalian, insect, yeast and / or bacteria act cells that the skilled person can produce using suitable techniques. In particular, this can also be a derivative of the target protein, the Ubi act quitin-related protein and / or the enzymes. Under the term As used herein, "derivative" means a protein or polypeptide that has a sequence homology, in particular a sequence identity to the aforementioned proteins of at least 50%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 95%. This also includes deletions from the respective protei ne as well as substitutions and additions (e.g. K48R Ubiquitin, which is  Target proteins can be linked, but the ubiqui linked via lysine 48 tin-ubiquitin no longer forms chains. This mutant therefore offers a clear one Advantage over wild-type ubiquitin in the FRET analysis). Accordingly The present disclosure also includes nucleic acids for the foregoing encode proteins or polypeptides. Examples of related nuclei Acids are nucleic acids from different human cells or Ge weave or allelic variants, as well as nucleic acids from different can come from human individuals. In a broader sense, one understands a nucleic acid encoding such a derivative, a nucleic acid encoding a Homology, especially a sequence identity to an above-mentioned pro a coding nucleic acid of at least 50%, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 95%. Stand by the specialist appropriate techniques and processes for the production and mutagenesis of Nu small acids as well as for gene expression and protein analysis are available (see the following manuals: 1. Molecular Cloning: A Laboratory Manual (3rd edition). Sambrook and Russell (2001). Cold Spring Harbor Press. 2. Current Protocol in Protein Science. Coligan, J.E. et al. (updated quarterly). John Wi ley & sons. 3. Current Protocols in Molecular Biology. Ausubel F. M. et al. (four updated every year). John Wiley & Sons.)

With regard to the enzymes contained in the respective test system, the Average expert can easily recognize which enzymes are suitable, to enable the corresponding enzymatic reaction to take place (for For an overview, see e.g. B. Hershko and A. Ciechanover, 1998; Yeh et al., 2000; Jentsch and Pyrowolakis, 2000.). As an illustration, it should be mentioned that this at for example when analyzing modifications of proteins with ubiquitin related proteins 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 Aos1 / Uba2 heterodimer as the E1 enzyme and the Ubc9 protein as the E2 enzyme. If an unidentified E3-  Enzyme exists, this z. B. by means of that described in this application Procedures are isolated and identified, and then, if necessary, in the proposed Test systems are used. In the case of analysis of deubiquitination the enzyme is one of the numerous ubiquitin isopeptidases (Review by Chung and Baek, 1999). For proteins with the Ubiqui tin-related proteins are linked to SUMO game for the previously known SUMO-specific isopeptidases Ulp1, Ulp2, Susp1 and / or Senp1 (original work for cloning the SUMO-specific Isopeptidases are cited in the review article Melchior 2000). Unless so far identified isopeptidases exist, these can e.g. B. by means of in this The procedure described in the application can be isolated and identified, and then may be used in the proposed test systems.

In a further aspect, the present invention relates to a method for Determining substances that modify a target protein with ubiquitin related proteins, the process following steps comprises: (a) providing a test system comprising (i) at least one target protein associated with a first chromophore of a donor-acceptor pair for FRET (Flurescence Resonance Energy Transfer) is covalently linked, (ii) min at least one ubiquitin-related protein, which with a second chromophore of Donor-acceptor pair is covalently linked, (iii) enzymes that form a Isopeptide bond between the at least one target protein and the minde cause at least one ubiquitin-related protein, (iv) ATP and / or an ATP Derivative, and (v) at least one substance to be tested; and bringing the Constituents (i) - (v) under conditions that affect the course of the enzymatic rea allow tion; and (b) detecting the formation of conjugate from target protein and Ubiquitin-related protein and / or determining the amount of formed Conjugate by determining FRET between the first and the second Chromophore of the donor-acceptor pair. In a preferred embodiment the enzymes according to step (a) (iii) are the corresponding ones  E1 and E2 enzymes, optionally in combination with at least one E3 Enzyme. In the case of SUMO, the E1 enzyme is the Aos1 / Uba2 heterodimer and the E2 enzyme 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 thereof as defined above, or a low molecular weight Substance, especially an inhibitor or activator. The "low molecular sub punch "can be any inorganic or organic molecule or ion be that its ability to test the enzymatic reaction in the to influence the test system described, in particular to activate this reaction four or inhibit. The low molecular weight substance has one in particular Molecular weight of at most 2500, in particular at most 2250, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1250, 1200, 1100, 1000, 900, 800, 750, 700, 600, 500, 450, 400, 350, 300, 250 and 200. For example, it can are molecules with a linear structure such as peptides, in particular Oligopeptides, peptoids, linear oligosaccharides, nucleotides, especially oligo act nucleotides, and their analogs, or z. B. monomers such as heterozy klen, especially nitrogen heterocycles, or molecules with a non-linear Structure, such as branched oligosaccharides. Low molecular weight inorganic ver For example, bonds can be arsenic and antimony compounds.

In view of the present description, it will be clear to the person skilled in the art that that the protein or the derivative thereof from an expression bank (Büssow et al., 2000) and / or the low-molecular substance or a combinatorial Bank, in particular a combinatorial peptide, non-peptide or "drug-like Small Molecule "bank (e.g. from Tecnogen S.C.p.A. Piana di Monte Verna, CE, Italy; ChemBridge Corporation, San Diego, USA; or Advanced ChemTech Europe Ltd, Cambridgeshire, GB), or from a cell extract and / or in a purified form, partially or to homogeneity. Suitable combinatorial strategies for the screening are  in particular the "high throughput screening" of substances to be tested known, especially the use of combinatorial banks, the 100, Can include 1,000 or even 1,000,000 different connections (see to overview z. B. Meyers, Encyclopedia of Molecular Biology and Molecular Medicine, 1997).

Another aspect relates to a method for determining substances that the Demodification of a protein related to at least one ubiquitin Protein is linked, the process involves the following steps comprises: (a) providing a test system comprising (i) an isopeptidase Substrate that contains a target protein that is linked to a first chromophore of a donor Pair of acceptors for FRET (Flurescence Resonance Energy Transfer) covalent and at least one ubiquitin-related protein associated with one second chromophore of the donor-acceptor pair is covalently linked; which is characterized by an isopeptide bond between the ε-amino group of a Ly sins of the target protein and the C-terminal carboxy group of at least one Ubiquitin-related protein are linked; (ii) at least one Enzyme that cleaves the isopeptide bond of the isopeptide substrate; and (iii) at least one substance to be tested; and contacting the inventory parts (i) - (iii) under conditions that affect the course of the enzymatic reaction allow; and (b) determining the amount of cleaved isopeptidase substrate, d. H. the conjugate of target protein and ubiquitin-related protein, by Be agree of FRET, in particular the acceptance of FRET, between the first and the second chromophore of the donor-acceptor pair. In a special one The embodiment according to step (a) is a Isopeptidase, especially Ulp1, Ulp2, SUSP1 and / or Senp1 (for overview see e.g. B. 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, e.g. B. ELISA, co-immunoprecipitation, microcalorimetri etc. are available to determine whether the protein or polypeptide binds non-specifically or specifically to the target protein, and thus represents a binding partner for the target protein (various techniques are e.g. described in Current Protocol in Protein Science, Coligan, JE et al. (updated quarterly. Publisher John Wiley & Sons.). In particular, binding is considered herein 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, the person skilled in the art has suitable methods and materials, e.g. B. combinatorial banks, for screening, in particular HTS screening, of substances to be tested available.

In a special embodiment of the two aforementioned methods for Determination of substances that influence the modification / demodification is the first chromophore is a first fluorescent protein and the second chromo phor is a second fluorescent protein, in particular the chromophore FRET donor CFP ("Cyan Fluorescent Protein") and the second chromophore FRET acceptor YFP ("Yellow Fluorescent Protein"). Conveniently, can the target protein and / or ubiquitin-related protein in the form of a fusion pro teins with a fluorescent protein can be used. The specialist are suitable methods for the production of such fusion proteins are known. In a special embodiment is the at least one target protein RanGAP1 and the at least one ubiquitin-related protein SUMO (Small Ubiquitin-related Modifier), especially SUMO1, SUMO2 and / or SUMO3. Here it can be preferred to use a fusion protein from RanGAP1 and CFP, in particular in particular where the C-terminal domain of RanGAP1 with the N- or C- terminal end of CFP is fused. Similarly, a merger protein from SUMO and YFP can be used, in particular the amino acids  1-91 of SUMO1, the amino acids of 1-92 of SUMO2 or the Amino acids 1-93 of SUMO3 fused to the C-terminal end of YFP are. The target protein, the ubiquitin-related protein and / or the enzymes can in the form of a purified protein, partially or to homogeneity, or of a cell extract can be used. Such an extract can be processed which are known to the person skilled in the art from mammalian, insect, yeast and / or bacteria cells are manufactured. If, for example, combinatorial banks are turned on be used to test substances for whether they affect the enzymatic reaction flow, it may be desirable in a further step to identify the identity of the to verify and / or determine the existing substance or the tested substance. The person skilled in the art has suitable molecular biological, biochemical for this purpose and / or biophysical processes, such as. B. the protein and / or nucleic acid resequence analysis and e.g. B. the various methods of mass spectrometry, like matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) are available. In certain cases it may also be desirable a derivative of the target protein, ubiquitin-related, as defined above Proteins and / or the enzymes to use, for example to certain Mutants of the target protein for their modification and / or demodification investigate.

A special embodiment is a so-called "cell based assay". Here is the Contacting the corresponding components in a cell, in particular a cell outside the human or animal body, whereby Mammalian or yeast cells are preferred. For example, the fusion protein from target protein and the first fluorescent protein and / or the Fu sionsprotein from the ubiquitin-related protein and the second fluoreszie protein are expressed in the cell. The at least one to be tested Substance can also be expressed in the cell, preferably by the cell is transfected 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 Detection of a defect in the modification of protein with ubiquitin related protein or in the demodification of proteins using Ubiqui tin-related proteins are linked. It can be assumed that defects in the Modification and demodulation of specific target proteins on a variety of Diseases are involved, being among the target proteins for this post-translational Modification are growth factors, tumor suppressors and proto-oncogenes. Defects in the ubiquitination of target proteins are already in a number of Diseases such as Von Hippel Lindau Syndrome, Angelman Syndrome, or Cervi calem cancer has been implicated (review: Schwartz and Ciechanover, 1999; Vu and Sakamoto, 2000) For example, mutations appear in Parkin, ei ner E3 ubiquitin ligase, for the development of a form of Parkinson's disease to contribute.

The method of detecting a defect in the modification of proteins with ubiquitin-related proteins involves the following steps: (a) Ready provide a test system comprising (i) at least one target protein which is associated with a first chromophore of a donor-acceptor pair for FRET (Flurescence Re sonance Energy Transfer) is covalently linked, and (ii) at least one ubiqui tin-related protein associated with a second chromophore of the donor-acceptor Pair is covalently linked; (iii) at least one cell extract to be tested; and Contacting the components (i) - (iii) under conditions that prevent formation allow conjugate of target protein and ubiquitin-related protein; and (b) Determine the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair.

The procedure for the detection of a defect in the demodification of protein Nes linked to ubiquitin-related proteins include the following the step: (a) providing a test system comprising (i) an isopeptidase  Substrate that contains a target protein that is linked to a first chromophore of a donor Pair of acceptors for FRET (Flurescence Resonance Energy Transfer) covalent and at least one ubiquitin-related protein associated with one second chromophore of the donor-acceptor pair is covalently linked; in particular, this being 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 are linked to one another; and (ii) at least one cell extract to be tested; and contacting the loading ingredients (i) - (ii) under conditions that target the cleavage of conjugate allow protein and ubiquitin-related protein; and (b) determining the Amount of cleaved conjugate by determining FRET, especially the Decrease in FRET, between the first and second chromophore of the Donor-acceptor pair.

The cell extract to be tested can in each case be examined from cells of tissue appropriate individual and / or from cells from cell culture. The subject Suitable methods for producing such cell extracts are known.

In yet another aspect there is a novel method of identification Target proteins for modification with ubiquitin-related proteins suggested that the following step includes: (a) Deploy a testsy stems, comprising (i) at least one target protein to be tested, which with a first Chromophore of a donor-acceptor pair for FRET (Flurescence Resonance Energy Transfer) is covalently linked, (ii) at least one ubiquitin-related Protein covalent to a second chromophore of the donor-acceptor pair (iii) Enzymes that form an isopeptide bond between the at least one target protein and the at least one ubiquitin-related Effect protein; (iv) ATP and / or an ATP derivative; and bringing the Constituents (i) - (iv) under conditions that affect the course of the enzymatic Re allow action; and (b) detecting the formation of conjugate from target protein and ubiquitin-related protein and / or determining the amount of formed  Conjugate by determining FRET between the first and the second Chromophore of the donor-acceptor pair.

This method allows screening for target proteins for certain ubiquitin related proteins. The contacting of the preceding Be components of the test system in a cell, especially in a cell outside of the human or animal body, a mammalian or yeast cell is preferred. The at least one target protein to be tested can be found in Form of a fusion protein with a first fluorescent protein, preferably as a fusion protein with the FRET donor CFP, can be used. In Such a fusion protein originates from a particularly practical embodiment from an expression bank and can e.g. B. by means of a fusion protein for this coding cDNA introduced into a cell and expressed therein. On in this way it is known to the person skilled in the art on the basis of those known in the prior art combinatorial techniques and procedures easily possible, a very large number screen 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 fluoresce related protein in the form of a fusion protein with the second the protein is expressed in a cell. An example of an ubiquitin related protein is SUMO (Small Ubiquitin-related Modifier), in particular SUMO1, SUMO2 and / or SUMO3. A fusion protein can advantageously be used comprising SUMO and YFP are used, in particular the Ami amino acids 1-91 from SUMO1, amino acids 1-92 from SUMO2 or the Ami noacids 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 above known method known (see also Hershko and Ciechanover, 1998; Hoch strasser, 1998; Jentsch and Pyrowolakis, 2000; Melchior, 2000; Yeh et al., 2000). So it becomes corresponding E1 and E2 enzymes, optionally in combination with  use at least one E3 enzyme. In the case of SUMO it can be used as an E1 enzyme the Aos1 / Uba2 heterodimer and as the E2 enzyme the Ubc9 protein the.

Conveniently, the target protein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein, either partially or cleaned to homogeneity, or a cell extract can be used, whereby an extract from mammalian, insect, yeast and / or bacterial cells is preferred.

The at least one target protein to be tested can also be a De act privately, as defined above, of a known target protein, for example wise about certain mutants. Alternatively or alternatively, a appropriately defined derivative of the ubiquitin-related protein and / or Enzymes can be used. Should it be desired to identify the target pro verify teins (for example, if a combinatorial bank made of mutan th target proteins is screened, which are obtained by means of the so-called "DNA shuffling" was determined) or to determine the identity of the target protein (e.g. during the screening an expression bank with potential target proteins), this can be done using the Molecular biological, biochemical and / or biophysics known to those skilled in the art lical process as described above.

In another aspect, the inventors propose a kit for the detection of Modifications of proteins with ubiquitin-related proteins that the comprises the following components: (i) at least one target protein and / or derivative of these, the one with a first chromophore of a donor-acceptor pair for FRET (Flurescence Resonance Energy Transfer) is covalently linked, and / or a nucleic acid coding therefor; and (ii) at least one ubiquitin related protein and / or derivative thereof with a second chromophore of the donor-acceptor pair is covalently linked, and / or a coding for this  Nucleic acid; and optionally (iii) enzymes that inhibit the formation of an isopep binding between the at least one target protein and the at least one egg cause an ubiquitin-related protein; and / or at least one for this coding nucleic acid; and / or (iv) ATP and / or an ATP derivative. benefit adhesive embodiments of the constituents mentioned are from the foregoing Description visible.

In another aspect, a fusion protein is provided that SUMO and comprises a fluorescent protein, preferably YFP, in particular the Amino acids 1-91 from SUMO1, amino acids 1-91 from SUMO2 or the Amino acids 1-93 of SUMO3 fused to the C-terminal end of YFP are. A fusion protein, RanGAP1, is also proposed and a fluorescent protein, preferably CFP, e.g. se the C-terminal domain of RanGAP1 with the N- or C-terminal end may be merged by CFP. Nu coding for the fusion proteins mentioned Small acids are also provided. A cell is also provided, especially a mammalian cell outside the human or animal body pers or a yeast cell that contains one or both of the aforementioned fusion proteins expressed.

In another aspect, there is provided an isopeptidase substrate comprising a target protein covalently linked to a first chromophore of a donor-acceptor pair for fluorescent resonance energy transfer (FRET), and an ubiquitin-related protein that modifies the target protein and is covalently linked to a second chromophore of the donor-acceptor pair, the target protein and the ubiquitin-related protein being linked via at least one isopeptide bond. The latter two proteins can also be derivatives in the sense of the preceding definition. In particular, the at least one isopeptide bond exists 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 can be one of the following proteins, or a protein still to be identified: RanGAP1, RanBP2, PML, Sp100, I _K B _α , D. melanogaster Dorsal, p53, c-Jun, HIPK2, S. cere visiae Cdc3 , S. cerevisiae Cdc11, S. cerevisiae Sep7 / Shs1, hCMV IE1, hCMV IE2, D. melanogaster Tramtrack, Topoisomerase I, Glut1, Glut4 and Mdm2 (see Melchior, 2000, Table I). In a particular embodiment, the first chromophore is a first fluorescent protein and the second chromophore is a second fluorescent protein, the first chromophore preferably being the FRET donor CFP (“cyan fluorescent protein”) and the second chromophore being the FRET acceptor YFP (“yellow Fluorescent Protein "). In particular, the target protein and / or ubiquitin-related protein is in the form of a fusion protein with a fluorescent protein, e.g. B. in the form of CFP-RanGAP1 and / or YFP-SUMO.

In yet another aspect of the invention, the inventors propose the ver using a kit, a fusion protein, and / or a cell, as before described above, for the analysis of modifications of proteins with Ubi quitin-related proteins, used to determine substances that modify of a target protein with ubiquitin-related proteins, for identification Decoration of target proteins for modification with ubiquitin-related pro stone and / or for diagnosis, in particular for the detection of a defect in the Modification of proteins with ubiquitin-related proteins. the same the use of the described isopeptidase substrate for analysis is measured demodification of proteins with ubiquitin-related proteins, to determine substances that demodify a target protein with Ubi affect quitin-related proteins, and / or for diagnosis, in particular for the detection of a defect in the demodification of proteins with ubiquitin related proteins.  

Finally, processes for the production of a pharmaceutical composition provision provided. The first method comprises in a first stage steps described above for determining substances that (i) the Mo affect identification of a target protein with ubiquitin-related proteins, or (ii) demodifying a protein containing at least one ubiquitin related protein is linked, and in a second stage that Mix the tested and / or identified substance with a subject known pharmaceutically acceptable carriers (see e.g. Sucker et al., 1991). The second method comprises in a first stage the above steps to identify a target protein for ubiquitin related proteins, and in a second stage mixing the identifi decorated target protein or a derivative thereof with a pharmaceutically ver inert carrier. The present disclosure thus provides effective methods for identifying agents, compounds or so-called "lead compounds" for Drugs ready to modify or demodify with ubiquitin relate directly or indirectly to related proteins. The identified substances and / or target proteins can be automated and cost-effective HTS Method for screening combinatorial banks to provide effective components of medicinal products are used. The latter will be then opti for normal activity and minimal toxicity mized. Clinical areas of application include the treatment of viral conditions diseases, neurodegenerative diseases, tumors, inflammation and Immune system diseases.

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

The following figures and examples are intended to explain the invention in more detail without to limit them to that. The following claims are hereby incorporated by Reference is incorporated into the text of this description.  

Description of the figures

Fig. 1 shows the principle and the possible uses of the test method 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 is formed between the ubiquitin-related protein and the target protein. Isopeptidases can break this bond again. Using "Fluorescence Resonance Energy Transfer" (FRET), both the linkage (modification) and the cleavage (demodification) can be followed kinetically 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 partners, 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. The identification of new target proteins for a known ubiquitin-related protein. 5. Diagnostics.

Fig. 2 shows the reconstitution of the SUMO1 modifier with fluorescent fusion proteins and recombinant enzymes.
Standard: the E1 enzyme heterodimer His-Aos1 / Uba2, the E2 enzyme Ubc9, CFP-RanGAP (amino acids (As.) 400-589), and YFP-SUMO1 (As. 1-97) were in the presence of ATP Incubated for 30 min at 30 ° C., then mixed with sample application buffer, separated by SDS-PAGE and by immunoblotting with anti-RanGAP1 antibodies (upper part of FIG. 1) or with anti-GFP antibodies (lower part) analyzed.
ATP: like standard, but without ATP.
SUMO: like standard, but with an excess of non-fluorescent SUMO1.
- Ubc9: like standard, but without adding Ubc9.
- E1: as standard, but without adding Aos1 / Uba2.
** indicates isopeptide binding.

Fig. 3 shows the detection of SUMO1 modification of fluorescent fusion proteins by FRET.
The E1 enzyme heterodimer His-Aos1 / Uba2, the E2 enzyme Ubc9, CFP-RanGAP (As. 400-589), and YFP-SUMO1 (As. 1-97) were mixed in microtiter plates (reaction volume 25 µl) and the modification reaction started by adding 1 mM ATP. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm over time. The primary data, ie emissions at 485 and 527 nm ( FIG. 3A), and the processed data, ie the quotient of the emissions at 527 nm and 485 nm ( FIG. 3B), are shown.

Fig. 4 shows the analysis of inhibitors of the modification in the FRET assay system.
His-Aos1 / Uba2, Ubc9, CFP-RanGAP (As. 400-589), and YFP-SUMO1 (As. 1-97) were mixed in microtiter plates (reaction volume 25 µl). In addition, increasing amounts of non-fluorescent RanGAP1 as competitor were added to the samples in FIG. 4A; increasing amounts of Gst-RanBP2 (As. 7634-8806), which binds to the enzyme Ubc9 and thereby inhibits it, were added to samples in FIG. 4B. The modification reactions were started by adding 1 mM ATP. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm over time. The addition of the competitor (see FIG. 4A) changes the rate and maximum level of the resulting signal. The addition of the non-competitive inhibitor (see FIG. 4B) changes the rate, but not the maximum level of the resulting signal.

Figure 5 shows the analysis of isopeptidase activity in the FRET test system.
25 µl each of isopeptidase substrate, consisting of the conjugate of CFP-RanGAP (As. 400-589) and YFP-SUMO1 (As. 1-97), were incubated with recombinant isopeptidase (Gst-Ulp1) at 37 ° C. The samples were excited at 430 nm and the emission was measured at 485 nm and 527 nm over time. Cleavage of the isopeptide bond leads to a decrease in the FRET signal.

Examples 1. Production of recombinant YFP-SUMO1, CFP-RanGAP, SUMO1- Activating enzyme Aos1 / Uba2, SUMO1-conjugating enzyme Ubc9 and the isopeptidase Ulp1

The C-terminal 20 kDa domain of the SUMO1 target protein RanGAP1 (mouse, As. 400-589; GenBank: U08111; Mahajan et al., 1997) was transformed into the vector pE- CFP (Clontech) cloned as a cyan fluorescent protein (CFP) fusion protein in E. coli expressed and partially purified via DEAE-Sepharose and gelation.

SUMO1 (human, As. 1-97; GenBank: U67122; Mahajan et al., 1997) was published in the Vector pE-YFP (Clontech) cloned as Yellow Fluorescent Protein (YFP) Fusi Onsprotein expressed in E.coil and from it via DEAE Sepharose and Gelfiltrati partially cleaned.

The coding region of Ubc9 (mouse; GenBank: U94402) was translated into the NdeI and BamHI interfaces of the vector pET23a (Novagen, Madison, USA) klo niert, expressed in E. coli and purified via S-Sepharose and gelation.

The E1 heterodimer Aos1 / Uba2 (human Uba2, GenBank: AF090384; human Aos1, GenBank: NM_005500) was obtained by simultaneous expression of both sub units in E. coli from the plasmids pET11d (Uba2) and pET28a (Aos1) (Plasmids from Novagen, Madison, USA) obtained. Aosl was with one N-terminal His6 tag expressed. Cleaning was initially carried out using bandages - and elution of Proßond ™ Resin (Invitrogen, Groningen, The Netherlands), then via gel filtration and then chromatography over Q-Sepharose.

Ulp1 was expressed as a Gst fusion protein in E. coli and expressed via glutathione Sepharose and gel filtration cleaned. The expression plasmid (Li and Hoch strasser. 1999) was developed by Dr. Mark Hochstrasser, Yale, USA provides.  

2. in vitro modification reaction

The recombinant proteins Aos1 / Uba2, Ubc9, YFP- described under 1. SUMO1 and CFP-RanGAP were combined in 386-well Mirotiter plates ben, mixed with ATP at the start of the reaction, and in the fluorescence measuring device Flu oroskan Ascent FL from Labsystems GmbH (Frankfurt, Germany) Measure 38 ° C every 30 or 60 seconds for up to 120 minutes. It was Light of the wavelength 430 nm (optimal excitation wavelength for CFP) turned on emits and the fluorescence at the wavelengths 485 nm (maximum of the emissi on of CFP) and 527 nm (maximum of the emission of YFP) measured. Alterna In addition, the samples were incubated for 30 min in SDS-PAGE Sample application buffer recorded and by Westerblot analysis with anti RanGAP1 (Mahajan et al., 1997) or anti-GFP antibodies (available from Santa Cruz Biotechnology, Santa Cruz, USA).

3. in vitro demodification reaction (isopeptidase test)

Isopeptidase substrate consisting of the conjugate of CFP-RanGAP (As. 400-589) and YFP-SUMO1 (or YFP-SUMO2), was created by first creating the Recombinant proteins Aos1 / Uba2, Ubc9, YFP-SUMO1 described under 1 (or YFP-SUMO2) and CFP-RanGAP in the presence of 1 mM ATP at 30 ° C were incubated for 30 min, and the reaction mix after linking of YFP-SUMO and CFP-RanGAP via gel filtration of remaining ATP was liberated. [Given this disclosure, those skilled in the art will be able to accordingly other isopeptidase substrates from the appropriate inventory manufacture parts.]

25 µl of this isopeptidase substrate were each in 386-well Microtiter plates with increasing concentrations of the isopeptidase Ulp1 ver sets, and in the fluorescence measuring device Fluoroskan Ascent FL from Labsystems  (Company, location, state) at 37 ° C for up to 120 minutes every 60 seconds. Light with a wavelength of 430 nm (optimal excitation wavelength for CFP) and the fluorescence at the wavelengths 485 nm (maximum the emission of CFP) and 527 nm (maximum of the emission of YFP) measured sen. Alternatively, the samples were incubated for 30 min, in SDS-PAGE Sample application buffer incubated, and by Westerblot analysis with anti-RanGAP1 (Mahajan et al., 1997) or anti-GFP antibodies (available from Santa Cruz Biotechnology, Santa Cruz, USA).

4. Quantitative evidence of modification / demodification of an Ubigui tin-related protein and the influence of inhibitors on this Re action

The general applicability of the FRET-based test procedure will hereinafter based on a specific pair of "target protein" and "ubiquitin" related protein ".

RanGAP1, a protein involved in the transport of proteins into the cell nucleus, is covalently linked to the ubiquitin-related protein SUMO1 (Mahajan et al., 1997; Mahajan et al., 1998). This modification changes the cytoplasmic localization of the unmodified RanGAP1 by allowing a stable association of the RanGAP1 with the nuclear pore complex protein RanBP2. The modification of RanGAP1 with SUMO1 can be reconstituted in vitro by using either extracts from mammalian cells or purified (recombinant) enzymes as the enzyme source and by starting and measuring the modification reaction as shown in Example 2. As demonstrated by Western blot analysis ( Fig. 2), the CFP-RanGAP fusion protein was quantitatively linked to the YFP-SUMO1 fusion protein. This reaction was dependent on energy (here ATP was used), on Ubc9, and on the heterodimer Aos1 / Uba2. The addition of an excess of unfused SUMO (As. 1-97) prevents the linkage with YFP-SUMO1 by competition.

The occurrence of FRET is characterized by a loss of acceptor fluorescence with a simultaneous increase in donor fluorescence, as can be observed after combining all of the components described in Example 1 and ATP, while in the absence of ATP FRET does not occur. FIG. 3A shows the data actually measured, and FIG. 3B shows the quotient of the emissions at 527 nm and 485 nm. An increase in the quotient correlated strictly with the occurrence of the conjugation under all the circumstances tested, and is therefore dependent on the presence and concentration the appropriate enzymes (either recombinant or in the form of cell extracts), and energy (in the form of ATP or other usable nucleotides).

A FRET signal could be prevented, for example, by an enzyme inhibitor (e.g. N-ethylmaleimide) or non-fluorescent competitors (e.g. recombinant RanGAP1 or SUMO1). An already generated FRET signal could be destroyed if cell extracts with isopeptidase activity were added after ATP had been removed by the hexokinase and glucose. Fig. 4 shows an example of the signal changes as a function of a competitive inhibitor (RanGAP1; Fig. 4A) and a non-competitive inhibitor (Gst-RanBP2, Fig. 4B; this protein binds very stably to Ubc9 and thereby removes it from the reaction).

In addition, mutants of SUMO1 and Ran GAP1 will be examined for its conjugation ability by the mutants were added as non-fluorescent competitors. Konjugationsfähige Mutants reduced / or prevented FRET, while certain mutants that are unable to conjugate, did not affect FRET.  

The reverse reaction, i.e. the cleavage of the isopeptide bond by isopeptidases, leads to a decrease in the FRET signal. Fig. 5 shows an example of the Demodifizierung by the yeast Ulp1 isopeptidase. Furthermore, the isopeptide activity in total extracts and in cell fractions of HeLA cells could be determined quantitatively using the FRET method (data not shown).

Taken together, these data demonstrate the suitability of FRET Test procedure for the kinetic analysis of the modification and / or the demodi Creation of RanGAP1 with SUMO1 and SUMO2. In principle, therefore for each pair of target protein and ubiquitin-related protein, such test ver build up driving. Care should be taken here by making the appropriate choice of chromophores, the protein domains used and the link to the chromophore the donor and acceptor chromophores according to covalent ver to bring knotting close enough for FRET. Given the present The disclosure will provide those skilled in the art with suitable materials and methods easily recognize.

5. Determine proteins that modify a known target affect proteins

The above demonstrated in vitro test procedures, but in principle also procedures in vivo can be used for the systematic identification of proteins that the modification of a defined ubiquitin-related protein with a de Inhibit or activate the defined target protein. In addition to putative E3- Ligases and isopeptidases also kinases, phosphatases and other binding partner.  

In vitro method: This can be done, for example, so that all for the in vitro Modification components to be provided including the acceptor labeled ubiquitin-related protein and the corresponding donor labeled target protein are presented in microtiter plates, and unknown recombinant proteins, e.g. B. obtained from expression banks in "High Throughput Screening "(HTS) procedure (e.g. when using N-terminal His tags on the fusion proteins), or cell extract fractions or purified Proteins are added. Changes in the reaction kinetics indicate an influence of the unknown protein on the modification.

In vivo method: The identification of such proteins in vivo can, for example that a cell line that contains the acceptor-labeled ubiquitin related protein and the donor-labeled target protein simultaneously stable or transparent sient is expressed, transfected with cDNAs, which are used to express unknown Lead proteins. The decrease or amplification of the (steady state) FRET signal indicate an influence of the protein to be tested on the modification.

6. Identification of low molecular weight substances that modify of a known target protein

The in vivo and in vitro test methods described in Example 5 can be the same are used for the systematic identification of substances, which is the modification of a defined ubiquitin-related protein with a inhibit or activate the defined target protein.

In vitro method: This can be done, for example, in such a way that all for the in components to be provided in vitro modification, including the acceptor labeled ubiquitin-related protein and the corresponding donor-labeled  Target protein are presented in microtiter plates, and unknown Substances, e.g. B. from a so-called combinatorial "Small Molecule Library", in HTS methods to be tested for an effect on reaction kinetics.

In vivo method: The identification of such substances in vivo can e.g. B. that a cell line that contains the acceptor-labeled ubiquitin related protein and the donor-labeled target protein simultaneously stable or transparent sient expressed, is incubated with these substances. The acceptance or Ver Strengthening of the FRET signal indicates an influence of the substance to be tested the modification.

7. Analysis and / or identification of mutants

The above-described in vivo and in vitro test methods can be the same for the analysis and / or identification of mutants of known Ubiquitin-related proteins, known target proteins, and / or the modifi ornamental / demodifying enzymes can be used.

8. Use of test methods in diagnostics

Variants of the test methods described above can ver be applied. This is e.g. B. then desirable if defects in the Modifi a certain (target) protein for certain clinical pictures wear or are associated with certain diseases and / or disorders. at for example, the ability of different cell extracts (e.g. made from Tissue or from cell cultures) to modify a specific target protein can be quantified by the corresponding pair of FRET donor and FRET acceptor-labeled proteins with defined amounts of one to be tested  Incubated extract and the occurrence of the FRET signal time-dependent is determined.

9. Identification of target proteins for modification with ubiquitin related proteins

The in vivo and in vitro test methods described herein can be used for systema tical identification of new target proteins for the modification of Ubi Quitin-related proteins can be used instead of a known target unknown and / or unidentified donor chromophore proteins to be tested labeled proteins, e.g. B. from an expression bank or a combinatorial Bank, in combination with the acceptor chromophore-labeled ubiquitin related protein can be used.

Identification using in vitro methods: This can e.g. B. done that all for the Components to be provided in vitro modification including a YFP labeled ubiquitin-related protein are placed in microtiter plates, and unknown recombinant CFP fusion proteins are added. FRET Signals should only arise here if the unknown protein is covalent to the YFF-labeled ubiquitin-related protein is linked (here arises FRET only depending on energy and enzyme addition) or not covalently (Energy and enzyme independent) interacts with him. Unknown recombinant CFP fusion proteins can, for example, from expression banks in HTS Methods can be obtained (e.g. when using N-terminal His tags on the fusion proteins).

Identification using in vivo methods: For example, this can be done gene that cells that the YFP-ubiquitin-related protein is stable or transient  express, be transfected with cDNAs that are used to express unknown Carry proteins in fusion with CFP. The analysis then takes place e.g. B. in microtiter plate fluorescence measuring devices and / or with fluorescence microscopes, which the corresponding FRET filters. The occurrence of FRET Signals indicate the linkage of the unknown protein with the known ubiquitin-related protein. This can also be done by appropriate Controls can be verified independently (e.g. mobility change in the SDS gel and use of mutant proteins that can no longer be linked NEN). For the in vivo modification, all cells come into question which are those for the Contain suitable enzymes (endogenous or due to Manipu simulations). An ubiquitination or modification with SUMO and its Homologues can therefore, for example, both in established cultures of Sau cells can also be examined in baker's or split yeast.

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Claims (55)

1. A method for analyzing modifications of proteins with ubiquitin-related proteins, the method comprising the following steps:

• 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 (fluorescence resonance energy transfer), (ii) at least one ubiquitin-related protein, the is 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) Determine the amount of conjugate formed from target protein and ubiquitin-related protein by determining FRET between the first and second chromophore of the donor-acceptor pair.

2. A method for analyzing demodifications of proteins linked to ubiquitin-related proteins, the method comprising the following steps:

• a) providing a test system comprising (i) an isopeptidase substrate, a target protein that is covalently linked to a first chromophore of a donor-acceptor pair for FRET (Fluorescence Resonance Energy Transfer), and at least one ubiquitin-related protein, which is covalently linked to a second chromophore of the donor-acceptor pair; wherein these are 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; and (ii) at least one enzyme that cleaves the isopeptide bond of the isopeptidase substrate; and contacting components (i) and (ii) under conditions which allow the enzymatic reaction to proceed; and
• b) Determine the amount of cleaved isopeptidase substrate by determining FRET between the first and the second chromophore of the donor-acceptor pair.

3. The method of claim 1 or 2, wherein the first chromophore is a first fluorescent protein and the second chromophore a second fluores ornamental protein, in particular where the first chromophore is the FRET donor CFP (cyan fluorescent protein) and the second chromophore the FRET acceptor is YFP (Yellow Fluorescent Protein). 4. The method according to any one of claims 1-3, wherein the target protein and / or the ubiquitin-related protein in the form of a fusion protein with a fluorescent protein can be used. 5. The method according to any one of the preceding claims, wherein the mind at least one target protein RanGAP1 and the at least one ubiquitin related protein SUMO (Small Ubiquitin-related Modifier), esp special SUMO1, SUMO2 and / or SUMO3. 6. The method according to any one of the preceding claims, wherein RanGAP1 and CFP in the form of a fusion protein can be used, in particular where the C-terminal domain of RanGAP1 with the N- or C terminal end of CFP is fused.   7. The method according to any one of the preceding claims, wherein SUMO and YFP can be used in the form of a fusion protein, especially where amino acids 1-91 of SUMO1, amino acids 1-92 of SUMO2 or amino acids 1-93 of SUMO3 with the C-terminal end merged by YFP. 8. The method according to any one of the preceding claims, wherein the Zielpro tein, the ubiquitin-related protein and / or the enzymes in the form of a purified protein and / or a cell extract, in particular an Ex tracts from mammalian, insect, yeast and / or bacterial cells are used become. 9. The method according to any one of the preceding claims, wherein a derivative the target protein, the ubiquitin-related protein and / or the enzymes is used. 10. The method according to any one of claims 1, 3-9, wherein the enzymes according to Step (a) (iii) of claim 1 are the E1 and E2 enzymes, optionally in Combination with at least one E3 enzyme, in particular where that E1 enzyme is the Aos1 / 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 claim 2 is an isopeptidase (what about C-terminal hydrolases), in particular Ulp1, Ulp2, SUSP1 and / or Senp 1. 12. A method of determining substances that affect the modification of a target protein with ubiquitin-related proteins, the method comprising the following steps:

• 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 (fluorescence resonance energy transfer), (ii) at least one ubiquitin-related protein, the is 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 the components (i) - (v) under conditions which allow the enzymatic reaction to proceed; and
• b) Detect the formation of conjugate from target protein and ubiquitin-related protein and / or determine the amount of conjugate formed by determining FRET between the first and the second chromophore of the donor-acceptor pair.

13. The method according to claim 12, wherein the enzymes according to step (a) (iii) of the E1 and E2 enzymes are, optionally in combination with at least an E3 enzyme, in particular the E1 enzyme comprising the Aos1 / Uba2 Heterodimer and the E2 enzyme is Ubc9. 14. The method according to claim 12 or 13, wherein the at least one to te stende a protein, in particular an E3 ligase, isopeptidase, Kina se or phosphatase, or derivative thereof, or a low molecular weight Substance, especially an inhibitor or activator. 15. A method for determining substances that affect the demodification of a protein linked to at least one ubiquitin-related protein, the method comprising the following steps:

• a) providing a test system comprising (i) an isopeptidase substrate, 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, which is covalently linked to a second chromophore of the donor-acceptor pair; wherein these are 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 cleaves 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) Determine the amount of cleaved isopeptidase substrate by determining FRET between the first and the second chromophore of the donor-acceptor pair.

16. The method according to claim 15, wherein the at least one enzyme according to Step (a) is an isopeptidase, in particular Ulp1, Ulp2, SUSP1 and / or SENP1. 17. The method according to claim 15 or 16, wherein the at least one to te constant substance is a protein, especially a binding partner for the target protein, a kinase or phosphatase, or derivative thereof, or one low molecular weight substance, especially an inhibitor or activator. 18. The method according to any one of claims 12-17, wherein the protein or De rivat thereof and / or the low molecular weight substance from an expressi onsbank or a combinatorial bank, especially a kombina toric peptide, non-peptide or small molecule library, or from one Cell extract originates and / or is present in a purified form.   19. The method according to any one of claims 12-18, wherein the first chromophore a first fluorescent protein and the second chromophore a second is fluorescent protein, especially where the first chromophore is the FRET donor CFP (cyan fluorescent protein) and the second chromophore the FRET acceptor is 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 in the form of a fusion protein can be used with a fluorescent protein. 21. The method according to any one of claims 12-20, wherein the at least one Target protein RanGAP1 and the at least one ubiquitin-related pro is a SUMO (Small Ubiquitin-related Modifier), especially SUMO1, SUMO2 and / or SUMO3. 22. The method according to any one of claims 12-21, wherein RanGAP1 and CFP be used in the form of a fusion protein, in particular wherein the RanGAP1 C-terminal domain with the N- or C-terminal end merged by CFP. 23. The method according to any one of claims 12-22, wherein SUMO and YFP in Form of a fusion protein can be used, in particular where the Amino acids 1-91 from SUMO1, amino acids 1-92 from SUMO2 or amino acids 1-93 of SUMO3 with the C-terminal end of YFP are merged. 24. The method according to any one of claims 12-23, wherein the target protein, the Ubiquitin-related protein and / or the enzyme in the form of a gerei extracted protein or a cell extract, in particular an extract Mammalian, insect, yeast and / or bacterial cells can be used.   25. The method according to any one of claims 12-24, wherein the substance to be tested in a further step using suitable processes, in particular moles biological, biochemical and / or biophysical processes, is identified. 26. The method according to any one of claims 12-25, wherein a derivative of the target proteins, the ubiquitin-related protein and / or the enzymes ver is applied. 27. The method according to any one of claims 12-14, and 18-26, wherein the in Bringing the components (i) - (iv) into contact in a cell outside the cell human or animal body, especially in one Mammalian or yeast cell; especially where the fusion protein from target protein and the first fluorescent protein and / or the fusion pro tein from the ubiquitin-related protein and the second fluorescence protein is expressed in the cell. 28. The method of claim 27, wherein the at least one substance to be tested is expressed in the cell, preferably by the cell with a for cDNA encoding this substance is transfected, and / or being the cell is incubated with the at least one substance to be tested. 29. A method for detecting a defect in the modification of proteins with ubiquitin-related proteins, the method comprising the following steps:

• 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 (fluorescence resonance energy transfer), and (ii) at least one ubiquitin-related protein, that is 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 that allow conjugation of the target protein and ubiquitin-related protein; and
• b) Determine the amount of conjugate formed by determining FRET between the first and second chromophore of the donor-acceptor pair.

30. A method of detecting a defect in the demodification of proteins linked to ubiquitin-related proteins, the method comprising the following steps:

• a) providing a test system comprising (i) an isopeptidase substrate, 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, which is covalently linked to a second chromophore of the donor-acceptor pair; which are linked by an isopeptide bond between the E-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 components (i) - (ii) under conditions that allow cleavage of the conjugate of target protein and ubiquitin-related protein; and
• b) Determine 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 from cells of Tissue of an individual to be examined and / or from cells Cell culture comes from.   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 covalently linked to a first chromophore of a donor-acceptor pair for FRET (fluorescence resonance energy transfer), (ii) at least one ubiquitin-related protein , which is 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) Detect the formation of conjugate from target protein and ubiquitin-related protein and / or determine the amount of conjugate formed by determining FRET between the first and the second chromophore of the donor-acceptor pair.

33. The method of claim 32, wherein contacting the stock divide (i) - (iv) in a cell outside the human or animal Body is effected, especially in a mammalian or yeast cell. 34. The method of claim 32 or 33, wherein the at least one to te target protein in the form of a fusion protein with a first fluo resected protein, preferably as a fusion protein with the FRET Donor CFP is used. 35. The method of claim 34, wherein the at least one fusion protein comes from an expression bank, in particular one for the fusion protein  coding cDNA introduced into a cell and expri therein is lubricated. 36. The method according to any one of claims 32-35, wherein the second chromo phor a second fluorescent protein, preferably the FRET Is acceptor YFP; in particular where the at least one ubiquitin related protein in the form of a fusion protein with the second fluores ornamental protein is expressed in a cell. 37. The method according to any one of claims 32-36, wherein the at least one Ubiquitin-related protein SUMO (Small Ubiquitin-related Modifier) is, in particular SUMO1, SUMO2 and / or SUMO3. 38. The method according to claim 37, wherein a fusion protein comprising SUMO and YFP is used, in particular where amino acids 1-91 from SUMO1, the amino acids 1-92 from SUMO2 or the amino acids 1-93 of SUMO3 are fused to the C-terminal end of YFP. 39. The method according to any one of claims 32-38, wherein the enzymes according to Step (a) (iii) of claim 24, the E1 and E2 enzymes, optionally in Combination with at least one E3 enzyme, in particular where the E1 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 enzyme in the form of a gerei extracted protein or a cell extract, in particular an extract Mammalian, insect, yeast and / or bacterial cells can be used. 41. The method according to any one of claims 32-40, wherein it is the min at least a target protein to be tested to a derivative of a known  Target protein, and / or wherein a derivative of the ubiquitin related protein and / or the enzyme is used. 42. The method according to any one of claims 32-41, wherein the target protein in a further step by means of suitable processes, in particular moles biological, biochemical and / or biophysical processes, is identified. 43. A kit for detecting modifications of proteins with ubiquitin-related proteins, comprising:

• a) at least one target protein and / or derivative thereof which is covalently linked to a first chromophore of a donor-acceptor pair for FRET (fluorescence resonance energy transfer) and / or a nucleic acid coding therefor; and
• b) at least one ubiquitin-related protein and / or derivative thereof, which is covalently linked to a second chromophore of the donor-acceptor pair, and / or a nucleic acid coding therefor; as well as optional
• c) 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 therefor; and or
• d) ATP and / or an ATP derivative.

44. Fusion protein, the SUMO and a fluorescent protein, preferably YFP, in particular wherein amino acids 1-91 of SUMO1, which Amino acids 1-92 from SUMO2 or amino acids 1-93 from SUMO3 are fused to the C-terminal end of YFP; or a ko for this nucleic acid.   45. Fusion protein, the RanGAP1 and a fluorescent protein, preferred wise CFP, especially where the C-terminal domain of RanGAP1 are fused to the N- or C-terminal end of CFP; or a nucleic acid coding therefor. 46. Cell, especially a mammalian cell outside the human or animal body or a yeast cell containing a fusion protein according to An pronounced 44 and / or 45. 47. Isopeptidase substrate comprising a target protein that is associated with a first Chromophore of a donor-acceptor pair for FRET (Fluorescence Re sonance Energy Transfer) is covalently linked, and an ubiquitin related protein that modifies the target protein and with a second Chromophore of the donor-acceptor pair is covalently linked, the Target protein and the ubiquitin-related protein via at least one Isopeptide bond are linked. 48. Isopeptidase substrate according to claim 47, wherein the at least one Isopeptide bond 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, especially where the isopeptidase substrate a derivative of the target protein and / or the ubiquitin-related protein includes. 49. Isopeptidase substrate according to claim 47 or 48, wherein the ubiquitin related protein is SUMO, especially SUMO1, SUMO2 or SUMO3. 50. Isopeptidase substrate according to claim 49, wherein the target protein for SUMO RanGAP1, RanBP2, PML, Sp100, I _K B _α , D. melanogaster dorsal, p53, c-Jun, HIPK2, S. cerevisiae Cdc3, S. cerevisiae Cdc11, S. cerevisiae Sep7 / Shs1, hCMV IE1, hCMV IE2, D. melanogaster Tramtrack, Topoi somerase I, Glut1, Glut4 or Mdm2. 51. Isopeptidase substrate according to any one of claims 47-50, wherein the first Chromophore a first fluorescent protein and the second chromo phor is a second fluorescent protein, in particular the first Chromophore of the FRET donor CFP (cyan fluorescent protein) and that second chromophore the FRET acceptor YFP (Yellow Fluorescent Prote in); especially where the target protein and / or the ubiquitin related protein in the form of a fusion protein with a fluorescent the protein, particularly preferably in the form of CFP-RanGAP1 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 Analysis of modifications of proteins with ubiquitin-related Proteins, for the determination of substances, the modification of a target pro affect proteins with ubiquitin-related proteins for identification of target proteins for modification with ubiquitin-related protein and / or for diagnosis, in particular for the detection of 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 demodification of proteins with ubiquitin related proteins, for the determination of substances, the demodification target protein with ubiquitin-related proteins, and / or for diagnosis, in particular for the detection of a defect in the Demodification of proteins with ubiquitin-related proteins. 54. Process for the preparation of a pharmaceutical composition, comprising the method according to any one of claims 12-28, and the  Mixing the tested and / or identified substance with a phar pharmaceutically acceptable carriers. 55. Process for the preparation of a pharmaceutical composition, comprising the method according to any one of claims 32-42, and the Mixing the identified target protein or a derivative thereof with a pharmaceutically acceptable carrier.