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  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
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  • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6478—Aspartic endopeptidases (3.4.23)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to methods for the identification of proteins with secretase activity and methods for the analysis of interactions between membrane proteins .
• Protein secretion is central to the proper development and function of eucaryotic organisms. Moreover, several pathophysiological processes such as neurodegeneration, oncogenesis, apoptosis and inflammation are associated with the malfunction or aberrant regulation of protein secretion. It has become clear that there is no single biosynthetic mechanism common to all secretory proteins. Secretion of proteins can occur through either the regulated or constitutive pathways and, in some cell types, this secretion can be polarized to distinct cellular domains. An increasing number of proteins are now recognized as being derived from integral membrane proteins of type I and type II topology and, in this case, the secretory event involves their selective post-translational hydrolysis from the cell surface.
• proteases known as secretases .
• the cleavage of membrane proteins generally occurs near the extracellular face of the membrane, although in some cases it has been shown also to occur within the transmembrane domain.
• Proteins secreted in this fashion include membrane receptors and receptor ligands, ectoenzymes, cell adhesion molecules and others .
• Examples of protein secretion through the action of secretases include the vasoregulatory enzyme ACE (angiotensin converting enzyme) , the tumor necrosis factor (TNF) ligand and receptor superfamilies, the transforming growth factor— ⁇ , certain cytokine receptors, the Alzheimer's amyloid precursor protein (APP) and others (Hooper, N.M.
• ACE angiotensin converting enzyme
• TNF tumor necrosis factor
• APP Alzheimer's amyloid precursor protein
• Proc.Natl.Acad.Sci.USA 96, 2885-2890 to use yeast for screening for cytoplasmic proteases of the group of caspases and their regulators .
• a fusion protein is created in which a transcription factor is linked to the intracellular domain of a transmembrane protein by caspase cleavage sites.
• the transcription factor is part of a reporter system thus that in the presence of a caspase the transcription factor is released and induces transcription of a reporter gene and thus allows identification of positive cells.
• the method allows only the isolation of members of the caspase group .
• a general object of the present invention to provide a method for the identification of a secretase wherein suitable host cells are transformed to express under suitable conditions a target membrane protein as a fusion protein with a secreted protein and said suitable host cells transformed to express said fusion protein are further transformed with a library encoding candidate secretases and said host cells transformed with said fusion protein and said library are cultivated under conditions allowing expression of both said fusion protein and said library and said conditions allowing cell survival only in presence of said secreted protein that has been released from said fusion protein by a secretase encoded by said library.
• the present invention furthermore provides a secretase that can be obtained by a method of the invention.
• secretase as used herein should be understood to include all types of proteins with protease activity.
• Another object of the present invention relates to a method for the identification of a membrane protein that is a substrate of a known secretase wherein suitable host cells are transformed to express under suitable conditions said secretase and said host cells transformed to express said secretase are further transformed with a library encoding fusion proteins with a secreted protein and a candidate substrate and said host cells are cultivated under conditions allowing expression of both the secretase and the library encoded fusion protein and allowing cell survival only in the presence of said secreted protein that has been released from said fusion protein by said secretase due to the interaction of said secretase with said library encoded substrate .
• Another object of the present invention concerns a method for screening for a protein interacting with a target membrane protein wherein suitable host cells are transformed to express under suitable conditions said membrane protein as a fusion protein with a secreted protein and the two moieties of said fusion protein are linked by a recognition sequence that is cleaved by a defined secretase.
• Said host cells transformed to express said fusion protein are further transformed with a library encoding fusion proteins with said defined secretase and candidate interaction partners of said known membrane protein and cultivated under conditions allowing expression of both the fusion protein and the library and allowing cell survival only in the presence of an interaction of the known membrane protein with the library encoded interaction partner protein.
• the secretase of said library fusion protein is brought into vicinity of said secretase recognition sequence linking the target membrane protein and the secreted protein and this vicinity allows the release of the secreted protein from said fusion protein and thus cell survival.
• Appropriate culturing conditions of the cells must be used such that said fusion protein is not efficiently cleaved by the secretase in the absence of a protein-protein interaction between said known membrane protein and said library encoded partner protein.
• said secretase is modified, preferably said modification is such that the membrane-anchoring domain of said secretase is deleted.
• This method allows the identification of interactions between membrane bound proteins or between ER/Golgi luminal proteins .
• the invention furthermore provides an interacting protein that can be obtained by a method of the invention.
• Another object of the present invention is a method for screening for a secretase wherein suitable host cells are transformed to express under suitable conditions a target membrane protein as a fusion protein comprising the known membrane protein, a secreted protein and a transcriptional activator which is part of a reporter system that is stably integrated into the genome of said host cells. Said host cells are further transformed with a library encoding candidate secretases and cultured under conditions allowing expression of both said fusion protein and said library and allowing cell
• the host cell is an eucaryotic cell, in particular a yeast cell.
• the target membrane protein preferably is a transmembrane protein, preferably a type I or a type II transmembrane protein.
• membrane protein as used herein comprises full length proteins as well as fragments thereof.
• the secreted protein moiety is fused to the N-terminus or the C-terminus of said protein, such that said moiety faces the ER lumen.
• General molecular biological and biochemical methods well known to the person skilled in the art are applied to determine the most suitable fusion protein of the target membrane protein and the secreted protein to result in a functional secreted protein after secretase cleavage (Maniatis et al . , Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Laboratory, 1989) .
• There are many strategies well known in the technical field to construct DNA libraries appropriate for the purposes of the present invention and to clone them in suitable vectors for expression in host cells Maniatis et al . , Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Laboratory, 1989).
• the introduction of the expression constructs of this invention into said suitable host cells can be performed by cotransformation or more preferably by sequential transformation, wherein in a much preferred embodiment of this invention said host cells are first transformed with the expression construct encoding said target protein or a fusion protein of said target protein and in a second transformation step said cells transformed to express said target protein or a fusion protein of said target protein are transformed with the second expression construct.
• the second construct can be a library encoding fusion proteins or a library encoding single proteins.
• the secreted protein is an protein with invertase activity or functional fragments of a protein with invertase activity, preferably a yeast invertase or functional fragments of a yeast invertase.
• a yeast invertase or functional fragments of a yeast invertase e.g. the yeast pheromone ⁇ peptide
• tissue culture cell lines whose growth is dependent on a protein growth factor. These cells can be manipulated such that they express in one embodiment of this invention the growth factor fused to a target membrane protein. Since the modified growth factor can not be secreted, these cells are dependent on a exogenous growth factor. Said host cells are then further transfected with a library and cultivated under conditions allowing the expression of both said fusion protein of the target membrane protein with said growth factor and the library and said conditions furthermore allowing growth of said host cells only in the presence of said growth factor that has been released from said fusion protein by a library encoded secretase.
• a suitable host cell line is for example PC12 whose growth is depending on the presence of NGF (nerve growth factor) .
• Fig 1 A Deletion strategy of the YAP3 gene of S . cerevisiae.
• Fig 1 B Confirmation of homologous recombination at the YAP3 locus by PCR genotyping.
• Fig 1 C Deletion strategy of the MKC7 gene of S . cerevisiae.
• Fig 1 D Confirmation of homologous recombination at the MKC7 locus by PCR genotyping.
• Fig 2 A Deletion strategy of the SUC2 gene in S . cerevisiae.
• Fig 2 B Confirmation of homologous recombination at the SUC2 locus by PCR genotyping.
• Fig 4 A Western blot analysis of SUC2 fusion with N-terminal truncated APP.
• Fig 4 B Colony formation of ULY 2 cells expressing SUC2-APP fusion proteins on sucrose plates.
• Fig 5 SUC2-APP (590-695) is activated in + background .
• Fig 6 ER retrieval signal in ⁇ + and ex- background .
• yeast such as Saccharomyces cerevisiae to utilize sucrose as a carbon source depends on the secretion of the enzyme invertase, which cleaves sucrose to yield glucose and fructose ( Carlson, M. et al. (1983) Mol. Cell. Biol. 3, 439-447). Indeed, deletion of the entire SUC2 gene, which encodes the invertase protein, or deletion of the signal peptide, which prevents secretion of the invertase, cripples the ability of yeast cells to grow on sucrose medium ( Perlman, D. , and Halvorson, H.O. (1981) Cell 25, 525-536; Carlson, M. , and Botstein, D.
• yeast strains deleted for the endogenous SUC2 gene to express human cDNAs fused to a modified SUC2 gene lacking its leader sequence, which encodes the secretion signal sequence.
• Heterologous secreted proteins appropriately fused to the N-terminus of the modified, non-secreted invertase could be identified through positive selection because they provided the necessary signals to restore invertase secretion, thus restoring cell growth on sucrose medium.
• invertase protein expression and localization of the invertase protein have also been monitored by alternative techniques such as colorimetric ( Goldstein, A. , and Lampen, O.J. (1975) Methods Enzymol . 42, 504-511) and immunodetection assays.
• colorimetric Goldstein, A. , and Lampen, O.J. (1975) Methods Enzymol . 42, 504-511
• immunodetection assays have been used to identify protein-sorting sequences that mediate localization to yeast mitochondria ( Emr, S.D. et al. (1986) J. Cell. Biol. 102, 523-533), vacuoles ( Klionsky, D.J. et al . (1988) Mol. Cell. Biol. 8, 2105- 2116; Tague, B.W. et al .
• the identification of a secretase activity expressed in yeast is based on its ability to cleave a specific target membrane protein fused to the invertase enzyme. Because of its fusion with the membrane-bound protein, this invertase is not secreted; consequently, these yeast cells, which lack the endogenous invertase, cannot grow on sucrose medium. However, in the presence of a secretase activity that specifically recognizes and cleaves the membrane-bound protein, the invertase enzyme is liberated from its anchor and it is secreted to the periplasm where it can hydrolyze sucrose, thus allowing cell growth on sucrose medium.
• chimerical proteins were used bearing the invertase enzyme fused to different portions of the membrane-bound Amyloid- ⁇ Precursor Protein (APP) .
• APP a type I transmembrane protein
• ⁇ , ⁇ - and ⁇ -secretases a type I transmembrane protein
• Cleavage by the latter two generates the 40 and 42 a ino acids A ⁇ peptides involved in Alzheimer's disease, while the ⁇ -secretase cleaves APP near the middle of the A ⁇ sequence.
• a yeast strain deleted for the endogenous SUC2 gene and expressing the invertase enzyme fused to the membrane- bound APP can efficiently grow on sucrose only when the Yap3p and Mkc7p secretases that cleave APP at the -site are co-expressed with the fusion protein.
• the experiments utilize two Saccharomyces cerevisiae strains, both of which have been deleted for the SUC2 gene, and one of them has been additionally deleted for the YAP3 and MKC7 genes.
• Fig. 1 shows the strategy that was used to delete the YAP3 and MKC7 genes.
• a kanamycin resistance cassette specifically constructed for yeast expression ( Steiner, S., and Philippsen, P. (1994) Mol. Gen. Genet. 242, 263- 271) was amplified by PCR using primers that possess at their 5 ' termini sequences homologous to YAP3 regions (Fig. 1A) .
• the yeast strain JPY9 ( Barberis, A. et al . (1995) Cell 81, 359-368) was transformed with this PCR product and plated on a selective agar medium containing the kanamycin analog G418. Only those cells that have steadily integrated the kanamycin resistance cassette could grow and form colonies on these selective plates.
• APP amino acid residues 590-695
• APP amino acid residues 590-695
• APP deletion mutant containing amino acid residues 590 to 695 is proteolytically processed in a proper manner in mammalian cells ( Citron, M. et al . (1995) Neuron 14, 661-670).
• invertase-HA-APP fusion protein bearing an APP sequence ending at the ⁇ site (aa 590-612) .
• Fig. 4A Western blot analysis using anti-HA antibodies
• Fig. 4B shows the effect that these fusion proteins had on the rate of ULY2 cell growth and colony formation on sucrose plates.
• SUC2 expression plasmid described above was used as a positive control; an empty vector, i.e. not expressing any SUC2 gene, was used as a negative control.
• sucrose plates of ULY2 cells expressing either the wild type SUC2 gene or the detruncated invertase-HA-APP (590-612) fusion protein was indistinguishable (Fig.
• yeast cells lacking endogenous invertase restored their ability to grow on sucrose plates to an extent very similar to the positive control, in which cells were transformed with a vector expressing wild type invertase.
• yeast cells that do not co-express the ⁇ -secretases with the invertase fusion protein have a reduced growth rate on sucrose plates, although, as previously shown, not to the extent observed with cells transformed with the empty vector.
• FIG. 6 shows that expression of this novel fusion protein in cells lacking the ⁇ -secretases did not confer them any ability to grow on sucrose plates above that observed upon transformation of an empty plasmid (negative control) .
• co- expression of this fusion protein with the ⁇ -secretases restored the ability of the transformed cells to grow on sucrose plates almost to the extend achieved by expressing wild type invertase.

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