WO2001081427A1 - Construction proteique « rapporteur » contenant partiellement une proteine gfp - Google Patents

Construction proteique « rapporteur » contenant partiellement une proteine gfp Download PDF

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WO2001081427A1
WO2001081427A1 PCT/SE2001/000921 SE0100921W WO0181427A1 WO 2001081427 A1 WO2001081427 A1 WO 2001081427A1 SE 0100921 W SE0100921 W SE 0100921W WO 0181427 A1 WO0181427 A1 WO 0181427A1
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ubiquitin
reporter
proteasome
gfp
lys
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PCT/SE2001/000921
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Maria Masucci
Nico Dantuma
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Karolinska Innovations Ab
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Priority to AU2001252837A priority Critical patent/AU2001252837A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a reporter protein construct partly comprising GFP .
  • the invention refers to a reporter protein construct, partly comprising GFP, for quantification of ubiquitin/proteasome-dependent proteolysis in living cells, and a method for the detection and screening of accumulated reporter constructs.
  • Ubiquitin/proteasome-dependent proteolysis has been implicated in the degradation of proteins that control vital processes such as cell cycle progression, signal transduction, differentiation and apoptosis 1 . It also participates in the clearance of misfolded and damaged proteins 2 ' 3 and in the generation of peptides for MHC class I restricted antigen presentation 4 .
  • These diverse roles in proteolysis place the ubiquitin/proteasome pathway at the core of pathologic processes such as inflammation, autoimmunity, neurodegenerative diseases and cancer (reviewed in 5), and motivates the large efforts devoted to elucidate its mechanism of action and identification of compounds that may allow modulation in living cells 6,7 . Indeed, proteasome inhibitors have been proposed as antitumor agents 8,9 and the cancer drugs epoxomicin and eponemycin interact specifically with the proteasome 10,11 .
  • the proteasome is a multicatalytic multisubunit protease composed of a proteolytic core, the 20S proteasome, sandwiched between 19S/PA700 or 11S/PA28 regulatory complexes (reviewed in 12).
  • the 19S regulator is involved in the ATP-dependent recognition and unfolding of proteins that are "marked” for degradation by covalent attachment of multiple ubiquitin molecules (reviewed in 1).
  • the specificity of this process is safeguarded by signals that identify the substrates for recognition and ubiquitin-dependent degradation 13 .
  • the 20S core consists of two heptameric outer ⁇ -rings and two inner ⁇ -rings that form a barrel-like structure.
  • ⁇ subunits Only three of the ⁇ subunits, ⁇ 5 (X), ⁇ 2 (Z), and ⁇ l (Y), are armno-terminal threonine hydrolases that execute proteolysis with chymotrypsin-like, trypsin-like and post glutamyl peptide hydro lysing (PGPH) specificity, respectively 14 .
  • PGPH glutamyl peptide hydro lysing
  • W099/11774 discloses a polypeptide- reporter molecule that may comprise a ubiquitin part, a GFP-part and a "coiled coir'-domain, as well as a method in which this molecule is used
  • W098/57978 refers to proteasome purification and discloses a fusion protein that may comprise a ubiquitin part and a signal producing part.
  • US- A-5132213 discloses a gene construct comprising a ubiquitin part, a reporter part such as ⁇ -galactosidase, as well as an interjacent part.
  • the interjacent part comprises lysine, which is regarded to be important for destabilisation.
  • a method for producing a fusion protein of the construct above is disclosed. This construct is adapted for use in yeast cells.
  • Application W099/11774 refers to a model protein which can be used for identification of proteins that interact specifically with proteins that undergo posttranslational modifications, such as ubiquitination.
  • Application W098/57987 is based on fusion of a reporter with different type of domains, often referred to as 'ubiquitin-like domain'. The inventors exploited the unique properties of ubiquitinrlike domains for the purification of proteasomes from cell lysates.
  • a reporter construct for proteasomal activity comprising GFP with, for example, a destabilising N-terminus
  • the inventors have succeeded in providing a system, in which proteasomal activity in living cells can be measured through the accumulation of the reporter protein. While the reporter protein preferably is GFP, the accumulation can easily be quantified.
  • the invention refers to a construct encoding a reporter protein comprising a reporter part at the carboxy-terminal and an amino-terminal part comprising ubiquitin (Ub) or a functional analogue.
  • the reporter is characterised by that it further comprises a linker peptide between the reporter part and the ubiquitin, whereby the linker comprises at least one Lys-residue, and whereby position 3 of the linker is occupied by a Lys-residue.
  • the invention concerns a method for the measurement of the activity of the ubiquitin/proteasome pathway, characterised by that the method is performed in living cells, and that it comprises the steps of (1) expressing a reporter construct of any of claims 1-9 into the cell, (2) optionally adding substances to inhibit the ubiquitin/proteasome dependent proteolysis, and (3) measuring the presence of accumulated reporter protein.
  • Such a method could for example be used for high-throughput screening of substances, which selectively modify proteolysis in- vivo.
  • the reporter can be used as a measure of the proteasome inhibiting potential of added substances, or to study whether a cell in it self has an defective proteasome function.
  • the idea of the invention is to use a modified GFP-reporter protein to measure the presence of ubiquitin/proteasome-activity in vivo, whereby the reporter protein accumulates in a cell with defective ubiquitin-proteasome dependent proteolysis or in a cell when substances inhibiting the proteolytic pathway are used.
  • Some of the advantages of the present invention is that it can be used to measure/study proteasomal activity in living cells, it provides a very sensitive detection system, it can be used to measure partial degradation and quantify destabilisation, as well as to screen for other (earlier) steps of the ubiquitin/proteasome pathway. Moreover, it can be used to distinguish between qualitative modification of the pathway and induction that leads to apoptosis. Furthermore, the construct of the invention is specifically aimed for higher eukaryotic cells, specifically mammalian cells.
  • a reporter protein construct here refers to a molecule having the ability to be detected when it accumulates in a cell, such as a molecule having a GFP (green fluorescent protein)- part, and which thus is detectable through fluorescent techniques.
  • GFP green fluorescent protein
  • the reporter part is the part of the fusion protein having the ability to give rise to detectable signals.
  • Reporters such as enzymes, for example ⁇ -galactosidase; bioluminiscence, luciferase, or fluorescent proteins, such as GFP, EGFP; or modified variants of these reporter proteins, can be used.
  • GFP is used.
  • the ubiquitin part refers to the part of the reporter protein construct comprising a ubiquitin molecule or any functional analogue, such as a ubiquitin having one or more mutations.
  • a functional analogue refers to any proteins or protein domains that once inserted at the amino terminus of a substance will target this substance for degradation by the proteasome.
  • GFP is a widely used reporter in studies of gene expression and protein localisation 26 .
  • the inventors have exploited these properties to generate the first reporter that allows in vivo quantification of ubiquitin-proteasome-dependent proteolysis in mammalian cells.
  • the inventors show that GFP can be directly targeted to the ubiquitin-proteasome pathway by insertion of N-end rule and UFD degradation signals.
  • N-end rule and UFD targeting signals were shown to generate artificial substrates with variable half-lives in yeast cells, depending on the identity of the N-end residue 17 or efficiency of cleavage of the N-te ⁇ ninal ubiquitin by ubiquitin hydrolases 18 .
  • the efficacy of these targeting signals depended on the presence of a specific linker sequence, e ⁇ , directly adjacent of the ubiquitin moiety.
  • these constructs were found to be stable in cells of higher eukaryotes. Only insertion of a linker derived from a viral protein resulted in destabilization of the substrate although even these optimized substrates remained more stable than the e ⁇ -linker counterparts in yeast (ref).
  • the inventors of the present invention developed a set of Ub-GFP fusions provided with a specific linker sequence that were efficiently destabilized in mammalian cells. This was confirmed in experiments performed by the inventors by the inverse correlation between the steady state expression of the reporters and the level of accumulation induced by treatment with inhibitors of the proteasome. Thus, while Ub G76v -GFP was not detected in stable transfectants, supporting the notion that proteins carrying this UFD targeting signal may be cotranslationally degraded 18 , blockade of the ubiquitin-proteasome pathway resulted in more then 100 fold increase in fluorescence intensity.
  • Positioning of the putative ubiquitination sites in Lys 3 and 17 is likely to be instrumental for this effect, as suggested by the effect of Lys 3 on the degradation of N-end rule-tagged ⁇ -galactosidase in yeast cells 27 .
  • the low background fluorescence and efficient accumulation induced by proteasome inhibitors provide a sensitive tool for quantification in individual live cells, without need for time-consuming biochemical assays.
  • proteasomes lacking one of the activities were shown to generate a new set of peptides from protein substrate in vitro 31 .
  • the inventors results suggest that a similar situation may be achieved in mammalian cells.
  • Three proteasome inhibitors, NP-LLG-VS, YL 3 -VS and ritonavir were unable to prevent the degradation of Ub G76V -GFP over a wide range of concentrations, while accumulation of the reporter was induced by mixtures of the inhibitors.
  • the cleavage of fluorogenic substrates by proteasomes isolated from treated cells demonstrates that these s ⁇ bstances have largely overlapping effects in vivo.
  • the inventors findings identify N-end rule and UFD-tagged GFPs as a new powerful tool for high throughput screening of inhibitors that may interfere with multiple steps of the ubiquitin-proteasome pathway; including ubiquitin tagging, unfolding and proteolysis.
  • the pharmacodynamics of the inhibitors may be readily evaluated by treating stable transfectants in 96 well plates where the efficiency of proteolysis can be analysed by monitoring changes of fluorescence intensity.
  • the possibility to directly correlate ubiquitin/proteasome-dependent proteolysis with toxicity may facilitate the deteirnination of therapeutic index and dose escalation in clinical trials.
  • GFP-reporters for discriminating between inhibitors with cytostatic or immunomodulating activities based on whether they do or do not block ubiquitin-proteasome dependent proteolysis.
  • this reporter system may help to achieve a more complete understanding of the distinct effects of proteasome inhibitors on protein degradation.
  • one main object of the invention is a reporter molecule having one reporter part, preferably GFP, and one of either two destabilising tags, targeting the molecule to a degradation pathway.
  • Another object of the invention is a linker between the reporter part and the Ub-part, whereby the linker functions as a separator that will enhance degradation.
  • the linker sequence should contain an N-end rule destabilizing amino acid at position 1, most preferably an Arg, Glu, Gin, Tip, Phe, Lys, His, Tyr, or Asp residue; less preferable an Ala, Ser, Leu, Thr, Val, He, Gly or Cys residue.
  • Said sequence should also contain a Lys residue at position 3.
  • Other Lys residues may be, but are not necessarily, included in any of the positions between the Lys residue in position 3 and the Lys residue in position 15, 16, or 17.
  • Nj N-end rule residue in position 1
  • X 2 any residue in position 2
  • K 3 Lys residue in position 3
  • X 8-10 polypeptide of 8, 9, or 10 amino acids
  • K b Lys residue in position 13, 14 or 15
  • N ! N-end rule residue in position 1
  • X n . 13 (K) polypeptide of 11, 12, or 13 amino acids with additional Lys residue(s)
  • the length of the linker peptide is normally in the interval from 3 to 30 amino acids, preferably 11-17 amino acids.
  • Still another object of the invention is a DNA construct, encoding the above mentioned peptides and proteins of the invention.
  • One further object of the invention is a method for the measurement of proteasomal activity characterised by that the method is performed in living cells, and that it comprises the steps of (1) introducing a reporter construct into a cell, (2) optionally adding substances to inhibit the proteasomal activity, and (3) measuring the presence of accumulated reporter construct, whereby the accumulation of the reporter construct is used as a measure of ubiquitin-proteasome dependent proteolysis.
  • the introduction of the reporter construct into the cell can be performed by use of a transfer vector, such as a recombinant vaccinia virus vector, adenovirus vector, retrovirus vector, Semliki forest virus vector, and any other type of viral and nonviral vector, comprising a liposome vector.
  • a transfer vector such as a recombinant vaccinia virus vector, adenovirus vector, retrovirus vector, Semliki forest virus vector, and any other type of viral and nonviral vector, comprising a liposome vector.
  • Substances inhibiting the proteasomal activity are for example: - vinyl sulfone-based inhibiotrs such as carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3-VS), nitrophenol leucyl-leucyl-glycine vinyl sulfone (NP-LLG-VS); tyrocyl-leucyl-leucyl-leucine vinyl sulfone (YL3-VS) 22,23 .
  • Z-L3-VS carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone
  • NP-LLG-VS nitrophenol leucyl-leucyl-glycine vinyl sulfone
  • YL3-VS tyrocyl-leucyl-leucyl-leucine vinyl sulfone 22,23 .
  • MG132 carboxybenzyl-leucyl-leucyl-leucinal
  • the presence of accumulated reporter molecule can for instance be measured by FACS-analysis or any other fluorescent-based detection method.
  • Blockage of the ubiquitin-proteasome pathway leads to induction of apoptosis in proliferating cells.
  • Reporter-expressing cells become fluorescent upon abrogation of proteasomal degradation. Therefore reporter-expressing cells can be used in high throughput screens for the identification of compounds that block proteasomal degradation in living cells. To the inventors knowledge there is no such screen available in living cells.
  • the commonly applied assays rely on the in vitro cleavage of different fluorogenic substrates for the three proteolytic activities of the proteasome. The inventors have demonstrated (fig 4B and 5) that information regarding the in vitro inhibitory activity of the proteasome inhibitors is insufficient to conclude whether the compound will block the proteasomal degradation in vivo.
  • One aspect of the invention is to incubate reporter-expressing cells in a multi-well plate with different compounds and monitor the fluorescence intensity of the cells by flow cytometry or fluorimetry.
  • This can be used in order to identify compounds that block the ubqiuitin-proteasome pathway by inhibition of the proteolytic activity, and thus have anti-tumor activity, anti-inflammatory activity and anti-angiogenic activity.
  • Proteasomes are involved in the generation of peptides, which in complex with
  • MHC class I can provoke an attack by cytotoxic T cells. This mechanism is crucial in the elimination of viruses or other intracellular pathogens. In addition this reaction may contribute to the elimination of malignant cells. In some autoimmunologic disorders this response is disturbed and reacted against normal cells. Others have demonstrated in yeast that inactivation of one of the three proteolytic activities of the proteasome leads to generation of a different set of peptides without affecting the overall ubiquitin-proteasome pathway. Recently it has been demonstrated that an HIV1 protease inhibitor, which cross reacts with the proteasome and changes the proteolytic activity of the proteasome, modifies the response to viral infection in mice.
  • Another aspect of the invention is to incubate reporter-expressing cells with an inhibitor, which does not or poorly blocks the ubiquitin-proteasome pathway (for example YLLL-VS) in combination with a library of compounds. Cells will become fluorescent if the mixture blocks the ubiquitin-proteasome pathway, which can be monitored as mentioned in 1. Both full inhibitors (as mentioned in 1) and partial inhibitors (potential immunomodulators) will be identified.
  • an inhibitor which does not or poorly blocks the ubiquitin-proteasome pathway
  • YLLL-VS ubiquitin-proteasome pathway
  • Partial inhibitors can also be used to abrogate an unwanted immune response, such as autoimmune disorders or uncontrolled inflammatory response. The same inhibitors may also be helpful to provoke an immune response (for example in cancer patients).
  • the inhibitors can be used to modify the quality of the peptide expressed by the target cell, so that the cell is recognised by T-cells specific for TAP-independent epitopes. 3) Screening for chemical agents/polypeptide sequences that, by inhibition in cis or trans, block the ubiquitin-proteasome pathway of a pre-proteolytic step, like ubiquitination or transport of ubiquitinated proteins to the proteasome.
  • inhibitors of the ubiquitin-proteasome pathway focus on the proteolytic activity of the proteasome.
  • inhibitors of proteolytic activity are relatively easy to find.
  • An important drawback of the usage of inhibitors of proteolytic activity is that each of these inhibitors inhibits also other cellular proteases.
  • a much more specific inhibition of the ubiquitin-proteasome pathway can be accomplished by blocking a step in the pathway before the actual degradation of the proteasome. For example, the ubiquitination step could be attacked.
  • Possible candidates are ubiquitin hydrolases, which are important for the recycling of ubiquitin, or one of the different enzymes involved in ubiquitin tagging, such as the ubiquitin activase, ubiquitin conjugases or ubiquitin ligases. To the inventors knowledge there is no assay available that allows screening of such compounds.
  • Still another aspect of the invention is an assay as described in 1. In the assay described in 1 all inhibitors of the ubiquitin-proteasome pathway regardless of the step at which they interfere will be identified.
  • this type of compounds are expected to be more specific for the inhibitors of the proteolytic activity and should be helpful if malignant cells become resistant to inhibitors of the proteolytic activity.
  • Ub-X-GFPs can be used as transcriptional reporters because of their short half life.
  • GFP is a very stable protein with an extremely long half-life (> 24 hr). For this reason, GFP is not very well suited as a transcriptional reporter. Leaky expression will result in very high background levels and switching off transcription cannot be monitored since it will take very long before the GFP is cleared from the cells. Others already anticipated that it would be helpful to have GFP variants with shorter half lives and the biotech company Clontech is selling destabilized GFPs as transcriptional reporters which are targeted for proteasomal degradation by introduction of a PEST sequence. The Ub-X-GFPs are similarly targeted for proteasomal degradation and can be used for this purpose.
  • the reporter molecule can be used as a reporter for transcriptional activation.
  • the measurement of proteasomal activity in live cells, e.g. cancer cells, before CTL-based immunotherapy, can be used to investigate antigen processing.
  • kits to be used in the different methods of the invention, whereby the kits comprise the chemicals and biomolecules to be used in the methods.
  • FIG. 1 Targeting of GFP for proteasomal degradation.
  • FIG. 1 Ub-X-GFP expression monitored by fluorescence intensity.
  • A) Flow cytometric analysis of transiently transfected HeLa cells expressing the Ub-X-GFP chimeras. Fluorescent cells are detected in the upper right quadrant. The mean fluorescence intensity and % positive cells are indicated.
  • FIG. 3 Accumulation of GFP reporter upon treatment with inhibitor.
  • FIG. 4 Correlation between proteasomal degradation and cell growth.
  • A) Cytosolic fractions were prepared from Ub G76V -GFP expressing cells treated with increasing concentrations of Z-L 3 -VS and analysed for chymotrypsin-like activity against the suc-LLVY-MCA substrate (closed circles), and GFP fluorescence (open circles). Relative fluorescence is expressed as fold induction over the background of untreated cells. The % hydrolysis of proteasomes from inhibitor treated samples relative to the untreated control is shown. Hydrolysis due to contaminating proteases was measured by addition of 10 ⁇ M Z-L 3 -VS and 25 ⁇ M YL 3 -VS during the assay and was subtracted from each sample. Mean of two samples.
  • C) The mean fluorescence intensity (open circles) and cell numbers (closed circles) were determined at the indicated times. The relative fluorescence is expressed as fold induction over the background of untreated cells. Mean ⁇ SD of three experiments.
  • Figure 5 Effect of different inhibitors on degradation of reporter.
  • the cells were counterstained with Hoechst H332258 (right panel).
  • FIG. 7 A viral polypeptide blocks in cis the degradation of the GFP reporters.
  • A) GAr polypeptides of 25 and 239 amino acids were inserted at the C-terminus of GFP reporters that had been targeted for ubiquitin-proteasome dependent degradation by insertion of N-end rule or UFD degradation signals.
  • the transfected cells were preincubated for 10 h without (-) or with (+) 10 ⁇ M of the proteasome inhibitor Z-L 3 -VS.
  • C) Flow cytometric analysis of HeLa cells transfected with GFP, Ub-M-GFP, Ub G76V - GFP and Ub-R-GFP expressing plasmids and their GA25 or GA239 counterparts. Data are expressed as the ratio between the number of fluorescent cells in untreated samples and samples treated with 10 mM Z-L 3 -VS. Mean and SD of three experiments. * Significantly different from the corresponding construct without GAr (paired T-test, P ⁇ 0.05).
  • N-end rule 19 and ubiquitin-fusion degradation (UFD) 18 signals were used to convert the stable jellyfish GFP into a substrate for ubiquitin/proteasome-dependent proteolysis.
  • Two N-end rule substrates were constructed using Arg (Ub-R-GFP) and Leu (Ub-L-GFP) as destabilising amino acids while two UFD substrates contained a poorly cleavable ubiquitin (Ub-P-GFP) or a mutated uncleavable ubiquitin moiety (Ub G76V -GFP).
  • the inventors constructed a Ub-M-GFP chimera that, upon ubiquitin cleavage, does not contain a degradation signal and is therefore expected to be as stable as unmodified GFP.
  • the degradation of N-end rule substrates is dependent on the presence of a Lys residue in position 15 or 17 (ref. 17). Therefore, a random sequence was introduced between the destabilising amino acid and the start codon of GFP, placing the first Lys of GFP in position 17 (Fig. 1A). An additional Lys was introduced in position 3 of the linker to further increase the efficiency of ubiquitination.
  • the ubiquitin moiety of the ubiquitin-GFP fusion constructs was obtained by amplifying with the polymerase chain reaction (PCR) the ubiquitin open reading frame from the Ub-Pro- ⁇ Gal plasmid 32 with the sense primer 5'-GCG GAA TTC ACC ATG CAG ATC TTC GTG AAG ACT-3' (£coRI restriction site, underlined; start codon, bold) and the antisense primers 5'-GC GGG ATC CTG TCG ACC AAG CTT CCC XXX CCC ACC TCT GAG ACG GAG TAC-3' for Ub-X-GFP, or 5'-GC GGG ATC CTG TCG ACC AAG CTT CCC CAC CAC ACC TCT GAG ACG GAG TAC-3' for Ub Gly76Val -Val-GFP (BamUI restriction site, underlined; variable codon, bold).
  • PCR polymerase chain reaction
  • EGFP-N1 encodes a GFP variant with an optimized codon usage for expression in mammalian cells and a red-shifted excitation spectrum 35 .
  • Human cervical epithelial carcinoma HeLa cells were cultured in Iscove's modified enriched medium supplemented with 10% fetal calf serum (Life Technologies).
  • Transient transfections were performed with Lipofectamine (Life Technologies) in 24 or 12 well plates by incubating the cells for 5 hr in the presence of a transfection mixture containing 0.5 ⁇ g plasmid DNA and 1.5 ⁇ l lipofectamine/well.
  • Fig. IB Western blot analysis
  • the transfected cells were harvested 24-48 post-transfection in 100 ⁇ l SDS-PAGE loading buffer and lysed by boiling. Total cell lysates of 10 5 HeLa cells were run on 10% SDS-PAGE gels and transferred to Protan BA 85 nitrocellulose filter (Schleicher & Schuell). Filters were blocked for 1 hr in phosphate buffered saline supplemented with 5% skim milk and 0.1% Tween-20 and incubated for 1 hr with rabbit polyclonal anti-GFP serum (Molecular Probes) diluted 1 : 1500 in the same buffer.
  • Example 2 Use of the reporters form measurement of ubiquitin-proteasome dependent proteolysis in living cells
  • HeLa cells were transfected as described in Example 1.
  • the transfected cells were preincubated in 50 ⁇ M of the reversible proteasome inhibitor MG132 (ref. 21) or 50 ⁇ M of the irreversible inhibitor Z-L 3 -VS (ref. 22) for 10 h and fluorescence intensity was measured by flow cytometry and fluorescence microscopy. For fluorescence microscopic analysis the cells were grown and transfected on coverslips.
  • the control and transfected cells were fixed in 4% paraformaldehyde in phosphate buffered saline 24-48 hr posttransfection and mounted with 50% glycerol in PBS.
  • the cells were analyzed with a LEITZ-BMRB fluorescence microscope (Leica) using a bandpass FITC filter setting. Images were captured with a Hamamatsu 4800 cooled CCD camera (Hamamatsu). All images were obtained with equal exposure and processed in parallel in Adobe Photoshop software.
  • HeLa cells were transiently transfected and harvested by trypsinization 24-48 hr posttransfection. The cells were spin down at 3000 rpm and washed in cold phosphate buffered saline (PBS) once.
  • PBS cold phosphate buffered saline
  • the cells were analysed directly after collection using a FACsort flow cytometer (Beckton & Dickinson). The data were analysed with Cellquest software.
  • the steady state fluorescence of the transfected cells corresponded to the ' intensity of the GFP specific band detected in Western blots (Fig. 2A).
  • Treatment with the proteasome inhibitors resulted in accumulation of fluorescence detected by FACS (not shown) and an increase in the number of GFP positive cells detected by and fluorescence microscopy (Fig. 2B). Note that fluorescence measurements detect solely mature GFP while Western blotting detects both mature and mimature/unfolded polypeptides.
  • Example 3 Production of stable transfectants for quantification of ubiquitin- proteasome dependent proteolysis
  • a stable transfectant expressing the strongly destabilised Ub G76v -GFP was produced in order to investigate the activity of the ubiquitin/proteasome pathway in living cells.
  • Stable transfectants were generated by selection of cells transfected as described in Example 1 in the presence of 500 ⁇ g/ ml of G418 (Sigma St. Louis, Missouri). HeLa cells stably transfected with Ub G76V -GFP were incubated with proteasome inhibitor for 10 hrs in a 24 well plate. The cells were harvested by trypsinization and spinned down for 3 minutes in 2000 rpm.
  • the cell pellet was resuspended in 500 ⁇ l PBS and the fluorescence was measured with a fluorimeter (LS50B, Perkin Elmer). Excitation wavelength is 480 nm and emission wavelength is 510 nm. The spontaneous fluorescence of the transfectant was only marginally above the autofluorescence of untransfected cells while a characteristic GFP emission peak was detected at 510 nm upon treatment with proteasome inhibitor (Fig. 3A). Fluorimetric analysis and fluorescence microscopy analysis showed that the increase was time- dependent (Fig 3B).
  • HeLa cells stably transfected with the Ub G76V -GFP construct were incubated with the proteasome inhibitor Z-L 3 -VS for 10 hrs in a 24 well plate.
  • the cells were harvested by trypsinization and transferred to a 96 well plate for immediate analysis of fluorescence with a multilabel plate reader (Victor, Wallac).
  • a dose dependent increase of fluorescence intensity was observed (Fig. 3C).
  • the cytosolic fraction was prepared by differential centrifugation at 100 000 g (ref 30; supernatant after 1 h 100 000 g)
  • Non-specific hydrolysis was assessed by preincubation for 10 min with 10 ⁇ M Z-L3-VS and 25 ⁇ M YL3-VS and was found to be less then 10% of the total activity.
  • Specific activity was expressed as the hydrolysis of untreated samples minus the hydrolysis of samples pretreated with inhibitors.
  • GFP fluorescence was monitored in parallel in the cytosolic fractions. In line with the effect of Z-L 3 -VS on purified proteasomes, a dose-dependent inhibition of the chymotrypsin-like activity.
  • the cells were cultured in the absence or presence of different concentrations of Z-L3-VS and fluorescence intensity was determined by flow cytometry at the indicated times.
  • a 7 fold increase of fluorescence was induced by preincubation for 2 h with 5 ⁇ M of the reversible inhibitor MG132 and clearance of the reporter was then monitored in medium alone or in the presence of 1 or 2 ⁇ M Z-L 3 -VS (Fig. 4B).
  • the fluorescence half-life was approximately 2 h in the absence of inhibitor and 3 and 4 h in the presence of 1 and 2 ⁇ M Z-L 3 -VS, respectively.
  • This dose-dependent delay confirms that the activity of the proteasome is affected also at low concentrations of the inhibitor and shows that the reporter system can be used to monitor suboptimal inhibition of ubqiuitin- proteasome-dependent proteolysis.
  • Example 4- Use of the reporter to assess induction of apoptosis by inhibition of the ubiquitin-proteasome pathway
  • Cells cultured in the presence of increasing concentrations of Z-L 3 -VS were compared for fluorescence emission, cell cycle distribution and induction of apoptosis.
  • HeLa cells stably expressing the UbG76V- GFP reporter were cultured in the presence of different concentrations of Z-L3-VS. At the different times cell number and viability were assessed by counting in trypan blue. For flow cytometric analysis of the fluorescence UbG76V-GFP expressing
  • HeLa cells were harvested by trypsinization, spun down at 3000 rpm and washed in cold phosphate buffered saline (PBS) once. The cells were analysed directly after collection using a FACsort flow cytometer (Beckton & Dickinson). The data were analysed with Cellquest software. The increase in fluorescence is expressed as induction of the mean fluorescence intensity over background. Cell cycle distribution and the percentage of apoptotic cells were determined with propidium iodide staining. The DNA content and GFP fluorescence were determined in parallel by flow cytometry (Fig. 4C). Failure to accumulate GFP in cells treated with 2 ⁇ M Z-L 3 -VS correlated with normal G1-G2/M distribution and undisturbed growth.
  • PBS cold phosphate buffered saline
  • Example 5 Use of the reporter to identify partial inhibitors of proteasomal activity
  • the inventors assessed the applicability of the reporters for the identification of the activity of compounds that selectively modify the catalytic function of the proteasome.
  • the inventors chose three recently identified inhibitors: carboxybenzyl- leucyl-leucyl-leucinal (MG132), carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L 3 -VS), nitrophenol leucyl-leucyl-glycine vinyl sulfone (NP-LLG-VS); tyrocyl- leucyl-leucyl-leucine vinyl sulfone; (YL 3 -VS), and the HIV-1 protease inhibitor ritonavir.
  • MG132 carboxybenzyl- leucyl-leucyl-leucinal
  • Z-L 3 -VS carboxybenzyl-
  • Each of these inhibitors was shown to preferentially affect the hydrolysis of certain fluorogenic substrates 23"25 .
  • YL 3 -VS preferentially inhibits the trypsin-like activity while NP-LLG-VS inhibits the chymotrypsin- and trypsin-like activities but has virtually no effect on the PGPH activity (23 and Hidde Ploegh, personal communication).
  • Ritonavir inhibits the chymotrypsin-like activity but induces the trypsin-like activity and affects the presentation of certain MHC class I restricted T-cell epitopes 24,25 .
  • HeLa cells stably expressing the reporter were treated with inhibitors and analyzed by fluorescence spectrometry as described in Example 3.
  • the fluorescence is expressed as fold increase of total fluorescence over background.
  • Treatment of Ub G76V -GFP-expressing cells with up to 100 ⁇ M YL 3 -VS did not induce accumulation of the reporter and a weak effect was observed at concentration of NP-LLG-VS above 50 ⁇ M (Fig. 5A).
  • Ritonavir had a very weak effect that reached a plateau at 60 ⁇ M.
  • the inventors evaluated in the treated cells the activity of each of the enzymatic activities of the proteasome.
  • this reporter system can be used to identify modifiers of the proteasome that do not affect block the ubiquitin-proteasome pathway individually. Such modifiers are expected to change the peptides generated from a protein substrate upon proteasomal degradation.
  • Example 6 Use of the reporter to identify polypeptides that inhibit in trans the ubiquitin/proteasome pathway.
  • the Ub +1 molecules may competitively inhibit binding of polyubiquitinated substrates to the proteasome, which is expected to result in a general blockage of the ubiquitin/proteasome pathway (ref).
  • the inventors addressed the question whether the postulated inhibitory activity of Ub +1 polypeptide could be revealed with the usage of the GFP reporters for proteasomal degradation. This question is difficult to address using conventional methods since polypeptides that block overall proteasomal degradation are expected to induce apoptosis in the transfected cells making it difficult to generate stable Ub +1 transfectants. Moreover analysis of the ubiquitin/proteasome pathway in transiently transfected cells is complicated by the presence of both transfected and untransfected cells within the population.
  • HeLa cells stably expressing the Ub G76V -GFP reporter were transiently transfected with a Ub+1 -encoding plasmid or with a control plasmid encoding wild type FLAG-tagged ubiquitin. Two days post-transfection the cells were stained with an anti-Ub +1 antibody and analyzed by fluorescence microscopy. It was observed that many of the cells expressing Ub +1 demonstrated a dramatic accumulation of the GFP reporter as measured by the GFP fluorescence indicating an obstruction in the ubiquitin/proteasome-dependent proteolysis in these cells (Fig 6A).
  • Example 7 Use of the reporter to identify polypeptide sequences that inhibit in cis the ubiquitin/proteasome pathway.
  • the inventors assessed the applicability of the GFP reporters in monitoring and quantifying the in cis inhibitory effect of proteasomal degradation mediated by the viral GAr.
  • GArs of different lengths were linked to the C-terminus of four GFP reporters varying in stability from completely stable to highly destabilized (Fig. 7A).
  • HeLa cells were transiently transfected in duplicate with the GAr-harbouring GFP reporters and from 16 until 26 hr post-transfection the cells were either left untreated or incubated with 10 ⁇ M Z-L 3 -VS proteasome inhibitor.
  • the effect of the short 25 and the 239 amino acid long GAr was compared by transfection and flow cytometric analysis.
  • the inventors used the ratio between the percentage of positive cells in the absence or presence of proteasome inhibitor as a measure for proteasomal degradation.
  • Two stable controls, GFP and Ub-M-GFP resulted values close to 1 confirming that these proteins are not processed by the proteasome and give equal percentage of fluorescent cells in the absence or presence of the inhibitor (Fig. 7C).
  • the destabilized Ub-R-GFP and Ub G76V -GFP significantly reduced this value as expected for a true proteasome substrate.
  • Insertion of the 25 amino acid and 239 amino acid GAr resulted in a length dependent increase of the ratio. This shows that the GAr does also protect the strongly destabilized reporters even though it does not reach a full protective effect.
  • the GFP reporter system can be used to identify and characterize polypeptide sequences that inhibit in cis degradation of proteasome substrates.
  • 25 ⁇ M YL 3 -VS were analysed by cleavage of the fluorogenic substrates Suc- LLVY-AMC, Boc-LRR-AMC and Cbz-LLE- ⁇ NA, respectively.
  • the data are presented as percentage residual enzymatic activities of proteasomes from treated cells relative to proteasomes from untreated controls. Mean ⁇ SD of triplicate tests. One representative experiment out of three performed with different proteasome preparations.

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Abstract

La présente invention concerne une construction protéique « rapporteur » dont l'extrémité carboxy comporte une partie « rapporter » et l'extrémité amino comporte une partie ubiquitine (Ub). Cette construction protéique est caractérisée en ce qu'elle contient également, entre la partie rapporteur et la partie ubiquitine, un peptide lieur comprenant au moins un résidu Lys. L'invention concerne également un procédé permettant de mesurer l'activité de la voie ubiquitine/protéasome dans des cellules vivantes.
PCT/SE2001/000921 2000-04-27 2001-04-27 Construction proteique « rapporteur » contenant partiellement une proteine gfp WO2001081427A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004066935A2 (fr) * 2003-01-24 2004-08-12 Becton, Dickinson And Company Constructions de detecteurs de proteasome sensibles et procedes de fabrication de celles-ci et utilisations
US7258981B2 (en) 2003-01-24 2007-08-21 Clontech Laboratories, Inc. Sensitive proteasome sensor constructs and methods for their design and use
WO2012097860A1 (fr) * 2011-01-18 2012-07-26 Helmholtz-Zentrum für Infektionsforschung GmbH Nouvelles sondes d'ubiquitine-isopeptide
EP2487183A1 (fr) * 2011-01-18 2012-08-15 Helmholtz-Zentrum für Infektionsforschung GmbH Sondes d'ubiquitine-isopeptide
CN112194729A (zh) * 2020-09-29 2021-01-08 浙江大学 一种用于检测泛素-蛋白酶体系统降解活性的生物探针

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WO1999011774A1 (fr) * 1997-08-30 1999-03-11 Fluorescience Limited Polypeptides a structure bispiralee comportant un site d'addition

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WO1999011774A1 (fr) * 1997-08-30 1999-03-11 Fluorescience Limited Polypeptides a structure bispiralee comportant un site d'addition

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NICO P. DANTUMA ET AL.: "Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells", NATURE BIOTECHNOLOGY, vol. 18, May 2000 (2000-05-01), pages 538 - 543, XP002945558 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004066935A2 (fr) * 2003-01-24 2004-08-12 Becton, Dickinson And Company Constructions de detecteurs de proteasome sensibles et procedes de fabrication de celles-ci et utilisations
WO2004066935A3 (fr) * 2003-01-24 2006-06-29 Becton Dickinson Co Constructions de detecteurs de proteasome sensibles et procedes de fabrication de celles-ci et utilisations
US7258981B2 (en) 2003-01-24 2007-08-21 Clontech Laboratories, Inc. Sensitive proteasome sensor constructs and methods for their design and use
WO2012097860A1 (fr) * 2011-01-18 2012-07-26 Helmholtz-Zentrum für Infektionsforschung GmbH Nouvelles sondes d'ubiquitine-isopeptide
EP2487183A1 (fr) * 2011-01-18 2012-08-15 Helmholtz-Zentrum für Infektionsforschung GmbH Sondes d'ubiquitine-isopeptide
CN112194729A (zh) * 2020-09-29 2021-01-08 浙江大学 一种用于检测泛素-蛋白酶体系统降解活性的生物探针
CN112194729B (zh) * 2020-09-29 2021-12-21 浙江大学 一种用于检测泛素-蛋白酶体系统降解活性的生物探针

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