WO2010040842A9 - Linking protein aggregation and yeast survival - Google Patents
Linking protein aggregation and yeast survival Download PDFInfo
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- WO2010040842A9 WO2010040842A9 PCT/EP2009/063213 EP2009063213W WO2010040842A9 WO 2010040842 A9 WO2010040842 A9 WO 2010040842A9 EP 2009063213 W EP2009063213 W EP 2009063213W WO 2010040842 A9 WO2010040842 A9 WO 2010040842A9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/9065—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5)
- G01N2333/90655—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1) in general
- G01N2333/90661—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1) in general with a definite EC number (1.5.1.-)
- G01N2333/90666—Dihydrofolate reductase [DHFR] (1.5.1.3)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- protein aggregation in cell factories represents a major bottleneck in recombinant protein production, narrowing the spectrum of polypeptides obtained by recombinant techniques and hampering the development of priority research areas such as structural genomics and proteomics. Therefore, there is an increasing interest in the development of protein solubility screening methods that allow foreseeing genes, chemical compounds or culture conditions that would modulate protein aggregation.
- Fluorescence resonance energy transfer has also been used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the E.coli cytoplasm (Philipps, B., Hennecke, J. & Glockshuber, R. FRET-based in vivo screening for protein folding and increased protein stability. JMo/ Biol 327, 239-249 (2003)).
- the system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue fluorescent protein (BFP) to the N terminus.
- GFP green fluorescent protein
- BFP blue fluorescent protein
- Another assay has been developed as a result of the identification of specific genes responding to protein misfolding.
- one of these genes promoters (IbpAB) is fused to ⁇ -galactosidase in order to quantify the response of the promoter to intracellular misfolding and aggregation (Lesley, S. A., Graziano, J., Cho, CY. , Knuth, M. W. & Klock, H. E. Gene expression response to misfolded protein as a screen for soluble recombinant protein. Protein Eng 15, 153-160 (2002)). In this way, ⁇ -gal expression (and activity) becomes linked to protein aggregation inside the cell.
- Chloramphenicol resistance has also been used as a readout to detect soluble mutants of an aggregating-prone protein in vivo in E. coli (Maxwell, K.L., Mittermaier, A.K., Forman-Kay, J.D. & Davidson, A.R. A simple in vivo assay for increased protein solubility. Protein Sci 8, 1908-1911 (1999)).
- the reporter protein is chloramphenicol acetyltransferase (CAT). Resistance to high levels of chloramphenicol will be equivalent to the expression of soluble mutant fusions of the target protein. The selection can be carried out growing the cells in plates with high concentration of the antibiotic.
- twin-arginine translocation pathway Methods based on the twin-arginine translocation pathway have also been used for detection of protein misfolding.
- the basis for these assays is the protein dependence on correct folding in order to be transported through the bacterial twin-arginine translocation (Tat) pathway (Fisher, A.C., Kim, W. & DeLisa, M. P. Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway. Protein Sci 15, 449-458 (2006)).
- a target protein is expressed as a tripartite fusion between a N-terminal Tat signal peptide and a C- terminal TEMl ⁇ -lactamase reporter protein (BIa).
- the protein folds correctly, it will be translocated through the TAT pathway to the periplasm. Due to the fact that the target protein is also fused to ⁇ -lactamase, it will confer ampicillin resistance to the bacteria. Then, survival of E. coli cells expressing a Tat-targeted test protein/ ⁇ - lactamase fusion on selective medium correlates with the solubility of the protein of interest. Using this assay, variants of the Alzheimer's A ⁇ 42 peptide with an enhanced solubility could be detected and isolated from a large combinatorial library.
- WO2007/103788 describes a method for determining protein aggregation in yeast. It is based on the capacity of the translational termination factor Sup35p to form self- propagating infectious amyloid aggregates. This factor manifests a prion phenotype referred to as [PSI+] and it is composed of three domains.
- the N-terminal domain (N) is dispensable for viability, and it is required and sufficient for the prion properties of Sup35p. While the function of the highly charged middle (M) domain remains unclear, the C-terminal RF (release factor) domain performs termination of protein translation and is essential for viability
- the activity of the termination factor Sup35p is conveniently assayed in vivo by examining the efficiency with which protein synthesis terminates at a premature stop codon (a nonsense-suppression assay).
- the assay uses the adel-14 nonsense allele. Strains carrying this mutation and bearing fully active NMRF produce only a truncated (inactive) version of Adelp, and as a result cannot grow on synthetic medium lacking adenine (-Ade), while they grow normally on synthetic medium supplemented with adenine (+Ade). In addition, these cells accumulate a red intermediate of the adenine synthesis pathway when grown on complex medium.
- This method provides a negative assay for determining protein folding, since the yeast which do not aggregate the protein of interest (due, for example, to the presence of an inhibitor of aggregation) are not viable.
- the invention is related to a method that could couple an easily measurable phenotype like cell survival to protein aggregation using yeast as a model of eukaryotic organism. It is based in the fusion of the target protein to the human dihydrofolate reductase (h-DHFR).
- DHFR is a key enzyme in thymidine synthesis that catalyses the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate with NADPH as a coenzyme and in the three-hybrid method.
- Prokaryotic and eukaryotic DHFRs are central to cellular one-carbon metabolism and are absolutely required for cell survival. And its activity can be specifically inhibited by the drug methotrexate (MTX).
- MTX drug methotrexate
- yeast cells can become insensitive to MTX if they express high levels of h-DHFR.
- Other specific inhibitors of the DHFR could be used (such as, for example, trimethoprim in case DHFR of bacterial origin was used).
- This enzyme is a very soluble protein and, in our approach, it is expressed at concentrations that allow cell survival in MTX concentrations that otherwise would be lethal. Therefore, the invention is based on the surprising effect that the fusion of h- DHFR to aggregation-prone polypeptides might inactivate the enzyme and render the cells expressing these kinds of fusions MTX susceptible.
- the approach discussed here aims at the easy and reliable evaluation of the effects of intrinsic and extrinsic factors on protein aggregation. And it is based on the correspondence between the intracellular activity and solubility of recombinant h- DHFR and cell growth in the presence of lethal concentrations of MTX. Furthermore, the use of fMTX (a MTX labeled with a fluorescent compound) enables to monitor simultaneously the cell viability and the localization of the aggregates inside the cell.
- the method is able to anticipate the intracellular aggregation propensity of genetic variants of three unrelated polypeptides linked to important human disorders.
- the system could become also a convenient platform for chemical screening of agents that interfere with protein aggregation in order to assist in the development of new therapeutic leader compounds targeting protein aggregation and toxicity.
- S. cerevisiae is compatible with these applications due to the availability of drug- permeable strains (i.e. erg ⁇ A), although any other yeast cell capable of expressing the gens of interest could be used.
- the invention relates to a method for the identification of compounds that are capable of decreasing aggregation of an aggregation-prone polypeptide comprising:
- yeast cells express a fusion protein which comprises an aggregation-prone polypeptide and an enzyme, wherein the enzyme is capable of modifying a compound which adversely affects yeast cell viability into a metabolite with a reduced adverse effect on said yeast cell viability,
- step (ii) adding the toxic compound to the yeast cell of step (i) in an amount which, without the presence of the activity of the enzyme forming part of the fusion protein used in step (i), would adversely affect the yeast cell viability and
- the invention relates to a polypeptide comprising an aggregation- prone polypeptide and a polypeptide having enzymatic activity wherein said polypeptide having enzymatic activity is capable of modifying a compound which adversely affects yeast cell viability into a metabolite of said compound with a reduced adverse effect on said yeast cell viability.
- the invention relates to a polynucleotide encoding a polypeptide of the invention, a vector comprising a polynucleotide of the invention, and a host cell comprising a polypeptide, a polynucleotide or a vector of the invention.
- the invention relates to the use of a host cell of the invention for the identification of compounds which are capable of inhibiting aggregation of an aggregation-prone polypeptide.
- the invention is related to a method of screening for a compound that decreases aggregation of aggregation-prone polypetides wherein the method comprises (a) contacting one or more yeast cells with a candidate compound, wherein the yeast cells express a fusion protein comprising an aggregation-prone polypeptide, such as a amyloidogenic protein, and an enzyme which inhibits a toxic compound which affects yeast cell viability, or which prevents the toxic compound from acting and affecting the cell viability (b) adding the toxic compound to the yeast cell in an amount which, without the presence of the enzyme, would affect cell viability.
- Saccharomyces uvae Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha
- Pichia pastor is, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., Geotrichum fermentans, and Saccharomyces cerevisiae.
- Saccharomyces cerevisiae More preferred is Saccharomyces cerevisiae FY384, since the lethality of Methotrexate (MTX) on S . cerevisiae FY384 cells above certain concentrations (1 mM of sulfonamide, which is used to promote MTX cell intake) can be overcome by heterologous expression of human DHFR under the control of Gall 10 promoter.
- MTX Methotrexate
- Different degrees of sensitivity to MTX may thus be correlated with the intracellular activity of the enzyme.
- the fused protein would promote, at least partially, its deposition lowering the intracellular activity and increasing sensitivity to MTX. Therefore, aggregation state of the fused protein is linked directly to yeast cell survival in the presence of methotrexate.
- the invention is based in the surprising effect that the fusion of an h-DHFR to an amyloidogenic protein, inactivate the enzyme and render these cells expressing these kinds of fusion susceptible to MTX.
- the fusion of an enzyme to an aggregation-prone polypeptide inactivate the enzyme and render these cells expressing these kinds of fusion susceptible to a toxic compound which would be inhibited or otherwise inactivated by the enzyme, as it is MTX with h-DHFR.
- the aggregation-prone peptides are amyloidogenic peptides, such as A ⁇ 42, PoIyQ expansions, or ⁇ -synuclein variants.
- the method of the invention can be used, in a more preferred way, to test compounds related to the prevention of treatment of a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Familial Amyloid Polyneuropathy, a Tauopathy, Trinucliotide disease, transmissible spongiform encephalopathies (TSEs), Alzheimer's (AD), and Huntingdon's Disease (HD), which are known to be caused by the aggregation of amyloidogenic peptides.
- a disease selected from the group consisting of Alzheimer's disease, Parkinson's disease, Familial Amyloid Polyneuropathy, a Tauopathy, Trinucliotide disease, transmissible spongiform encephalopathies (TSEs), Alzheimer's (AD), and Huntingdon's Disease (HD), which are known to be caused by the aggregation of amyloidogenic peptides.
- the invention is related to a kit useful for screening compounds that inhibit protein aggregation which uses the method of the invention.
- Figure 1 shows a scheme of the plasmid used in this assay.
- the target protein fused to DHFR was cloned between the restriction sites CIaI i BgIII in the plasmid pESC.
- Figure 2 shows A) Visualization of intracellular wild-type and A ⁇ 42(F19D) distribution with GFP expressed in S. cerevisiae B) Co-staining of the cell nucleus with Hoechst (blue). The aggregated A ⁇ 42-GFP has a juxtanuclear position.
- Figure 3 shows cell viability (spotting) assays for yeast expressing DHFR, peptide A ⁇ 42-DHFR or peptide A ⁇ 42 F19D-DHFR at different temperatures and MTX concentrations. Four- fold serial dilutions starting with equal number of cells are shown.
- Figure 4 shows growth assays of FY834 yeast cells expressing DHFR (empty circles), peptide A ⁇ 42-DHFR (empty squares) or peptide A ⁇ 42(Fl 9D)-DHFR (solid circles) in the presence of 0 ⁇ M (left) and 20 ⁇ M of MTX (right).
- Figure 5 shows filter trap assay of cells expressing DHFR, A ⁇ 42(Fl 9D)-DHFR or
- a ⁇ 42-DHFR Protein aggregates were detected by immunoblot analysis using specific antibody against DHFR.
- Figure 6 shows the addition of a fluorescent inhibitor of DHFR (fMTX) enables the visualization of the intracellular distribution of wild-type and F19D mutant A ⁇ 42 fused to DHFR.
- fMTX fluorescent inhibitor of DHFR
- Figure 7 shows A) Fluorescence microscopy of yeast cells expressing different polyQ expansions (Q25, Q72 or Q103) fused to GFP B) Growth assays of yeast cells expressing the different polyQ expansions fused to DHFR in the presence of 20 ⁇ M MTX.
- Figure 8 shows the addition of a fluorescent inhibitor of DHFR (fMTX) enables the visualization of the intracellular distribution of the different poliQ expansions fused to DHFR.
- fMTX fluorescent inhibitor of DHFR
- Figure 9 shows A) Fluorescence microscopy of yeast cells expressing different ⁇ - synuclein variants fused to GFP.
- B Growth assays of yeast cells expressing ⁇ -synuclein variants fused to DHFR in the presence of 100 ⁇ M MTX.
- Figure 10 shows the addition of a fluorescent inhibitor of DHFR (fMTX) and how it enables the visualization of the intracellular distribution of the different ⁇ -synuclein variants fused to DHFR.
- fMTX fluorescent inhibitor of DHFR
- Figure 11 shows A) Growth restoration of ergo A yeast cells expressing in A ⁇ 42-DHFR in the presence of 20 ⁇ M MTX, ImM sulfanilamide and selected concentrations of quercetin (30 ⁇ M and 100 ⁇ M) and CR (10 ⁇ M). Growth is normalized to 0 ⁇ M compound. Significant differences are marked with an asterisk. B) Fluorescence microscopic assessment of A ⁇ 42-GFP aggregation in control or compound treated ergo A cells.
- Figure 12 shows A) Growth of yeast FY834 strains overexpressing a chaperone and co- expressing peptide A ⁇ 42-DHFR in the presence of MTX in liquid media. Growth is normalized to the same strain expressing the corresponding chaperone and DHFR. Significant differences are marked with an asterisk. B) Cell viability (spotting) assays for the different strains overexpressing a chaperone and DHFR or A ⁇ 42-DHFR. In each case, four- fold serial dilutions starting with equal number of cells are shown.
- Figure 13 shows A) Fluorescence microscopy of different strains overexpressing a chaperone and peptide A ⁇ 42 fused to GFP.
- Figure 14 shows Growth of yeast FY834 strains with a deletion in one chaperone and expressing peptide A ⁇ 42-DHFR in the presence of MTX in liquid media. Growth is normalized to the same strain expressing the corresponding chaperone and DHFR. Significant differences are marked by an asterisk.
- Figure 15 shows A) Fluorescence microscopy of different strains with chaperone knockouts and expressing peptide A ⁇ 42 fused to GFP.
- An objective of the invention is to develop a method to detect protein aggregation using simultaneously yeast survival and fluorescence emission as reporter signals.
- Alzheimer's amyloid ⁇ (A ⁇ ) peptide polyglutamine expansions in the huntingtin protein (poliQ) and alpha-synuclein ( ⁇ -Syn).
- a second objective is to study the ability of the method to detect the effect of different factors that modulate protein aggregation in vivo, such as chemical compounds, overexpression of chaperones, deletion of chaperones and growth conditions (i.e. temperature).
- the invention relates to a method for the identification of compounds that are capable of decreasing aggregation of an aggregation-prone polypeptide comprising:
- yeast cells express a fusion protein which comprises an aggregation-prone polypeptide and an enzyme, wherein the enzyme is capable of modifying a compound which adversely affects yeast cell viability into a metabolite with a reduced adverse effect on said yeast cell viability,
- step (ii) adding the toxic compound to the yeast cell of step (i) in an amount which, without the presence of the activity of the enzyme forming part of the fusion protein used in step (i), would adversely affect the yeast cell viability and (iii) determining the viability of the yeast cells wherein an increased viability of the cells with respect of the cells which have not been exposed to the candidate compound is indicative that the compound is capable of decreasing aggregation of the aggregation-prone polypeptide.
- aggregation-prone polypeptide refers to a polypeptide which is able to adopt a beta-pleated sheet conformation and/or to form oligomers, fibrils and plaques.
- peptides having a potential for self- assembling and f ⁇ brillogenesis are fibrillaric proteins derived from at least one of the following precursor proteins: Tau, alpha-synuclein, huntingtin, ataxin, superoxide dismutase, TDP-43, SAA (Serum- Amyloid-Protein A), AL (k or Might chains of Immunoglobulins), AH (gl Ig-heavy chains), ATTR (Transthyretin, Serum-Prealbumin), AApo-A-1 (Apolipoprotein Al), AApoA2 (Apolipoprotein A2), AGeI (Gelsolin), ACys (Cystatin C), ALys (Lysozyme), AFib (Fibrinogen), Beta-a
- the aggregation-prone polypeotide is selected from the group of mutant-huntingtin, beta-amyloid, tau, alpha-synuclein, mutant androgen receptor, mutant SODI, mutant ataxin and the like.
- the aggregation-prone polypeptide is an amyloidogenic peptide.
- the aggregation-prone polypeptide is a polypeptide selected from the group of A ⁇ 42, a peptide comprising a Poly-glutamine expansions and ⁇ -synuclein or a variant thereof.
- yeast cells includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
- the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
- the yeast host cell is selected from the group of Saccharomyces increasinglyvisiae, Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp. and Geotrichum fermentans
- contacting a cell with a candidate compound includes any possible way of taking the candidate compound inside the cell expressing the fusion protein.
- the candidate compound in the event that the candidate compound is a molecule with low molecular weight, it is enough to add said molecule to the culture medium.
- the candidate compound in the event that the candidate compound is a molecule with a high molecular weight (for example, biological polymers such as a nucleic acid or a protein), it is necessary to provide the means so that this molecule can access the cell interior.
- conventional trans fection means can be used, as described previously for the introduction of the the polynucleotide.
- the cell can be put in contact with the protein directly or with the nucleic acid encoding it coupled to elements allowing its transcription / translation once they are in the cell interior.
- any of the aforementioned methods can be used to allow its entrance in the cell interior.
- a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV-I TAT protein, the third helix of the Antennapedia homeodomain protein from D.melanogaster, the VP22 protein of the herpes simplex virus and arginine oligomers (Lindgren, A. et al, 2000, Trends Pharmacol. Sci, 21 :99-103, Schwarze, S.R. et al., 2000, Trends Pharmacol. Sd., 21 :45- 48, Lundberg, M et al., 2003, MoI. Therapy 8:143-150 and Snyder, EX. and Dowdy, S.F., 2004, Pharm. Res. 21 :389-393).
- a variant of the protein to be studied which has been modified with a peptide which can promote the translocation of the protein to the cell interior such as the Tat peptide derived from the HIV
- the compound to be assayed is preferably not isolated but forms part of a more or less complex mixture derived from a natural source or forming part of a library of compounds.
- libraries of compounds which can be assayed according to the method of the present invention include, but are not limited to, libraries of peptides including both peptides and peptide analogs comprising D-amino acids or peptides comprising non-peptide bonds, libraries of nucleic acids including nucleic acids with phosphothioate type non-phosphodiester bonds or peptide nucleic acids, libraries of antibodies, of carbohydrates, of compounds with a low molecular weight, preferably organic molecules, of peptide mimetics and the like.
- the library can have been preselected so that it contains compounds which can access the cell interior more easily.
- the compounds can thus be selected based on certain parameters such as size, lipophilicity, hydrophilicity, capacity to form hydrogen bonds.
- the compounds to be assayed can alternatively form part of an extract obtained from a natural source.
- the natural source can be an animal, plant source obtained from any environment, including but not limited to extracts of land, air, marine organisms and the like.
- fusion protein or "chimeric protein”, as used herein, comprises a polypeptide of the invention operatively linked to another polypeptide.
- operatively linked is intended to indicate that the polypeptide(s) according to the invention and the other polypeptide(s) are fused in- frame to each other.
- yeast cell viability refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations.
- an compound which adversely affects yeast cell viability or toxic compound is any compound whose presence in the host cell will prevent the host cell in culture from achieving the normal logarithmic growth it would have achieved but for the expression of the compound.
- the enzyme which adversely affects yeast cell viability is human dehydrofolate reductase (h-DHFR) and the toxic compound is methotrexate (MTX).
- the method according to the invention is carried out using a toxic compound whcih is fluorescently labelled.
- Suitable fluoresenct compounds that can be used to label teh toxic compound include, without limitation, FAMTM, TETTM, JOETM, VICTM, SYBR(R) Green; 6 FAM, HEX, TET, TAMRA, JOE, ROX, Fluorescein, Cy3, Cy5, Cy55, Texas Red, Rhodamine, Rhodamine Green, Rhodamine Red, 6-CarboxyRhodamine 6G, Oregon Green 488, Alexa Flour, Oregon Green 500 or Oregon Green 514.
- the fluorescently labelled toxic compound is fluoresencently labelled MTX and, in particular, fMTX.
- the method further comprises detecting the fluorescence in the yeast cell wherein an increased intracellular fluorescence is indicative that the compound is capable of decreasing aggregation of the aggregation-prone polypeptide.
- Suitable method for detecting the fluorescence in the yeast cell includes, without limitation, FACS, immunofluorescence, immunohistochemistry and the like.
- the method is carried out using yeast strains showing an increased membrane permeability. Strains having an increased cell permeability are widely known to the skilled person and can be identified using standard technology. In a preferred embodiment, the yeast strain carries an inactivating mutation in the ergo gene.
- the method of the invention is carried out in a cell which carries an inactivating mutation in one or more molecular chaperones.
- Molecular chaperones refers to any of a group of proteins that are involved in the correct intracellular folding and assembly of polypeptides without being components of the final structure.
- “molecular chaperones” and “chaperones” are used interchangeably.
- the molecular chaperone is selected from the group of a member of the HsplOO protein family, a member of the Hsp90 protein family, a member of the Hsp70 protein family, a member of the Hsp40 protein family or a small heat shock protein.
- the member of the HsplOO protein family is HsplO4
- the member of the Hsp90 protein family is selected from the group of Hsc82 and Hsp82
- the member of the Hsp70 protein family is selected from the group of Ssal , Ssa2, Ssa3 and Ssa4
- the member of the Hsp40 protein family is selected from the group of Ydjl and Sisl
- the small heat shock protein is selected from the group of Hsp26 and Hsp42.
- the invention also relates to polypeptides comprising an aggregation-prone polypeptide and a polypeptide having enzymatic activity wherein said polypeptide having enzymatic activity is capable of modifying a compound which adversely affects yeast cell viability into a metabolite of said compound with a reduced adverse effect on said yeast cell viability.
- the polypeptide of the invention further comprises a reporter polypeptide.
- reporter polypeptide refers to a polypeptide gene product, which, can be quantitated either directly or indirectly.
- Suitable reporter genes include, without limitation, a beta-galactosidase (lacZ), beta-glucuronidase (GUS), luciferase, alkaline phosphatase, nopaline synthase (NOS), chloramphenicol acetyltransferase (CAT), horseradish peroxidase (HRP).
- the reporter polypeptide is a fluorescent protein.
- fluorescent protein as used herein is a protein that has intrinsic fluorescence when excited with electromagnetic radiation at the appropriate wave length.
- Representative fluorescent proteins can include, but are not limited to, sgGFP, sgBFP, BFP blue-shifted GFP (Y66H), Blue Fluorescent Protein, CFP ⁇ Cyan Fluorescent Protein, Cyan GFP, DsRed, monomeric RFP, EBFP, ECFP, EGFP, GFP (S65T), GFP red shifted (rsGFP), GFP wild type, non-UV excitation (wtGFP), GFP wild type, UV excitation (wtGFP), GFPuv, HcRed, rsGFP, Sapphire GFP, sgBFP.TM., sgBFP.TM. (super glow BFP), sgGFP.TM., sgGFP.TM. (super glow GFP), wt GFP,
- the invention relates to a polynucleotide encoding a polypeptide of the invention.
- polynucleotide(s), means a single or doublestranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and corresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as wholly or partially synthesized polynucleotides
- the invention in another aspect, relates to vector comprising the polynucleotide according to the invention.
- a "vector”, as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- yeast expression vector refers to DNA expression constructs, e.g., nucleic acid segments, plasmids, cosmids, phages, viruses or virus particles capable of synthesizing the subject proteins encoded by their respective recombinant genes (carried by the vector in a yeast.
- nucleic acid segments may also be used to create transgenic yeast cells, using non-directional or homologous recombination, in which the gene or genes of interest are stably integrated into the yeast genome.
- the polynucleotides of the invention or the gene constructs forming them can form part of a vector.
- the invention relates to a vector comprising a polynucleotide or a gene construct of the invention.
- a person skilled in the art will understand that there is no limitation as regards the type of vector which can be used because said vector can be a cloning vector suitable for propagation and for obtaining the polynucleotides or suitable gene constructs or expression vectors in different heterologous organisms suitable for purifying the conjugates.
- suitable vectors include expression vectors in prokaryotes such as pUC18, pUC19, Bluescript and their derivatives, mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phages and shuttle vectors such as pSA3 and pAT28, expression vectors in yeasts such as vectors of the type of 2 micron plasmids, integration plasmids, YEP vectors, centromeric plasmids and the like, expression vectors in insect cells such as the pAC series and pVL series vectors, expression vectors in plants such as vectors of expression in plants such as pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors and the like and expression vectors in superior eukaryotic cells based on viral vectors (adenoviruses, viruses
- Vectors for use with the invention are, for example, vectors capable of autonomous replication and/or expression of nucleic acids to which they are linked in yeast cells.
- plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of a vector.
- the invention is intended to include such other forms of expression vectors that serve equivalent functions and which become known in the art subsequently hereto.
- Said yeast expression vector may be a yeast episomal expression vector or a yeast integrative expression vector, and they can be obtained by conventional techniques known for the skilled person in the art.
- said yeast expression vector is a yeast episomal expression vector.
- yeast episomal expression vector refers to an expression vector that is maintained as an extra-chromosomal DNA molecule in the yeast cytoplasm.
- said yeast episomal expression vector in addition to the nucleotide sequence coding for TF protein or a fragment thereof having pro-coagulant activity operatively linked to a yeast-functional promoter, further comprises: (i) a yeast selection gene; (ii) a yeast replication origin; (iii) a bacterial selection gene; and (iv) a yeast transcription termination signal.
- said yeast episomal expression vector further comprises a unique restriction t greater than Ue for cloning the selected gene (l"b protein or a fragment thereof having pro-coagulant activity) under the control of the yeast-functional promoter and followed by the yeast transcription termination signal.
- yeast-functional promoter any yeast-functional promoter, yeast selection gene, yeast replication origin, bacterial selection gene, yeast transcription termination signal, and restriction site for cloning, can be used in the manufacture of said yeast episomal expression vector; nevertheless, in a particular embodiment, the glyceraldehyde-3-phosrholiatc dehydrogenase promoter (pGPDj is used as the yeast-functional promoter; in another particular embodiment, the I R A3 gene (UR A3) a , used as yeast selection gene; in another particular embodiment, the yeast 2 microns (2mu) replication origin is used as the yeast replication origin; in another particular embodiment, the am pi oil Hn resistance gene S Amp) is used as the bacterial selection gene; and in another particular embodiment, the transcription termination signal of the phosphogSyceraie kinase (PGKt) is used art the specific yeast transcription termination signal.
- PGKt phosphogSyceraie kinase
- the yeast episornal expression vector comprises (i) the IJRA 3 gerse; (U) the Amp gene for selecting and propagating the vector in E. roll; fiiij the yeat greater than t 2mu replication origin; (iv) the pGPIX (v) the specific yeat greater than t transcription termination signal of PGKt: and (vi) a unique BawHl restriction site that allows cloning of selected genes under the control of the pGPD, and followed by the PGKt sequence.
- said yeast expression vector is a yeast integrative expression vector.
- yeast integrative expression vector refers to a vector which is capable of integrating into the yeast genome.
- said yeast integrative expression vector comprises: (i) a bacterial selection gene; and (ii) an expression cassette inserted in a yeast selection gene, said expression cassette further comprising a yeast-functional promoter, a yeast transcription termination signal and a unique restriction site for cloning the selected gene (TF protein or a fragment thereof having pro-coagulant activity).
- any bacterial selection gene, expression cassette inserted in a yeast selection gene, yeast-functional promoter, yeast transcription termination signal, and unique restriction site for cloning the selected gene can be used in the manufacture of said yeast integrative expression vector; nevertheless, in a particular embodiment, the ampicillin resistance gene (Amp) is used as the bacterial selection gene.
- Amp ampicillin resistance gene
- the vector of the invention can be used to transform, transfect or infect cells which can be transformed, transfected or infected by said vector.
- Said cells can be prokaryotic or eukaryotic.
- the vector wherein said DNA sequence is introduced can be a plasmid or a vector which, when it is introduced in a host cell, is integrated in the genome of said cell and replicates together with the chromosome (or chromosomes) in which it has been integrated.
- Said vector can be obtained by conventional methods known by the persons skilled in the art (Sambrok et al., 2001, mentioned above).
- the invention relates to a cell comprising a polynucleotide, a gene construct or a vector of the invention, for which said cell has been able to be transformed, transfected or infected with a construct or vector provided by this invention.
- the transformed, transfected or infected cells can be obtained by conventional methods known by persons skilled in the art (Sambrok et al., 2001 , mentioned above).
- said host cell is an animal cell transfected or infected with a suitable vector.
- Host cells suitable for the expression of the conjugates of the invention include, without being limited to, mammal, plant, insect, fungal and bacterial cells.
- Bacterial cells include, without being limited to, Gram-positive bacterial cells such as species of the
- Bacillus, Streptomyces and Staphylococcus genus and Gram-negative bacterial cells such as cells of the Escherichia and Pseudomonas genus.
- Fungal cells preferably include cells of yeasts such as Saccharomyces, Pichia pastoris and Hansenula polymorpha.
- Insect cells include, without being limited to, Drosophila cells and Sf9 cells.
- Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbous plants.
- Suitable mammal cells in the present invention include epithelial cell lines (porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), hepatic cell lines (from monkey, etc.), CHO (Chinese Hamster Ovary) cells, COS cells,
- BHK cells HeLa cells, 911, AT1080, A549, 293 or PER.C6, NTERA-2 human ECC cells, D3 cells of the mESC line, human embryonic stem cells such as HS293 and BGVOl, SHEFl, SHEF2 and HS181, NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.
- Suitable host cells includes those showing enhanced membrane permeability as well as having inactivating mutation in one or more molecular chaperones and have been described in detail above in respect to the method of the invention.
- the invention relates to the use of a host cell according to the invention for the identification of compounds which are capable of inhibiting aggregation of an aggregation-prone polypeptide.
- the invention relates to:
- Method of screening for a compound that decreases aggregation of aggregation-prone polypeptides comprises (a) contacting one or more yeast cells with a candidate compound, wherein the yeast cells express a fusion protein comprising an aggregation-prone polypeptides and an enzyme which inhibits a toxic compound which affects yeast cell viability (b) adding the toxic compound to the yeast cell in an amount which, without the presence of the enzyme, would affect cell viability.
- yeast cell is selected from the group consisting of Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp. and Geotrichum fermentans.
- TSEs Alzheimer's (AD), and Huntingdon's Disease (HD).
- Kit useful for screening compounds that inhibit protein aggregation characterized in that it uses the method according to [I].
- strain FY834 was used in the preliminary assays and in the study related to overexpression of chaperones whereas drug testing was done in the drug permeable strain ergo A in the BY4741 parental background.
- Strains with a deletion of specific chaperone were provided by Euroscarf and were also based in the BY4741 strain.
- Methotrexate (MTX), sulfanilamide, quercetin and Congo Red were purchased from Sigma Aldrich.
- Plasmids encoding for the different yeast chaperones are listed in the following table.
- Cells were grown overnight at 30 0 C in a selective synthetic complete (SC) media containing raffinose. They were inoculated at OD 6 Oo of 0.02 in minimal galactose medium containing the appropriate MTX concentration (20-100 ⁇ M) and 1 mM of sulfanilamide. Growth was followed measuring OD 6 Oo using Cary 400Bio spectrophotometer. In drug screenings, the protocol was the same being the cells inoculated in a minimal galactose medium containing 20 ⁇ M MTX, ImM sulfanilamide and the tested compound: quercetin (30 ⁇ M) or CR (10 ⁇ M).
- SC selective synthetic complete
- Yeast cells were grown overnight in minimal media at 30 0 C containing raffinose. Cell density was determined by measuring the OD 600 and cells were diluted to a final OD 600 of 0.18 using PBS. Afterwards, 10 ⁇ l of each dilution (1/10, 1/10 and 1/100) was spotted in plates of selective synthetic complete media with galactose, MTX (20 ⁇ M) and sulfanilamide (1 mM). The Petri dishes were incubated for 48 hours at 30 0 C. Images of the plates were taken using the molecular imager Gel Doc XR system from Bio-Rad.
- Yeast cells transformed with plasmids encoding the target proteins fused to DHFR were grown overnight in minimal media at 30 0 C containing raffinose. They were inoculated at OD 600 of 0.02 in minimal galactose medium. Yeast cells were grown during 24 hours and then, they were incubated with sulfanilamide (1 mM) and 10 ⁇ M of
- MTX labeled with the fluorescent molecule Alexa (Invitrogen) for another period of 24 hours. Afterwards, the medium was removed and the cells were washed with PBS and reincubated for 30 min in the selective medium to allow for efflux of unbound flvlTX.
- both variants were expressed in S. cerevisiae as fusion proteins with GFP. While the GFP-fusion with mutant A ⁇ 42(F19D) did not aggregate intracellularly and its fluorescence was distributed diffusely throughout the cell, the A ⁇ 42-GFP protein fluorescence was concentrated in a single large aggregate in a juxtanuclear position, as shown by co-staining with Hoechst ( Figure 2). Immunob lotting of total cellular protein indicated that both proteins were expressed at similar levels, demonstrating that the degree of coalescence exhibited by A ⁇ 42 forms is more dependent on their sequence than on the level of protein expressed.
- the lethality of MTX on S. cerevisiae FY384 cells above certain concentrations can be overcome by transformation with a plasmid encoding human DHFR (h-DHFR) under the control of GaIl O promoter.
- h-DHFR human DHFR
- Different degrees of sensitivity to MTX may thus be correlated with the intracellular activity of the heterologously expressed enzyme, which is likely to vary depending on its expression alone, as a fusion with soluble molecules or as a fusion with an aggregation-prone polypeptide.
- the fused protein would promote, at least in part, its deposition lowering the intracellular DHFR activity and causing a higher sensitivity to MTX.
- both peptides were fused to h-DHFR.
- the differential growth abilities of cells expressing these fusions were compared with that of cells expressing h-DHFR alone.
- the system should allow also monitoring the influence of culture conditions on polypeptide aggregation. It is assumed that high temperatures promote in vivo and in vitro protein aggregation by reinforcing hydrophobic intermolecular interactions among polypeptides. Increasing the growth temperature from 30 0 C to 37°C resulted in a decrease in viability of cells expressing A ⁇ 42-DHFR at all the MTX concentrations assayed, but also of cells expressing A ⁇ 42(F 19D)-DHFR at high MTX concentrations (Figure 3). No such phenotypic effect was observed in cells expressing h-DHFR alone. This result suggested that temperature was specifically increasing, directly or indirectly, the aggregation propensity of the A ⁇ moiety and that the system was sensitive enough to detect such effect.
- h-DHFR activity as reflected in cell growth, could be used as a reporter to monitor the influence of both intrinsic and extrinsic factors on the aggregation of a given polypeptide.
- yeast growth was inversely proportional to polyQ expansions length.
- yeasts expressing Q25-DHFR exhibited the highest growth rate. Therefore, the survival of yeast cells expressing different polyQ fused to DHFR correlated with the observed solubility of the GFP fusions. This link indicated that, under the conditions of the assay, the presence of properly folded DHFR moiety determined the cell growth.
- the protein ⁇ -synuclein ( ⁇ -Syn) forms the fibrous portion of Lewy Bodies, cytoplasmatic inclusions present in Parkinson's disease (PD).
- PD Parkinson's disease
- A53T and A30P Two rare early-onset forms of PD are linked with mutations in the ⁇ -Syn gene: A53T and A30P. Both variants have distinct physical properties: A53T is accumulated at the plasma membrane or in cytoplasmatic foci like wild-type ⁇ -Syn; whereas A30P is dispersed through the cell.
- SynA3 OP-DHFR was higher and no aggregates were observed.
- punctuated nuclei usually close to the cytoplasmic membrane, were observed for the wild-type and A53T mutant.
- HsplO4 allows rescuing proteins from aggregated states regaining their function. It is a key protein in the chaperone network. Puzzlingly, Sisl or specially HsplO4 promoted an increase of fluorescence signal in the cytoplasm and in the aggregates. This coincides with their effect in the yeast model of HD, where it was shown that the protein in those aggregates was more loosely packed. Taking into account the relationship between packing of A ⁇ - GFP aggregates and the activity of the embedded protein that was established in bacteria, these chaperones might promote loosely packed and probably more active A ⁇ - DHFR aggregates.
- Hsp82 promoted viability seems to be dependent on direct reduction of A ⁇ 42 aggregation, resulting in smaller intracellular foci.
- human homolog interacts with amyloid precursors in AD and its upregulation protects neurons from A ⁇ toxicity.
- Hsp35, Hsp26 and Hsp42 chaperones promoted cell growth, implying lower A ⁇ aggregation.
- Hsp35 is a member of the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) family. And it is supposed to be a chaperone because of its heat inducibility and its high abundance in yeast.
- GPDH glyceraldehyde- 3-phosphate dehydrogenase
- Hsp26 and Hsp42 are small heat shock proteins (sHsp), which trap misfolded proteins into aggregates that are subsequently reactivated by the Hspl04/Hsp70/Hsp40 chaperone system. Nevertheless, larger substrate/sHsp relationships result in larger (and maybe tighter) complexes, which are poorly reactivated by other chaperones. The knockout of sHsp might prevent the incorporation of overexpressed A ⁇ 42 into sHsp aggregates reducing indirectly its deposition.
- deletion of the members of the Hsp70 cytosolic family reduced cell viability and increased the number of fluorescent foci within the cells.
- mutation of the SSAl, SSA2 genes also inhibited the expansion of small aggregate foci into a large inclusion body.
- the huge impact on both viability and aggregation of SSa4 deletion suggest that it could play an important role in protein folding and/or deposition. Accordingly, under conformational stress the amounts of Ssa4 mRNA increase several fold.
- the erg6 mutation inhibits ergosterol biosynthesis, which enhances membrane fluidity and permeability to various chemical compounds. This results in a four-fold higher sensitivity to MTX when compared to the FY384 strain and in a concomitant decrease in the viability of erg ⁇ A cells expressing A ⁇ 42-DHFR relative to those expressing h-DHFR.
- Quercetin is a flavonoid compound shown to inhibit in vitro A ⁇ fibril formation and to reduce the toxicity of A ⁇ fragments in neuroblastoma cells.
- a ⁇ 42-DHFR were engineered.
- table 4.3 there is a classification off the all chaperones used in this section according to their family.
- Genotype MAT a ura3-52 leu2- ⁇ trpl-63 his3-200 fys2-202
- Genotype MAT a his3 ⁇ l; leu2A0; met 15A0; ur ⁇ 3 ⁇ 0
- Genotype MAT a his3Al leu2A0 metl ⁇ AO ura3A0 erg ⁇ A: :kanMX4.
- This strain (based in the BY4741 parental background) has a mutation that affects cell permeability, specifically, in the gene codifying C-24 sterol methyltransferase Erg6p.
- the following Saccharomyces cerevisiae vectors were used: pESC vectors (Stratagene)
- the pESC vectors are a series of epitope-tagging vectors designed for expression and functional analysis of eukaryotic genes in the yeast S. cerevisiae. These vectors contain the GALl and GALlO yeast promoters and the yeast 2 ⁇ origin, which enables autonomous replication of the plasmids in S. cerevisiae. They have a selectable marker gene (HIS3, TRPl, LEU2, or URA3) to select and maintain the expression vector in yeast cells.
- a selectable marker gene HIS3, TRPl, LEU2, or URA3
- Saccharomyces cerevisiae media has been used:
- YDP Yeast Extract Peptone Dextrose
- SC Synthetic Complete drop-out media Dissolve the following compounds in 1 liter ddH 2 O 6.7 g Yeast nitrogen base without amino acids 100 ml of the appropriate sterile 10x Drop Out Solution Adjust the pH to 5.8 if necessary, and autoclave. Add the appropriate sterile carbon source, usually dextrose (glucose) to 2%.
- yeast competent cells The preparation and of yeast competent cells will be known by the skilled person in the art, and can be, for example, those described in Gietz, R.D. & Schiestl, R.H. Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2, 1-4 (2007), which are the method used in the present invention.
- FCC frozen competent cell
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