AT8820U1 - USE OF A COMPOUND TO INCREASE THE EXPRESSION OF MEMBRANE PROTEINS ON THE CELL SURFACE - Google Patents

Abstract

The present invention is directed to the use of a compound that stimulates deubiquitin activity in a cell for the manufacture of a medicament for enhancing the expression of integral membrane proteins on the cell surface. In particular, the invention is directed to the use of such a compound for the manufacture of a medicament for the treatment of a disease or condition selected from the group consisting of cystic fibrosis, diabetes insipidus, hypercholesterolemia and long QT syndrome-2.

Description

2 AT 008 820 U1

Membrane proteins, in particular integral membrane proteins, must be cotranslationally introduced into the endoplasmic reticulum. This is done via the translocon, which is a channel formed by the Sec61 subunits. During and after the synthesis of membrane proteins in the endoplasmic reticulum, they are subjected to a rigorous quality control to ensure proper folding before being transported to their final site of action.

Several aspects of this quality control are not yet fully understood; however, it is clear that incorrect folding of a membrane protein is sensed by the mechanism of the endoplasmic reticulum (ie, presumably chaperone). This leads to activation of ubiquitinating enzymes on the cytoplasmic side. These transfer ubiquitin to the cytoplasmic peptide chain of the incorrectly folded protein, which is retrotranslocated through the Sec61 channel and degraded by the 26S proteasome (Kostova and Wolf, 2003). It must be emphasized that this scheme is predominantly based on observations 15 made in Saccharomyces cervisiae. However, based on several experimental evidence, it can reasonably be assumed that the higher eukaryotes utilize a related mechanism for eliminating misfolded proteins.

Increasingly, it has been recognized that many human diseases can be linked to mutations that result in the retention of the abnormal protein in the endoplasmic reticulum (ER). Cystic fibrosis is most frequently cited as a model disease: more than 1000 mutations were identified in the gene encoding the CFTR (cystic fibrosis transmembrane conductance regulator) (Rowntree and Harris, 2003), the majority of patients (~ 70%). ), however, has the AF508 mutation of the CFTR. 25

The resulting protein can function properly when it reaches the plasma membrane; however, it does not reach the plasma membrane due to an over-cautious ER quality control mechanism (Pasyk and Foskett, 1995). There are numerous other examples that lead to erroneous ER export of membrane proteins; these include mutations of the 30V2 vasopressin receptor (associated with diabetes insipidus, Oksche and Rosenthal, 1998), the LDL receptor (leading to hypercholesterolemia, Hobbs et al., 1990, Jörgensen et al., 2000). or the HERG K + channel (leading to a long QT syndrome-2; Kupershmidt et al., 2002) etc. It is unclear why these mutant proteins are retained and eventually degraded, although they are functionally active, at least in part are (see Pasyk and Foskett, 1995). The available evidence, however, suggests that the quality control mechanism in the endoplasmic reticulum is overly cautious. It is an object of the present invention to provide means for enhancing the expression of membrane proteins, particularly integral membrane proteins, at the cell surface. In particular, it is an object of the present invention to provide means for enhancing expression of a protein selected from the group consisting of CFTR (cystic fibrosis trans-membrane conductance regulator), V2-vasopressin receptor, LDL- Receptor and HERG K + channel, and further in the provision of a medicament for the treatment of a disease or condition selected from the group consisting of cystic fibrosis, diabetes insipidus, hypercholesterolemia and long QT syndrome-2. So this object is fulfilled by the subject of the main claims. Preferred embodiments are disclosed in the subclaims.

It has been found that stimulating deubiquitinating activity in a cell, in particular by increasing the amount of deubiquitinating enzymes in the cell or by stimulating it, enhances the expression of integral membrane proteins on the cell surface 3 AT 008 820 U1. It appears that deubiquitinating enzymes are able to reduce the degree of over-careful quality control in the endoplasmic reticulum.

Several therapeutic concepts that could overcome the stringent quality control have been proposed (see, e.g., Cohen & Kelly, 2003). However, enhancement of deubiquitinating activity has not yet been proposed as a strategy that would allow for enhanced surface expression of membrane proteins and mutated versions thereof. In particular, an increase in the amount of deubiquitinating enzymes in the cell can be achieved by introducing into the cell a compound selected from the group consisting of a deubiquitinating enzyme 15 - a nucleic acid sequence encoding a deubiquitinating enzyme.

Specifically, the cell can be transfected with a suitable plasmid containing DNA 20 encoding the deubiquitinating enzyme, followed by expression of the enzyme in the cell.

The modes of introducing a deubiquitinating enzyme or the nucleic acid sequence encoding it, as well as identifying appropriate amounts of a verbin compound to be introduced, are known to those skilled in the art or can be determined using knowledge readily available to those skilled in the art.

Preferably, the deubiquitinating enzyme is selected from the group consisting of ubiquitin carboxy-terminal hydrolases (UCH) and ubiquitin-specific proteases (USP). Thirty USPs are also referred to as ubiquitin-processing proteases (UBPs, Wing, 2003).

Deubiquitinating enzymes are thiol proteases that hydrolyze the amide bond between Gly76 of ubiquitin and the substrate protein. There are two classes of deubiquitinating enzymes; the ubiquitin-specific processing protease or USP class is one of these two known classes of deubiquitinating enzymes (Papa and Hochstrasser, 1993). Although catalytic activity has been tested using artificial substrates, very little is known about their physiological substrates and thus their physiological functions. It has been shown that USPs in the determination of cell fate (fat facets, Huang et al (1995), transcriptional silencing (UBP3, Moazed and Johnson, D. (1996)), 40 responses to cytokines (DUB1 and 2 Zhu et al, 1996) and the bulking transformation (tre-2, USP4; Gilchrist and Baker, 2000), but the mechanical details remained enigmatic.

In a particularly preferred embodiment, the deubiquitinating enzyme is USP-4. The sequence of the murine USP-4 enzyme is described, for example, in Strausberg, R.L., et al .; Proc. Natl. Acad. Be. U.S.A. 99 (26), 16899-16903 (2002). Human USP-4 exists in two variants, cf. Puente, X.S. et al., Nat. Rev. Genet. 4 (7), 544-558 (2003).

Preferably, to enhance the expression of integral membrane proteins on the cell surface, the medicament additionally comprises a compound selected from the group consisting of a proteasome inhibitor and a nucleic acid sequence encoding a proteasome inhibitor. 55 4 AT 008 820 U1

It has been found that the additional influence of a proteasome inhibitor in conjunction with deubiquitinating enzymes results in even more significant expression of the membrane proteins at the cell surface. The fact that proteasome inhibitors can enhance the expression of membrane proteins on the cell surface is known per se, cf. e.g. 5 Jensen TJ et al .; Cell. 1995 Oct 6; 83 (1): 129-35.

Preferably, the proteasome inhibitor is MG132. MG132 is a tripeptide aldehyde with the structure leucyl-leucyl-norleucinal (LLnL). In particular, the method of the present invention enables the expression of a protein selected from the group consisting of CFTR (regulator of transmembrane conductance in cystic fibrosis), V2-vasopressin receptor, LDL-receptor and HERG-K * - Channel. Furthermore, the method of the present invention may be used to treat conditions or diseases associated with or associated with the lack of expression of membrane proteins at the cell surface.

In particular, the method of the present invention enables the treatment of a disease or condition selected from the group consisting of cystic fibrosis, diabetes insipidus, hypercholesterolemia and long QT syndrome-2.

The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound that stimulates a deuteribucinating activity in a cell.

Preferably, this compound is selected from the group consisting of a deubiquitinating enzyme 30 - a nucleic acid sequence encoding a deubiquitinating enzyme. In addition, the pharmaceutical composition of the invention preferably additionally comprises a therapeutically effective amount of a compound selected from the group consisting of a proteasome inhibitor and a nucleic acid sequence encoding a proteasome inhibitor.

Brief description of the figures 40

Figure 1 shows the coexpression of A 1 receptor and USP 4 in HEK293 cells: HEK293 cells were transiently transfected with the following sets of plasmids: CFP-labeled A 1 receptor (= A2aR) (Figures A, E); CFP-labeled A2aR and GFP-labeled 45 USP4 (Figures B, F); CFP-labeled A2AR (1-311) (Figure C); CFP-labeled A2AR (1-311) GFP-labeled USP4 (Figure D).

The cells were incubated in the presence of the proteasome inhibitor MG132 (50 μΜ) for 3 h (FIG. E, F). 24 hours later pictures were taken with the appropriate filter settings. The experiments were carried out three times with comparable results.

Fig. 2 shows the deubiquitination of the A ^ receptor by USP4:

Immunoprecipitation of the A2A receptor RR) was performed from HEK293 cells transfected transiently with the following sets of plasmids: Flag-labeled 5 AT 008 820 U1

AzaR, HA-labeled ubiquitin (lanes 1, 2); Flag-labeled A ^ R, HA-labeled ubiquitin and GFP-labeled USP4 (lanes 4, 5); GFP-labeled USP4 and / or HA-labeled ubiquitin (lanes 6, 3 = control lanes). Cells were collected 48 hours after transfection and membrane preparation and immunoprecipitation were performed as described below. After electrophoretic transfer, membranes were stained with proteins with an anti-Flag antibody (1: 500 dilution) to visualize A2A receptor immunoreactivity (upper panel), then stripped at 50 ° C for 30 min incubated anti-HA antibody to stain io ubiquitin (lower panel). The data come from a representative experiment that was repeated 3 times.

Figure 3A shows saturation isotherms for specific binding of [3H] ZM241385 to membranes from transiently transfected HEK293 cells expressing the full length A 2A receptor:

Membranes were prepared from HEK293 cells transfected with plasmids expressing the expression of the full-length flagged A2A receptor and the enhanced green fluorescent protein (pEGFP) or the full length A2a receptor and 20 GFP-labeled USP4 (= UBP4 = ENP-GFP) pushed ahead; these membranes were incubated in a buffer containing the indicated concentrations of [3H] ZM241385 in the presence of 100 μM GTPgS. Data A & B are means of duplicate determinations in a representative experiment that was repeated three times (the mean parameters are tabulated). 25

Figure 3B shows saturation curves for specific binding of [3H] ZM241385 to membranes from transiently transfected HEK 293 cells containing the truncated versions of the A2A receptor [A2AR (1-311) and A ^ Ri 1-360)] with or express without USP4. The analysis conditions were as described in Fig. 3A. 30

Figure 3C shows the summary of Bmax values from panels A & B and saturation experiments performed on membranes from cells incubated in the absence and presence of the proteasome inhibitor MG132 (50 μΜ) for 3 hours: 35 The results are mean ± standard deviation from 4 independent experiments performed in parallel and with duplicate determinations. The asterisk indicates a significant deviation from full length A2aR at p = 0.001 (unpaired t-test):

Figure 4 shows the stimulation of cAMP accumulation in transiently transfected 40 HEK293 cells:

Cells expressing only the full length A ^ receptor (circles) or the combination of A2A receptor and USP4 (triangles) were inoculated in 6-well dishes, the cellular adenine nucleotide pool was metabolically metabolized with [for 16 h]. 3H] adenine pre-marked. After 45 minutes pre-incubation in a fresh medium containing adenosine deaminase (2 U / ml), cAMP production was stimulated by the indicated concentrations of the A ^ -selective agonist CGS 21680. Data are means ± standard deviation from 4 independent experiments performed in triplicate; in each individual experiment, the receptor alone and cotransfected with USP4 were always analyzed in parallel. 50

Figure 5 shows saturation curves for specific binding of [3H] ZM241385 to membranes from PC12 cells (expressing the A2A receptor endogenously):

Membranes were prepared from PC12 cells incubated in the presence or absence of 55 μM MG132 or 100 μL chloroquine for 3 hours and were resuspended in a 6 AT 008 820 U1

Buffer containing the indicated concentrations of [3H] ZM241385, incubated in the presence of 100 μM GTPyS.

Examples 5

In the following examples, the A 1 adenosine receptor was used as a model protein for the following reasons: (i) The A 1 adenosine receptor is a prototypical G protein-coupled receptor and thus one of a class of> 1000 receptors (many of which are of obvious therapeutic interest as they serve as drug targets). (ii) It has been documented that G protein-coupled receptors have a folding problem; in other words, much of the newly synthesized protein (£ 50%) undergoes degradation in the endoplasmic reticulum and does not reach the plasma membrane (Petaja-Repo et al., 2000 &2001; Pankevych et al., 2003). This is similar to the situation with many other membrane proteins with multiple transmembrane transponders, especially with CFTR (Jensen et al., 1995, Rowntree and Harris, 2003). 20 (iii) There is at least one disease in which mutations cause the retention of a G protein-coupled receptor in the endoplasmic reticulum: in some cases, diabetes insipidus arises from point mutations of the gene encoding the V2 vasopressin receptor, which is associated with a ER retention of the receptor (Oksche and Rosenthal, 1998). 25

Materials and Methods Radioligand Binding Analyzes: Thirty membranes (100 pg / analysis) prepared from PC12 cells or HEK293 cells transiently transfected with the appropriate plasmids were obtained in a final volume of 0.3 ml containing 50 mM Tris. HCI (pH 8.0), 1mM EDTA, 5mM MgC12, 8 pg / ml adenosine deaminase and concentrations of [3H] ZM241385 (specific activity ~ 20 Ci / mmole) covering a range of 0.2 to 20 nM, in the presence of 35 100 μM GTPyS incubated (Klinger et al., 2002). After 60 minutes at room temperature, the

Reaction terminated by rapid filtration over glass fiber filters. Non-specific binding was detected in the presence of 10 μΜ XAC and was 40% at the highest concentration of [3H] ZM241385. The data points were fit by nonlinear regression to the equation describing a rectangular hyperbola. The analyzes were carried out in duplicate.

Agonist-mediated cellular cAMP accumulation:

Cells were grown in 6-well dishes. The adenine nucleotide pool was metabolically labeled 45 by incubating confluent monolayers as described with [3H] adenine (1 pCi / well) for 16 h (Kudlacek et al., 2001). After preincubation, fresh medium containing 100 μM RO201724 (a phosphodiesterase inhibitor) and adenosine deaminase (2 U / ml) was added to remove any endogenously produced adenosine. After 1 h, cAMP formation was induced by the A2A-selective agonist CGS21680 (1 nM to 1 μΜ) for 15 minutes, and the reaction was stopped by adding 2.5% perchloric acid to 100 μΜ cAMP (1 ml / dish). were added. The supernatant (0.9 ml) was aspirated, neutralized with 100 μl of 0.4 μl KOH and diluted with 1.5 ml of 50 mM Tris-HCl, pH 8.0. [3H] cAMP was isolated by sequential chromatography on Dowex AG 50W-X4 and neutral alumina columns (Salomon (1991)). The analyzes were carried out in triplicate. 55 7 AT 008 820 U1

Immunoprecipitation of epitope-tagged A ^ adenosine receptor: HEK293 cells stably expressing the FLAG-labeled A2A adenosine receptor were washed three times with phosphate buffered saline; then membranes 5 were added to ice-cold lysis buffer [50mM Tris.HCl, pH 7.5, 1mM EDTA, 150mM NaCl containing 1% Nonidet P-40 (vol / vol), protease inhibitors (Complete, Roche Molecular Biochemicals ) and, if indicated, 10 mM N-ethylmaleimide (NEM)] for 1 hour on ice. The insoluble material was collected by centrifugation at 16,000 x g and 4 ° C for 10 minutes. The supernatant was treated for immunoprecipitation in which each step was carried out with constant io rotation at 4 ° C. Thereafter, 40 μl of a 50% (vol / vol) suspension of Anti-Flag M2 affinity gel (Sigma Chemical) was added and the sample was incubated overnight. The beads were collected by centrifugation and washed three times in 1 mL Tris-buffered saline. Immune complexes were dissociated in an SDS-polyacrylamide sample buffer containing 20 mM dithiothreitol by incubation at 37 ° C for 1 hour or alternatively incubation at 95 ° C for 5 minutes. Using a semi-dry transfer system, proteins were transferred to nitrocellulose membranes (Immobilon-P, Millipore); using monoclonal peroxidase-conjugated anti-FLAG and anti-HA antibodies, immunodetection was achieved to detect the FLAG epitope of the A2aR and the HA epitope of ubiquitin, respectively. The GFP portion in USP4 was detected with an anti-GFP-20 antiserum (Living colors A.v.) and a horseradish peroxidase-conjugated secondary anti-rabbit IgG antibody. The immunoreactive bands were developed with the improved chemiluminescence detection kit (Pierce SuperSignal).

Fluorescence microscopy: 25

Transiently transfected HEK-293 cells were examined 1 day after transfection with a reverse epifluorescence microscope (Zeiss Axiovert 200M) using a 63x oil immersion objective and filter sets that discriminated between CFP and YFP fluorescence (Chroma Technology Corp.). Brattleboro, VT). Photographs were taken with a cooled CCD camera (CoolSNAP fx; Photometrics, Roper Scientific, Tucson, AZ) and stored and processed with MetaSeries software (4.6 Metafluor and Metamorph, Universal Imaging).

Results USP4 enhances cell surface expression of the A2AA adenosine receptor

To visualize the A2A adenosine receptor in living cells, the receptor was labeled at its carboxyl terminus with the cyan fluorescent protein (CFP, a spectral aberrant variant of the green fluorescent protein of Aequoria victoria). This receptor binds ligands and activates its downstream signaling cascade in a manner that does not differ from that of the unlabeled receptor (data not shown). Fluorescence microscopy showed that much of the receptor accumulates within the cell when expressed in HEK293 cells (Figure 1A). 45

The fluorescently labeled A 1 adenosine receptor was predominantly found on the plasma membrane when the cells are cotransfected with a plasmid that promotes the expression of the deubiquitating enzyme USP4 (Figure 1B). It is believed that in the current model, endoplasmic reticulum quality control requires ubiquitination of the carboxyl end (Kostova and Wolf, 2003). Therefore, it was investigated whether truncation of the carboxyl end of the A ^ receptor should render the receptor insensitive to the action of USP4. This was the case: a comparison of Figures 1C and 1D shows that the absence and presence of USP4 does not affect that part of the fluorescent receptor trapped in the cell. 8 AT 008 820 U1

Finally, it was investigated whether inhibition of proteosomal degradation would also attenuate quality control and thus allow the receptor to exit the endoplasmic reticulum. The addition of the proteasome inhibitor MG132 actually increased the amount of receptor on the cell surface (see Figures 1E and 1A); in the presence of 5 of both, USP4 and MG132, substantially all of the receptor was found on the cell surface (Figure 1F).

Co-expression of USP4 leads to the accumulation of deubiquitinated A ^ receptor To demonstrate that USP4 employed the A 1 receptor as substrate, HEK293 cells were transiently cotransfected with plasmids encoding the flag-labeled A 1 adenosine receptor, HA-labeled ubiquitin and GFP-labeled USP4 encoded.

The A2A adenosine receptor was probed with anti-Flag antibodies from cell purification lysates containing either only HA-labeled ubiquitin (Figure 2A, lanes 1, 2) or the combination of HA-labeled ubiquitin and USP4 (Figure 2B , Lanes 4, 5) coexpressed, immunoprecipitated: Receptor bands were detected with anti-Flag antibody (blots shown above); in the absence of USP4, FLAG-reactive immunostaining was in the range of -48-50 kDa (Figure 2A above, lanes 1, 2); in the presence of USP4, the FLAG-labeled receptor 20 migrated at -40-42 kDa (Figure 2B above, lanes 1, 2).

Lanes 3 and 6 represent the negative controls, i.e., immunoprecipitation was performed with cell lysates lacking the A2A adenosine receptor but containing HA-labeled ubiquitin and -bit 6 - USP4. Regardless of the conditions, no immunoreactivity was recovered in either the 25-40-42 kDa or the -48-50 kDa range. Thus was the

Immunostaining specific.

The nitrocellulose membranes were stripped and stained with anti-HA antibodies (Figures 2A & B, bottom blots). In cells cotransfected with those plasmids encoding the Flag-30 labeled A ^ adenosine receptor and HA-labeled ubiquitin, the HA antibody stained a -48-50 kDa band. This corresponded to the ubiquitinated form of the A2A receptor since this band was also stained with the anti-HA antibody (see Fig. 2A, upper and lower blots). In contrast, the A2A receptor, which migrated as a band of 40-42 kDa (Figure 2B, supra, lanes 4 & 5), was not detected with the anti-HA antibody when coexpressed with USP4 , This band therefore represents the deubiquitinated type of receptor.

Coexpression of USP4 Enhances Expression of Functional A ^ Receptors 40 As documented in Figure 1, USP4 caused redistribution of the CFP-labeled A2a receptor to the cell surface. It is conceivable that attenuating quality control by co-expressing USP4 allowed leakage of unfolded receptors from the endoplasmic reticulum. In order to rule out this possibility, binding assays were performed with [3H] ZM241385, a specific and selective A ^ receptor antagonist (Palmer et al., 1995). Figure 3A shows a set of representative saturation curves for specific binding of [3H] ZM241385 to membranes from HEK293 cells transfected exclusively with a plasmid expressing (either the CFP or FI_AG labeled) A2A -50 receptor or receptor and USP4 propel. Co-expression of USP4 (Figure 3, red symbols) increased Bmax, but did not affect the affinity of the radioligand. This effect of USP4 depended on the carboxyl end of the A ^ receptor, as in the truncated forms A ^ receptor-O-SH or A ^ receptor 1-360), the last 100 and the last 50th, respectively Amino acids were missing, not seen; representative saturation curves are shown in Fig. 3B 55; Bmax, whose average was determined from several saturation attempts, is in 9 AT 008 820 U1

Bar chart of Fig. 3C shown.

The quality control model in the endoplasmic reticulum leads to the assumption that all steps are reversible, provided that the carboxyl end of the membrane protein has not yet been enveloped by the proteasome (Kostova and Wolf, 2003). Accordingly, it was investigated whether the effect of USP4 and proteasome inhibition is additive. This was the case. As can be seen from the Bmax averages summarized in Figure 3C, the sole addition of MG132 caused a marked increase in the amount of functional receptors, but the combined presence of both, USP4 and MG132, resulted in a dramatic increase in the number of receptors.

The A2A adenosine receptor is a prototypical Gs-coupled receptor, thus activation of the receptor leads to the stimulation of adenylyl cyclase. Binding data showed that co-expression of USP4 increased the number of functional receptors. This conclusion was independently verified by measuring the accumulation of agonist-induced cellular cAMP. In cells expressing USP4, the agonist CGS21680 elicited a greater maximal effect than in cells expressing only the A 1 adenosine receptor (Figure 4). It should be noted that this is not a non-specific effect, which may be explained, for example, by an increased responsiveness of the catalytic portion of adenylyl cyclase in the presence of USP4. Control experiments showed that cells expressing only the A ^ receptor or the A ^ receptor and USP4 did not differ in their responsiveness to forskolin.

All experiments so far relied on transient transfection to demonstrate the ability of USP4 to enhance expression of the A 1 receptor. Therefore, PC12 cells, a rat pheochromocytoma cell line in which the A2A receptor is physiologically expressed at high levels, have also been used. Addition of the proteasome inhibitor MG132 also increased the membrane concentration of the A2A receptor (▲ in Figure 5). In contrast, the lysosomal inhibitor chloroquine did not affect the A2A receptor levels (▼ in Fig. 5).

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Gilchrist, C.A., Baker, R.T. (2000) Characterization of the ubiquitin-specific protease activity of the mouse / human unp / unph oncoprotein. Biochim Biophys Acta 1481, 297-309.

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Kupershmidt S, Yang T, Chanthaphaychith S, Wang Z, Towbin JA, Roden DM. (2002) Defective human ether-a-go-go-related gene trafficking linked to endoplasmic reticulum retention Signal in the C terminus. J. Biol. Chem. 277; From 27,442 to 27,448. 15 Moazed, D., Johnson, D. (1996) A deubiquitinating enzyme interacts with SIR4 and regulate silencing in S. cerevisiae. Cell 86: 667-677.

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Pankevych H, Korkhov V, Freissmuth M, Nanoff C. (2003) Truncation of the aradenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling. J. Biol. Chem. 278: 30283-30293. 30 Papa F.R, Hochstrasser M. (1993) The yeast DOA4 gene encodes a deubiquitinating enzyme related to a human tre-2 oncogene. Nature 366: 313-319.

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Claims (6)

11 AT 008 820 U1 ubiquitin proteaseome pathway. IJBCB 35: 590-605. Zhu Y, Pless M, Inhorn R, Mathey-Prevot B, D'Andrea AD. (1996) The murine DUB-1 gene is specifically induced by the beta subunit of interleukin-3 receptor. Mol Cell Biol. 76: 4808-4817. Claims 1. Use of a compound selected from the group consisting of io - a ubiquitin-specific protease (USP) and a nucleic acid sequence encoding a ubiquitin-specific protease (USP) for the manufacture of a medicament for the treatment of a disease or condition which which is selected from the group consisting of cystic fibrosis, diabetes insipidus, hypercholesterolemia and long QT syndrome-2. 15 2. Use according to claim 1, characterized in that the ubiquitin-specific protease is USP-4. Use according to any one of the preceding claims, characterized in that the medicament additionally comprises a compound selected from the group consisting of a proteasome inhibitor and a nucleic acid sequence encoding a proteasome inhibitor. 4. Use according to claim 3, characterized in that the proteasome inhibitor MG 132. A pharmaceutical composition comprising a therapeutically effective amount of a ubiquitin-specific protease (USP) and 3o - a nucleic acid sequence encoding a ubiquitin-specific protease (USP). A pharmaceutical composition according to claim 5 additionally comprising a therapeutically effective amount of a compound selected from the group consisting of 35 - a proteasome inhibitor and a nucleic acid sequence encoding a proteasome inhibitor. For this purpose 4 sheets of drawings 40 45 50 55