WO1996040939A2 - Expression of functional vertebrate phospholipases in yeast - Google Patents
Expression of functional vertebrate phospholipases in yeast Download PDFInfo
- Publication number
- WO1996040939A2 WO1996040939A2 PCT/US1996/010002 US9610002W WO9640939A2 WO 1996040939 A2 WO1996040939 A2 WO 1996040939A2 US 9610002 W US9610002 W US 9610002W WO 9640939 A2 WO9640939 A2 WO 9640939A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- receptor
- phospholipase
- cell
- heterologous
- yeast
- Prior art date
Links
Classifications
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- 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
- C07K14/46—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
- C07K14/4722—G-proteins
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
Definitions
- This invention relates to functional expression of heterologous phospholipases in yeast, the recombinant yeast cells, and methods for using the engineered cells, including, inter alia, the identification of agonists and antagonists of signal transduction pathways which involve phospholipases.
- Eukaryotic phospholipases are known to be involved in cellular signal transduction processes important in regulation of cellular growth, differentiation and secretory events in numerous cell types.
- the binding of agonists to certain cell surface receptors leads to the generation of a variety of signaling molecules which include phospholipid derived second messengers.
- the phospholipid-derived second messengers mediate both transient and sustained responses and are implicated in a wide variety of physiological responses including growth regulation, immune modulation and neurotransmission.
- membrane lipids serve as substrates for the variety of different phospholipases that are responsible for the generation of phospholipid derived second messengers.
- phosphatidylinositols the substrate for phospholipase C enzymes, see below
- phosphatidylcholine the substrate for phospholipase C enzymes, see below
- phosphatidylcholine the substrate for phospholipase C enzymes, see below
- phosphatidylcholine the substrate for phospholipase C enzymes, see below
- phosphatidylcholine phosphatidylcholine
- phosphatidylethanolamine phosphatidylethanolamine
- sphingolipids e.g. sphingomyelin, sphingosine, sphingosine phosphate and sphingosine phosphocholine. See Dicheva and Irvine (1995) Cell 80:269-278 for a recent review.
- phospholipase activities known to be responsible for the generation of phospholipid second messengers, namely phospholipase A (including phospholipases Ai and A2), phospholipase B, phospholipase C, phospholipase D and sphingomyelinase.
- phospholipase A including phospholipases Ai and A2
- phospholipase B including phospholipases Ai and A2
- phospholipase C phospholipase C
- phospholipase D phospholipase D
- sphingomyelinase sphingomyelinase
- PLAi and PLA2 catalyze the release of fatty acids, in particular arachidonic acid (AA), from the sn ⁇ and snl positions of 1,2-diacylglycerol, 3- phosphocholines, generating AA and lysophosphorylcholine (lyso-PC) respectively.
- AA arachidonic acid
- AA has at least four known metabolic fates, namely, generation of proinflammatory eicosanoids (leukotrienes and prostaglandins), reincorporation into existing membrane phospholipids, reincorporation into soluble phospholipid metabolic pathways, and direct secretion from cells.
- lyso-PC serves as the precursor for the production of lipid second messengers like platelet activating factor (PAF).
- PAF platelet activating factor
- cDNA and genomic clones encoding the pancreatic form of PLA2 have been isolated from a variety of eukaryotic sources including bovine (Tanaka et al. 1987), rat (Ohara, O. et al. 1986 and 1990; and Kusunoki, et al. 1990), dog (Kerfelec et al. 1986),and pig (Seilhamer et al. 1986).
- cPLA.2 cellular PLA2
- cPLA.2 cellular PLA2
- the 85kDa gene product encoded by these cDNAs is expressed in cells types involved in the inflammatory response (neutrophils, platelets, monocytes and macrophages) and its expression is regulated by, for example, tumor necrosis factor, IL-1, TGF- ⁇ , MCSF and glucocorticoids.
- PLB Phospholipase B
- PLB isoforms have several catalytic properties including the release of fatty acids from the sn ⁇ and sn2 positions of phospholipids (as with PLAj and PLA2), the release of fatty acids from lysopholipids, and in the yeast Saccharomyces cerevisiae, acyltransferase activity that catalyzes the synthesis of phospholipids from lysophospholipids (Kuwabara et al. 1989; Gassama-Diagne 1989; and Lee et al. 1994). Genomic clones for PLB-like enzymes have been isolated from several sources including S. cerevisiae (Lee et al. 1994) and Penicillium notatum (Masuda et al. 1991).
- PLC Phospholipase C enzymes
- PLC- ⁇ , - ⁇ , - ⁇ , and - ⁇ catalyze the hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP2) to form two second messengers, inositol 1,4,5-trisphosphate (IP3) which is involved in the mobilization of intracellular stores of calcium, and diacylglycerol (DAG), which binds to and activates selected isoforms of protein kinase C (PKC; E.C. 2.7.1.37).
- Phospholipase D catalyzes the hydrolysis of phosphatidylcholines to yield phosphatidic acid (PA) and free choline (see Bishop, Pachter and Pai 1992; and Cook and Wakelam, 1992a).
- PA or its lyso derivative, lyso- PA may function to stimulate hydrolysis of polyphosphoinositides, activation of PKC, inhibition of adenylyl cyclase and stimulation of DNA synthesis (Moolenaar et al, 1986; Murayama and Ui, 1987, Van Corven et al, 1989; and Plevin et al, 1991b. refs in Cook and Wakelam, 1992b), as well as activate kinases directly in a manner similar to DAG (Eppand and Stafford, 1990, and Bocckina and Exton, 1990, refs in Cook and Wakelam, 1992b).
- phosphatidic acid formed by the action of PLD can be converted to DAG by the action of phosphatidic acid phosphohydrolase (E.C. 3.1.3.4), and thus serve as a secondary reservoir for the production of DAG by PLC-independent mechanisms (Bonser et al, 1989 cited in Divechia and Irvine 1995, review; and Cook and Wakelam, 1992b).
- cDNA clones for phospholipase D have been isolated from a number of different species including Arcanobacterium haemolyticum (Cuevas, W.A., and J.G.L.
- Phospholipase C enzymes are intracellular enzymatic mediators of a wide variety of cellular responses to extracellular stimuli. Binding of hormones, growth factors, neurotransmitters, and other agonists to specific cell surface receptors initiates the activation of PLC isozymes, which in turn result in the production of at least two well characterized active second messengers, inositol- 1,4,5-trisphophate (l.,4,5-IP3) and sn- ⁇ ,2- diacylglycerol (DAG).
- PLC Phospholipase C enzymes
- 1,4,5-IP3 binds to a specific family of intracellular receptors localized on modified portions of the endoplasmic reticulum that bears homology to the ryanodine receptor family of intracellular calcium channels (the IP3 receptor family, reviewed in Berridge, 1993). In addition to functioning as the IP3 receptor, these receptors also function as IP3-sensitive calcium channels which cause release of intracellular stores of calcium upon liganding with IP3.
- PLC protein kinase C
- MAPK Mitogen Activated Protein Kinase
- JNK Jun Kinase
- DAG forms a complex with phosphatidylserine and calcium and activates protein kinase C (PKC) isozymes, which in turn activate signaling pathways by direct phosphorylation of a variety of substrates which include adenylyl cyclases and RAF-1 (PJ. Parker et al, 1986; Coussens et al. 1986a, and Kolch et al. 1993).
- PLCs The division of PLCs into four classes, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ , originally corresponded to immunologically distinct proteins of 56 kDa, 150 kDa, 145 kDa, and 85 kDa purified from bovine brain (PLC- ⁇ , - ⁇ , and - ⁇ ) and other tissues including liver, seminal vesicles or uterus (PLC- ⁇ ). (Described in Suh et al. 1986; and summarized in A.J. Morris et al 1990).
- PI phosphatidyl inositol
- PIP phosphatidyl inositol 4-monophosphate
- PIP2 phosphatidyl inositol- 4,5,bisphosphate
- these immunologically distinct isozymes are distinguishable in several regards, not the least of which is that they are all unique gene products encoded by different genes, as discussed in further detail below. At the enzymatic level, these enzymes differ with respect to both their catalytic properties and their modes of regulation.
- polyphosphoinositides are the preferred substrates for the PLC- ⁇ and - ⁇ classes at low or physiological intracellular calcium concentrations, while PLC- ⁇ isozymes prefer PI and PIP2 at physiological intracellular calcium concentrations.
- the modes of regulation of PLC isozymes also differ considerably with respect to one another.
- the PLC- ⁇ isozymes are activated in response to ligand binding to G-protein coupled receptors and are directly stimulated by G ⁇ and G ⁇ components of heterotrimeric G-proteins.
- PLC- ⁇ isozymes are activated directly by phosphorylation via receptor and non-receptor tyrosine kinases.
- PLC- ⁇ regulation While little is known about the regulation of PLC- ⁇ activity in vivo or in vitro, recent studies of PLC- ⁇ regulation suggest that it may be regulated by a novel class of regulatory proteins (pl22-RhoGAP) that show similarity to the GTPase activating protein homology region of bcr, display GAP activity towards RhoA, and bind to and directly stimulate PLC- ⁇ 1 (Homma, Y., and Y. Emori, 1995).
- pl22-RhoGAP novel class of regulatory proteins
- PLC- ⁇ cDNA clones have been isolated from a variety of sources including rat basophilic leukemia cells (Bennett et al. 1988), mouse lymphocytes (Hempel and DeFranco, 1991), and bovine tissue (Hirano et al, GenBank Accession number D 16235).
- PLC- ⁇ encoding cDNAs encoding subtypes - ⁇ l-4 (Suh et al 1988; Bahk et al. 1994; Park et al. 1992; Lee et al. 1993; and Kim et al. 1993) have been reported.
- PLC- ⁇ encoding cDNAs Two PLC- ⁇ encoding cDNAs have been reported, which correspond to PLC- ⁇ l and - ⁇ 2 subtypes (Emori et al. 1989; Suh, et al, 1988; and Burgess et al. 1990). Three PLC- ⁇ encoding cDNAs encoding subtypes PLC- ⁇ 1-3 have been published (reviewed in Cook and Wakelam, 1992b and Boyer et al, 1994).
- PLC- ⁇ , - ⁇ ,and - ⁇ isozymes share considerable sequence homology, however, in two regions, variously referred to as the X and Y regions (Rhee et al, 1989) or regions I and II (Meldrum, et al. 1991).
- the X region is approximately 150 amino acid residues in length, while the Y region is approximately 260 residues in length.
- the X regions share approximately 50% identity, while the Y regions share approximately 40% identity in pairwise comparisons amongst PLC- ⁇ , - ⁇ , and - ⁇ isozymes. Based on the high degree of conservation of these two domains among the PLC- ⁇ , - ⁇ ,and - ⁇ isozymes, it has been argued that these regions contribute to the catalytic activity of these enzymes.
- An additional feature of the spacer region between the X and Y regions is that in PLC- ⁇ , and - ⁇ classes, these regions are separated by 50-100 residues rich in serine, threonine and acidic residues with no particular homology to known regulatory motifs, while in the PLC- ⁇ class the X and Y regions are separated by more than 400 residues.
- This region of the PLC- ⁇ isoforms encodes two src homology 2 (SH2) domains and one src homology 3 (SH3) domain, which are not present in either PLC- ⁇ or - ⁇ enzymes.
- SH2 domains are involved in regulation of PLC- ⁇ activation by receptor and non-receptor tyrosine kinases.
- PLC- ⁇ encodes neither the highly conserved X or Y regions found in all other PLC isoforms nor SH2 or SH3 domains found in PLC- ⁇ isoforms.
- Yeast PLC In addition to the mammalian PLC- ⁇ isoforms discussed in the preceding sections, various PLCs have been isolated from several other organisms. Of considerable importance in this regard is the molecular cloning of the gene (PLC1) encoding a protein with homology to the PLC class of phospholipases from the yeast Saccharomyces cerevisiae (Yoko-O et al. 1993; Payne and Fitzgerald-Hayes, 1993; and Flick and Thorner, 1993).
- Sequence analysis of the independently identified clones reveals first that all three clones are from the same genetic locus and that the protein coded for by this gene most resembles mammalian PLC- ⁇ isozymes both with respect to size of the enzyme encoded by it and with respect to structural features such as the relative distance between the X and Y domains, the size of the carboxyl terminal extension beyond the Y domain, and the absence of SH2 and SH3 domains between the X and Y motifs (this last point is to be expected as yeast show no evidence of phosphotyrosine modification by biochemical analyses).
- yeast secretory pathway A wide variety of foreign proteins have been produced in S. cerevisiae, that remain in the yeast cytoplasm or are directed through the yeast secretory pathway (Kingsman et al. TIBTECH 5, 53 (1987). These proteins include, as examples, insulin-like growth factor receptor (Steube et al. Eur. J. Biochem. 198, 651 (1991), influenza virus hemagglutinin (Jabbar et al. Proc. Natl. Acad. Sci. 82, 2019 (1985), rat liver cytochrome P-450 (Oeda et al. DNA 4, 203 (1985) and functional mammalian antibodies (Wood et al. Nature 314, 446 (1985).
- insulin-like growth factor receptor Steube et al. Eur. J. Biochem. 198, 651 (1991
- influenza virus hemagglutinin Jabbar et al. Proc. Natl. Acad. Sci. 82, 2019 (19
- yeast secretory pathway Use of the yeast secretory pathway is preferred since it increases the likelihood of achieving faithful folding, glycosylation and stability of the foreign protein.
- expression of heterologous proteins in yeast often involves fusion of the signal sequences encoded in the genes of yeast secretory proteins (e.g., ⁇ -factor pheromone or the SUC2 [invertase] gene) to the coding region of foreign protein genes.
- yeast expression vectors have been designed to permit the constitutive or regulated expression of foreign proteins.
- Constitutive promoters are derived from highly expressed genes such as those encoding glycolytic enzymes like phosphoglycerate kinase (PGK1) or alcohol dehydrogenase I (ADH1) and regulatable promoters have been derived from a number of genes including the galactokinase (GAL1) gene.
- PGK1 phosphoglycerate kinase
- ADH1 alcohol dehydrogenase I
- GAL1 galactokinase
- supersecreting yeast mutants can be derived; these strains secrete mammalian proteins more efficiently and are used as "production" strains to generate large quantities of biologically ac' /e mammalian proteins in yeast (Moir and Davidow, Meth. in Enzymol. 194, 491 (1991).
- Yoko-o et al. ((1993) Proc. Natl. Acad. Sci. USA 90:1804) claim to achieve complementation of a yeast PLC defect with an uncoupled rat PLC ⁇ l .
- Yoko-o et al. report the cloning of PLC from S. cerevisiae by PCR methodology using primers designed to incorporate sequences from two conserved amino acid sequences found in the X-region of mammalian PLC enzymes After cloning the gene, they proceeded to make gene disruptions at the PLC1 locus.
- Heterozygous diploid transformant (PLCl/plcl ::HIS3) were sporulated and tetrad analyses were performed.
- TY1 background all four spores were found to be viable at a range of temperatures including 18°C, 30°C, and 37°C.
- TY4 background Yoko-o et al. observed lethality at 30°Cin tetrad analyses. These phenotypes are in contradiction to those described herein, and those reported by Flick and Thorner in the art, where conditional lethality at 37°C is observed. This is important because it is not possible to measure complementation of a growth defect under the nonselective conditions of the Yoko-o et al experiments.
- Arkinstall et al ((1995) Mol. Cell. Biol. 15:1431) expressed a mammalian platelet- derived growth factor ⁇ receptor and PLC ⁇ 2 in Schizosaccharomyces pombe. Arkinstall et al. found that in cells coexpressing the mouse PDGF ⁇ receptor and PLC ⁇ 2, levels of [-1H]inositol phosphates in radioactively labeled yeast cells were increased. Coexpression of c-src was also found to result in phospholipid hydrolysis. Because these experiments only measure the immediate biochemical consequence of phospholipase C ⁇ stimulation, this work also fails to teach the functional integration of a mammalian PLC ⁇ into a yeast cell.
- Figure 1 is a graphic representation showing human phospholipases and G ⁇ l6 complementation of the plcl growth defect on high salt medium: S. cerevisiae strain CY1630 (MAT ⁇ plcl ⁇ l::His3 ade2-101 his3 ⁇ 200 leu2 ⁇ l lys2-801 trpl ⁇ l ura3-52) transformed with plasmids carrying each of the following as described in the text was streaked out in sectors 1-6: (1) Gallp-rat PLC- ⁇ l and GPAlp-G ⁇ l6; (2) CUPl-human PLC- ⁇ 2 and no G protein; (3) CUPl-human PLC- ⁇ 2 and GPAlp-G ⁇ i2-Q205L; (4) CUPl-human PLC- ⁇ 2 and GPAlp-G ⁇ S-Q227L; (5) CUPl-human PLC- ⁇ 2 and GPAlp-G ⁇ l6; (6) No PLC and GPA1-G ⁇ l6.
- (+) represents growth and (-) represents no
- the present invention relates to the functional expression of mammalian phospholipases in yeast cells.
- the use of the engineered yeast cells in identifying potential inhibitors or activators of the mammalian phospholipase, or of other proteins which belong to a phospholipase signal transduction pathway, i.e., proteins which are naturally or artificially "coupled” to the mammalian phospholipase in the engineered yeast cell is also within the scope of the invention.
- the term “coupled” here means that inhibition or inactivation of the coupled protein results in inhibition or activation (not necessarily respectively) of the phospholipase.
- coupled is meant to include both "natural” coupling of heterologous signaling molecules into a yeast signal transduction pathway and “artificial” coupling of heterologous signaling molecules into a yeast pathway in a manner not normally operative in yeast cells. Functional expression of human phospholipases is especially desirable.
- the yeast cells of the subject invention are characterized as including a heterologous gene encoding a heterologous phospholipase C (PLC), e.g., a polypeptide having a phospholipase C activity.
- PLC is a mammalian PLC such as a human PLC.
- the PLC can be a phospholipase selected from the group consisting of PLC ⁇ 's, PLC ⁇ 's, PLC ⁇ 's and PLC ⁇ 's.
- Preferred PLC's are of the ⁇ subtype, such as PLC ⁇ l, PLC ⁇ 2, PLC ⁇ 3 and PLC ⁇ 4.
- the heterologous phospholipase C can be constutitively activated. This may occur by way of mutations to the PLC itself which result in a higher level of activity, relative to the wild- type protein, in the absence of signaling to the PLC.
- the constitutive activation of the PLC can occur due to overexpression and/or consitutively activating mutations to upstream regulators of the PLC, such as receptors, G proteins and the like.
- the recombinant cell is characterized by the ability of the heterologous PLC to hydrolyze phosphatidylinositol 4,5-bisphosphate.
- the yeast cell has a loss-of-fun ⁇ ion mutation to an endogenous phospholipase gene, giving it a first detectable phenotype, and the heterologous PLC complements the loss-of-function mutation and confers a second detectable phenotype on the cell.
- the heterologous PLC is functionally integrated in a phospholipase-dependent signal pathway of the cell.
- the heterlogous PLC may complement a loss-of-function mutation of an endogenous phospholipase gene of the yeast cell, e.g., the heterologous PLC may complement the plcl ⁇ mutation.
- Expression of the heterologous PLC may confer a phenotype to the host cell which is detectably different than the phenotype of the host cell in the absence of the heterologous PLC protein. For instance, complementation of a loss-of-function mutation, such as the plcl .
- the heterologous PLC can rescue/overcome such phenotypes as temperature- sensitivity and/or salt sensitivity.
- the heterolgous PLC via a phospholipase-dependent signal pathway, can modulate such downstream effectors as calcium mobilization and/or PKC activity.
- the recombinant cell may also be engineered to include a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the phospholipase-dependent signal pathway, expression of the reporter gene providing the detectable signal.
- reporter genes encode a gene product that gives rise to a detectable signal, such as color, fluorescence, luminescence, cell viability relief of a cell nutritional requirement, cell growth, or drug resistance.
- the reporter gene encodes a gene product selected from the group consisting of chloramphenicol acetyl transferase, beta- galactosidase, luciferase, fluorescent protein, and alkaline phosphatase.
- the reporter gene encodes a gene product which confers the ability to grow in the presence of a selective agent, e.g., canavanine.
- the yeast cell further comprises a heterologous regulatory protein which is coupled to the phospholipase signaling pathway, e.g., the regulatory protein modulates the activity of the heterologous PLC or is modulated by the PLC activity.
- exemplary regulatory proteins include, for example, G-protein heterotrimers; G ⁇ subunits; G ⁇ subunits; proteins known to interact functionally with PLC substrates (e.g., profilin, gelsolin, cofilin, and alpha-actinin); and other effectors whose activity is dependent on PLC activity (i.e., IP 3 receptors/calcium channels, and IP 3 or calcium sensitive enzyme activities such as PKC or calcium/calmodulin kinase (CaM kinase)).
- the yeast cell comprises a heterologous receptor or ion channel or other surface protein capable of transducing signals via the phospholipase- dependent signal pathway, e.g., which is known to couple to PLC activity either directly or via a regulatory protein.
- This aspect of the invention provides for more general readout systems to assay modulation of the activity of a variety of different cellular signaling components which effect phospholipase C activation.
- the host cell further comprises a heterologous gene encoding a heterologous G-protein coupled receptor.
- Preferred G-protein coupled receptors include chemoattractant peptide receptors, neuropeptide receptords, cytokine, light receptors, neurotransmitter receptors, cyclic AMP receptors, and polypeptide hormone receptors.
- the receptor may be any one of: an ⁇ lA-adrenergic receptor, an ⁇ lB-adrenergic receptor, an ⁇ lC-adrenergic receptor, an M j AChR receptor, an M 3 AChR receptor, an M 5 AChR receptor, a D dopamine receptor, a D 3 dopamine receptor, an Al adenosine receptor, a 5HTl-like receptor, a 5HT Id-like receptor, a 5HTld beta receptor, a substance K (neurokinin A) receptor, a f- Met-Leu-Phe (FMLP) receptor, an angiotensin II type 1 receptor, a mas proto-oncogene receptor, an endothelin ETA receptor,
- the receptor protein is a receptor tyrosine kinase.
- the recombinant cells are further engineered to include an expressible recombinant gene encoding a heterologous test polypeptide. In preferred embodiments, such cells are provided as mixed culture collectively expressing a variegated population of test polypeptides.
- the present invention also relates to a rapid, reliable and effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of a phospholipase.
- the subject assay enables rapid screening of large numbers of test compounds to identifying those compounds which modulate, e.g., agonize or antagonize, phospholipase bioactivity.
- the assay is characterized by the use of the recombinant cells described herein as engineered to express heterologous phospholipase activities which produce, in the engineered cell, a detection signal.
- the ability of particular test compounds contacted with the instant cells to modulate the phospholipase activity can be scored for by detecting up or down-regulation of the detection signal. For example, second messenger generation via the heterologous phospholipase activity can be measured directly.
- an assay for identifying a modulator of a phospholipase C activity comprising the steps of: (i) contacting the cell of claim 1 with a test compound under conditions appropriate for detecting an intracellular signal transduced via the phospholipase-dependent signal pathway; and (ii) measuring a level of signal transduced by the phospholipase-dependent signal pathway in the presence of the test compound.
- a statistically significant difference in the level of signal in the presence of the test compound indicates that the test compound is a modulator of the heterologous phospholipase C activity.
- the intracellular signal can be detected, for example, by expression of a reporter gene operably linked to transcriptional regulatory elements sensitive to the phospholipase- dependent signal pathway.
- the intracellular signal is detected by measuring products of the hydrolysis of phosphatidylinositol 4,5-bisphosphate; by measuring Ca 2+ mobilization; and/or by measuring the activation of a protein kinase such as PKC.
- the intracellular signal can be detected by measuring a change in phenotype conferred by the heterologous phospholipase, such as a change in temperature sensitivity or NaCl sensitivity.
- the assay makes use the ability of a heterologous PLC to complement a loss-of-function mutation to an endogenous phospholipase activity.
- This embodiment of the subject assay generally includes the steps of: (i) contacting the cell with a test compound under conditions appropriate for detecting one or both of the phenotypes of the loss-of-function mutation or the complemented phenotype; and (ii) measuring the level or degree of one or both of these phenotype in the presence of the test compound.
- the ability of the test compound to alter the phenotype, relative to the absence of test compound or phospholipase C can be used to identify test compounds that are modulators of the heterologous phospholipase C activity.
- the assay utilizes a cell which is further engineered with a reporter gene construct which is sensitive to a phospholipase-dependent signal pathway from a heterologous PLC activity.
- Such assay formats generally include the steps of: (i) contacting the cell with a test compound under conditions appropriate for expression of the reporter gene; (ii) detecting expression of the reporter gene in the presence of the test compound; and (iii) comparing the level of reporter gene expression in the presence of the test compound to the level of reporter gene expression in the absence of the test compound or the absence of the heterologous phospholipase C.
- a statistically significant difference in the level of reporter gene expression in the presence of the test compound, relative to the absence of test compound or phospholipase C indicates that the test compound is a modulator of the heterologous phospholipase C activity.
- Still another embodiment of the assay provides a differential screening format for identifying an agent which selectively inhibits a non-mammalian phospholipase C relative to a mammalian phospholipase.
- Such assay formats can include the steps of: (i) providing a first yeast cell containing a non-mammalian phospholipase gene; (ii) providing a second yeast cell expressing a mammalian phospholipase gene; (iii) contacting each of the first and second yeast cells with a test compound; and (iv) detecting or quantitating the activity of the phospholipases of the first and second cell in the presence of the test compound.
- Such differential screening format can be used to identify inhibitors which are selective for phospholipases of mammalian pathogens relative to the host mammalian phospholipase.
- the mammalian PLC can be a human PLC
- the non-mammalian PLC can be derived from a human fungal pathogen, e.g.
- a pathogen which causes a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
- the phospholipase of the first cell can be cloned from a human pathogen selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
- a human pathogen selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Asper
- the assay can be carried out, e.g., repeated, for a library of at least 100 different test compounds, though more preferably at least 10 3 , IO 4 , IO 5 , IO 6 , or IO 7 different (variegated) compounds.
- the test compound can be, to illustrate, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extracts, synthetic compounds, or a mixture of combinatorial compounds.
- Phospholipases C are a family of enzymes which hydrolyze the sn-3 phosphodiester bond in membrane phospholipids producing diacylglycerol and a phosphorylated polar head group. Mammalian PLC enzymes exhibit specificity for the polar head group which is hydrolyzed, i.e., phosphatidylcholine, phosphatidylinositol, etc.
- PLC activation in cell proliferation is evident from the fact that the hydrolysis of PtdIns(4,5)P 2 is one of the early events that follow the interaction of many growth factors and mitogens with their respective receptors.
- the importance of PLC activation is not restricted to proliferation; it is one of the most common transmembrane signaling events elicited by receptors that regulate many other cellular processes, including differentiation, metabolism, secretion, contraction, and sensory perception.
- PLC- mediated transmembrane signaling events include, for example, signaling via receptor tyrosine kinases and G protein coupled receptors.
- the diacylglycerol released may be further metabolized to free arachidonic acid by sequential actions of diglycerol lipase and monoglycerol lipase.
- phospholipases may be further metabolized to free arachidonic acid by sequential actions of diglycerol lipase and monoglycerol lipase.
- C are not only important enzymes in the generation of second messenger molecules, but may serve an important role in making arachidonic acid available for eicosanoid biosynthesis in select tissues.
- the present invention is based on the development of yeast strains which express a functional, heterologous phospholipase and makes available a rapid, effective assay for screening and identifying pharmaceutically effective compounds that specifically interact with and modulate the activity of the heterologous phospholipase.
- the present invention provides a convenient format for discovering drugs which can be useful to modulate cellular function, as well as to understand the pharmacology of compounds that specifically interact with phospholipases.
- Signal transduction is the process by which chemical signals are relayed from the cellular environment to cellular targets via the cell membrane, and may occur through one or more mechanisms, such as phosphorylation, activation of ion channels, effector enzyme activation via guanine nucleotide binding protein intermediates, activation of phospholipase, and/or direct activation (or inhibition) of a transcriptional factor.
- a “mammalian phospholipase” is a protein which is either identical to an phospholipase occurring naturally in a mammal, or is a mutant which is substantially homologous with such a mammalian phospholipase and more similar in sequence to it than to the yeast phospholipase.
- Related terms, such as “primate phospholipase”, or “human phospholipase” are analogously defined.
- a mammalian phospholipase is "functionally homologous" to a yeast protein if, either alone, or in concert with other exogenous proteins, or after being modified by a drug, it is able to provide an phospholipase activity within the engineered yeast cell. It is not necessary that it be as efficient as the yeast protein, however, it is desirable that it have at least 10% of the activity of the analogous yeast protein.
- an “activator” or “agonist” of a phospholipase is a substance which causes the phospholipase to become more enzymatically active, e.g., elevates the rate at which phospholipid hydroylsis occurs under particular conditions.
- the mode of action of the activator may be direct, e.g., through binding the phospholipase, or indirect, e.g., through binding another molecule which otherwise interacts with the phospholipase.
- an "inhibitor or antagonist" of an phospholipase is a substance which causes the enzyme to become less active, and thereby reduces the rate or production of the hydrolytic second messengers to a detectable degree. The reduction may be complete or partial, and due to a direct or an indirect effect.
- phospholipase-dependent signal pathway refers to a signal transduction pathway including a phospholipase activity as a step in the transduction of the intracellular signal.
- a signal transduction pathway is meant to include components of a pathway whose activity is modulated by a phospholipase, whether their activity is directly modulated by the phospholipase or indirectly modulated by the phospholipase or its second messengers. This term also includes components of the pathway which are "upstream" of the phospholipase which regulate an activity of the phospholipase.
- the term "functional integration” as used herein refers to the ability of an exogenous phospholipase to function in a phospholipase-dependent signal transduction pathway such that a distal effect of a phospholipase activity can be determined, i.e., an activity regulated by the products produced from phospholipase dependent phospholipid hydrolysis. Such distal signaling effects can be observed in yeast expressing either a functional or mutant phospholipase.
- Another example of functional integration is complementation by a heterologous phospholipase C of one of the pleiotropy of effects seen in phospholipase loss- of-function yeast strains (e.g., reduced growth or NaCl sensitivity).
- Another assay of functional complementation is the ability of the heterologous phospholipase to modulate gene transcription.
- Yet another assay of functional integration is the reconstitution of regulated phospholipase activity, i.e., phospholipase activity requiring stimulation, for example phospholipase activity upon receptor-ligand activation or upon activation by a G protein subunit or subunits.
- This term can also include functional integration of a phospholipase into an endogenous yeast pathway or a chimeric pathway including components not naturally found in yeast cells.
- modulation of a phospholipase activity in its various grammatical forms, as used herein, designates induction and/or potentiation, as well as inhibition of one or more signal transduction pathways involving of a phospholipase. Such modulation may involve effects of a compound directly on a phospholipase, or on a component of a signal transduction pathway involving a phospholipase, for example upstream regulatory elements, or downstream elements, including substrates.
- substantially homologous when used in connection with amino acid sequences, refers to sequences which are substantially identical to or similar in sequence, giving rise to a homology in conformation and thus to similar biological activity. The term is not intended to imply a common evolution of the sequences.
- substantially homologous sequences are at least 50%), more preferably at least 80%, identical in sequence, at least over any regions known to be involved in the desired activity. Most preferably, no more than five residues, other than at the termini, are different.
- the divergence in sequence, at least in the aforementioned regions is in the form of "conservative modifications".
- recombinant cells include any cells that have been modified by the introduction of heterologous DNA.
- Control cells include cells that are substantially identical to the recombinant cells, but do not express one or more of the proteins encoded by the heterologous DNA, e.g., do not include or express one or more of the exogenous phospholipase, regulatory protein, test polypeptide, or the reporter gene construct.
- heterologous DNA or “heterologous nucleic acid” is meant to include DNA that does not occur naturally as part of the genome in which it is present or DNA which is found in a location or locations in the genome that differs from that in which it occurs in nature.
- Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell, i.e., is exogenous to the cell.
- such DNA encodes RNA and proteins that are not normally produced by the cell in which it is expressed.
- Heterologous DNA may also be referred to as foreign DNA.
- heterologous DNA any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by the term heterologous DNA.
- heterologous DNA include, but are not limited to, DNA that encodes a phospholipase, test polypeptides, regulatory proteins, reporter genes, transcriptional and translational regulatory sequences, or selectable or traceable marker proteins, such as a protein that confers drug resistance.
- recombinant protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.
- a “heterologous phospholipase” refers to a non-yeast phospholipase enzyme, having a phospholipase activity.
- phospholipase C in the presence of calcium, can convert phosphatidyl inositol (PI), phosphatidyl inositol 4-monophosphate (PIP) or phosphatidyl inositol (4,5) biphosphate (PIP2) into at least inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG).
- PI phosphatidyl inositol
- PIP3 phosphatidyl inositol
- DAG diacylglycerol
- an "endogenous, mutant phospholipase gene” as used herein refers to a yeast phospholipase gene that encodes an endogenous, mutant or impaired yeast phospholipase protein.
- an endogenous, mutant phospholipase may have a deletion, insertion, point mutation or other mutation that results in a conditional impairment or a constitutive impairment, making them ideal for selection or complementation studies.
- An example of an endogenous, mutant phospholipase gene is a deletion mutant in the S. cerevisiae PLC1 locus (plcl).
- plcl strains are sensitive to high osmolarity (0.5M NaCl or 1.2M sorbitol) at 30°C and do not grow well in the presence of carbon sources other than glucose (i.e. glycerol/ethanol, raffinose, and galactose).
- plcl strains have a temperature sensitive phenotype; at temperatures above 35°C, plcl strains do not grow, while at temperatures below 35°C, they grow, albeit slower than wildtype strains.
- a "loss of function" mutation is one which leads to a change in the activity of a phospholipase gene. Such a mutation may alter one or more of the coding sequence, the transcription, or the translation of an endogenous yeast phospholipase. Such a mutation will lead to the lack of expression of a yeast phospholipase, or the expression of an inactive yeast phospholipase or a phospholipase of reduced activity.
- a "reporter gene construct” is a nucleic acid that includes a “reporter gene” operatively linked to a transcriptional regulatory sequences. Transcription of the reporter gene is controlled by these sequences. The activity of at least one or more of these control sequences is directly or indirectly regulated by a signal transduction pathway involving a phospholipase, e.g., is directly or indirectly regulated by a second messenger produced by the phospholipase activity.
- the transcriptional regulatory sequences can include a promoter and other regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or regulatory sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or regulatory sequences that are recognized by effector molecules, including those that are specifically induced upon activation of a phospholipase.
- modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.
- Such sequences are herein collectively referred to as transcriptional regulatory elements or sequences.
- the construct may include sequences of nucleotides that alter the stability or rate of translation of the resulting mRNA in response to PLC second messages, thereby altering the amount of reporter gene product.
- a heterologous regulatory protein refers to a protein that regulates or is coupled to a heterologous phospholipase. Such a regulatory protein may be upstream or downstream of a phospholipase and is capable of modulating the activity of a phospholipase or being modulated by the phospholipase.
- a preferred regulatory protein is a GTP binding protein (G protein), which as used herein refers to heterotrimeric G proteins, G protein subunits thereof, e.g.
- a regulatory protein as used herein may also be a receptor that modulates a signal transduction pathway involving a phospholipase.
- preferred regulatory receptors are those that directly activate a phospholipase, such as the G protein coupled receptors, receptor tyrosine kinases (RTK's), receptors that activate phospholipases via the activation of a non-receptor kinases, such as those that signal via src kinases and the like.
- Other regulatory proteins can bind PIP 2 , the preferred substrate for many PLC isozymes (e.g. profilin, cofilin, gelsolin, and alpha- actinin) as discussed in Homma and Emori, 1995 and references therein. Regulatory proteins are discussed in further detail below.
- cell surface receptor refers to molecules that occur on the surface of cells, interact with the extracellular environment, and transmit or transduce the information regarding the environment intracellularly in a manner that ultimately modulates transcription of specific promoters, resulting in transcription of specific genes.
- extracellular signals include a molecule or a change in the environment that is transduced intracellularly via cell surface proteins that interact, directly or indirectly, with the signal.
- An extracellular signal or effector molecule includes any compound or substance that in some manner specifically alters the activity of a cell surface protein. Examples of such signals include, but are not limited to, molecules such as ions, acetylcholine, growth factors, and hormones, that bind to cell surface and/or intracellular receptors and ion channels and modulate the activity of such receptors and channels.
- Extracellular signals also include as yet unidentified substances that modulate a signal transduction pathway involving a phospholipase, and thereby influence intracellular functions. Such extracellular signals are potential pharmacological agents that may be used to treat specific diseases by modulating phospholipase activity.
- the present invention relates to recombinant yeast cells expressing a heterologous phospholipase and the methods of using these cells to identify compounds capable of modulating the signal transduction activity of such phospholipases.
- the subject assay is characterized by the use of recombinant cells which express a heterologous phospholipase protein whose signal transduction activity can be modulated in the recombinant cell so as to generate a detectable signal.
- these cells may (optionally) also express one or more of: (i) an expressible recombinant gene encoding an exogenous test polypeptide from a polypeptide library, (ii) a regulatory protein coupled to a phospholipase activity, and (iii) a reporter construct.
- the functional expression of a mammalian phospholipase in yeast provides for the design of inexpensive screens useful in the identification of modulators of the enzyme, the activity which is required for the generation of a central signaling molecule in mammalian cells. Any chemical entity, or combination of chemical entities, whether natural or synthetic, may be screened for the ability to modulate the mammalian phospholipase.
- modulators may act directly on the enzyme to alter the activity of the enzyme, e.g., they can competitively, noncompetitively, and/or allosterically potentiate or inhibit the enzyme's activity, or may affect the ability of a regulatory protein to alter phospholipase activity.
- the engineered cell is used to screen for drugs which, like G ⁇ or in some cases G ⁇ , can directly activate the phospholipase, or increase the activity of a partially activated phospholipase. In certain embodiments a chimeric G ⁇ or G ⁇ may be expressed for this purpose.
- the engineered cell is used to screen for drugs which inhibit mammalian phospholipase. In this situation, the phospholipase must first be activated. This can be done by engineering the cell to express G ⁇ or G ⁇ , as appropriate.
- the cell may be engineered to co-express both a G protein and a G protein coupled receptor, or any other protein which regulates the activity of the phospholipase, and the receptor stimulated either by externally added ligand or by a co-expressed ligand.
- the ligand is a known activator used merely to stimulate activation of the phospholipase, and the drugs are screened for inhibition of this phospholipase.
- the engineered cell is used to screen for drugs which inhibit or activate phospholipase indirectly, e.g., by their action upon a regulatory protein, for example, a G protein-coupled receptor.
- the receptor activates the G protein subunits which act on the phospholipase.
- a compatible G protein-coupled receptor and a compatible G protein would be provided with the mammalian phospholipase in the same yeast cell.
- test compounds to modulate the signal transduction activity of the target phospholipase can be scored for by detecting up or down-regulation of the detection signal.
- second messenger generation e.g. phospholipid hydrolysis, Ca + mobilization or PKC activation
- a reporter gene can provide a convenient readout.
- a statistically significant change in the detection signal can be used to facilitate identification of those compounds which are effectors of the target phospholipase.
- the assay may be repeated and modified by the introduction of a step in which the recombinant cell is first contacted with a known activator of the phospholipase, and the test compound can be assayed for its ability to inhibit the activity of the phospholipase, e.g., to identify phospholipase antagonists.
- the cell can be engineered with an activated phospholipase.
- test compounds can be screened for members which potentiate the response to a known activator of the phospholipase.
- a phsopholipase or a phospholipase regulatory protein may be constituatively active to facilitate screening for inhibitors of an activated phospholipase.
- constituatively active forms of the heterotrimeric Gq family members such as GI 1 and G16 have been described, such as G ⁇ qQ209L and G ⁇ 16Q212L.
- a constituatively actived form of a phospholipase activating receptor may be expressed, such as an ⁇ lB-adrenergic receptor which has been mutated to render it constituatively active (Perez et al.
- the subject reagent cells can be used to perform differential screening assays, e.g., which can be used to identify a compound that selectively effects a non-mammalian (e.g. non-human) phospholipase.
- Such compounds by selective inhibition, can be used for treating or preventing a pathogen infection (e.g. viral, fungal, bacterial or protozoan) in a mammal (e.g. human).
- a pathogen infection e.g. viral, fungal, bacterial or protozoan
- Preferred heterologous phospholipase enzymes for performing differential screening assays against a mammalian phospholipase include phospholipases from viral, fungal or protozoan pathogens.
- fungal pathogens include fungi that cause Candidiasis, Aspergillosis, Mucormycosis, Blastomycosis, Geotrichosis, Cryptococcosis, Chromoblastomycosis, Coccidioidomycosis, Conidiosporosis, Histoplasmosis, Maduromycosis, Rhinosporidosis, Nocaidiosis, Para-actinomycosis, Penicilliosis, Monoliasis, and Sporotrichosis.
- the ability of a compound to modulate the activity of one phospholipase and not another phospholipase may be measured.
- compounds which are agonists or antagonists of PLC ⁇ and not PLC ⁇ or are capable of modulating PLC ⁇ 2 and not PLC ⁇ 3 may be selected.
- compounds which are capable of modulating a heterologous phospholipase while not affecting another member of a signal transduction protein which may or may not be a member of a phospholipase pathway, such as PKC may be tested.
- the intracellular signal that is transduced can be initiated by the specific interaction of an extracellular signal, particularly a ligand, with a cell surface receptor which is coupled to the phospholipase.
- This interaction sets in motion a cascade of intracellular events, the ultimate consequence of which is a rapid and detectable change in the transcription or translation of a gene.
- transcriptional regulatory sequences that are responsive to the phospholipase-dependent intracellular signals and operatively linking the selected promoters to reporter genes, whose transcription, translation or ultimate activity is readily detectable and measurable
- the transcription based assay provides a rapid indication of whether a specific test compound influences intracellular transduction. Expression of the reporter gene, thus, provides a valuable screening tool for the development of compounds that act as agonists or antagonists of phospholipase activities.
- Reporter gene based assays of this invention measure the end stage of the above described cascade of events, e.g., transcriptional modulation and are thus ideal for assays in which a heterologous phospholipase is functionally integrated into a signal transduction pathway.
- a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on 2nd messengers generated by the phospholipase activity.
- the reporter gene construct will include a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the target phospholipase, with the level of expression of the reporter gene providing the phospholipase-dependent detection signal.
- the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expressio may be detected using Northern blots or specific protein product may be identified by a characteristic stain or an intrinsic activity. The amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound or it may be compared with the amount of transcription in a substantially identical cell that lacks the target phospholipase. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the target phospholipase.
- the gene product of the reporter is detected by an intrinsic activity associated with that product.
- the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
- the reporter or marker gene provides a selective growth advantage, e.g., the reporter gene may enhance cell viability, relieve a cell nutritional requirement, and/or provide resistance to a drug.
- second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium or phospholipid metabolism are quantitated, for instance, the products of phospholipid hydrolysis IP3 or DAG could be measured
- one of the downstream effects of PLC activation in mammalian cells is Ca " * ⁇ mobilization; because the teachings of the present invention demonstrate that a mammalian phospholipase can be functionally integrated into a yeast cell, it is likely that this effect may be expected in yeast cells expressing functional, recombinant PLCs.
- mammalian PLC is known to modulate PKC via its second messenger products (including DAG) functional expression of mammalian PLCs in yeast may be expected to couple to other signal pathway effectors including IP3-receptor, CaM kinase, calmodulin, PKA and PKC, or other proteins which interact with the phospholipases.
- DAG second messenger products
- the host yeast cell of the present invention may be of any species which is cultivable and in which an exogenous phospholipase can be modulated. Suitable species include Kluyverei lactis, Schizosaccharomyces pombe, and Ustilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeast which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha.
- yeast includes not only yeast in a strictly taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi or filamentous fungi.
- reporter constructs can provide a selectable or screenable trait upon transcriptional activation (or inactivation) in response to a signal transduction pathway coupled to the target receptor
- the reporter gene may be an unmodified gene already in the host cell pathway, such as the genes responsible for growth arrest in yeast. It may be a host cell gene that has been operably linked to a "phospholipase-responsive" promoter. Alternatively, it may be a heterologous gene that has been so linked. Suitable genes and promoters are discussed below. Accordingly, it will be understood that to achieve selection or screening, the host cell must have an appropriate phenotype. For example, introducing a pheromone-responsive chimeric HIS3 gene into a yeast that has a wild-type HIS3 gene would frustrate genetic selection. Thus, to achieve nutritional selection, an auxotrophic strain will be desired.
- Inactivation with respect to genes of the host cell, means that production of a functional gene product is prevented or inhibited. Inactivation may be achieved by deletion of the gene, mutation of the promoter so that expression does not occur, or mutation of the coding sequence so that the gene product is inactive. Inactivation may be partial or total.
- yeast genetics The field of yeast genetics is advanced and techniques for disrupting yeast genes are known in the art. For example, transplacement may be used to replace the endogenous yeast phospholipase gene with a modified form, as described by Flick and Thorner in art.
- a plcAl::HlS3 mutation can be created by replacing the internal Pvul ⁇ fragment of PLCl (nucleotides +1069 to +1482) with HIS3, as follows.
- the 3.7-kb Eco RV fragment containing most of PLCl can be inserted into Ec ⁇ RV-digested YCp50 to (Rose et al. 1987. Gene. 60:237) to yield pJFlO, which can be cleaved with Pvull and ligated with the
- HIS3 has replaced residues 357 to 494 of Plclp.
- the plcl l::HIS3 allele can be introduced into yeast cells by DNA mediated transformation
- a plclA2::LEU2 mutation can be created by replacing the Hpal-Clal segment of PLCl (nucleotides +277 to +2333) with LEU2, as follows.
- pJF58 which consists of a 4.3-kb Hindlll fragment containing PLCl inserted in pUC19 can be digested with Hpal and Clal and incubated with the Klenow fragment of E. coli DNA polymerase I.
- the resulting 5.4-kb linearized vector can be gel purified and ligated with a 2.2 kb SaR-Xhol fragment containing LEU2 that can be excised from pJJ283 (Jones and Prakash, supra) and converted to flush ends by treatment with Klenow enzyme, yielding pJF96.
- the plcl A2::LEU2 allele which is almost a complete deletion of the PLCl gene, can be introduced into S. cerevisiae by DNA- mediated transformation of the Ieu2/leu2 diploid strain, YPH501, using HmrflH-digested pJF96 and selecting for leucine prototrophy.
- any mutants generated will have a selectable phenotype, such as, for example, cessation of growth at 37°C, or failure to grow on a particular carbon source, will exhibit sensitivity to hypertonic stress (e.g., to salt or sorbitol), or will fail to grow on medium lacking a nitrogen source.
- a yeast cell expressing an endogenous mutant PLC will be capable of growth under one set of experimental conditions and will fail to grow or exhibit growth impairment under a second set of experimental conditions, i.e., would be conditionally lethal.
- “Complementation” with respect to genes of the host cell, means that at least partial function of inactivated gene of the host cell is supplied by an exogenous nucleic acid.
- yeast cells can be “mammalianized”, and even “humanized”, by complementation of receptor and signal transduction proteins with mammalian homologs.
- inactivation of a yeast phospholipase gene can be complemented, as described in the appended examples, by expression of a human phospholipase gene.
- PLC- ⁇ agonists can be identified in a screen for compounds that stimulate growth of plcl A yeast strains expressing PLC- ⁇ 2 but not expressing any G ⁇ q family G ⁇ -subunit under non-permissive growth conditions, plcl A strains expressing wildtype yeast PLCl could serve as an appropriate counterscreening strain.
- PLC- ⁇ antagonists could be screened for in two formats.
- plcl ⁇ yeast strains whose growth under non-permissive conditions is not dependent upon co-expression of G ⁇ q subunits (i.e. strains expressing mammalian PLC- ⁇ l, - ⁇ 2, or - ⁇ 3 on high copy plasmids) could be screened for compounds that block growth at 37°C or on 0.5M NaCl at 30°C but not at 30°C.
- An additional counterscreen for this format would be to screen for compounds that block growth of the plcl A strains but not of PLCl strains under non-permissive conditions. This counterscreen would serve to distinguish between compounds that act on fungal and mammalian PLC isoforms as opposed to those that act on one or the other or both.
- plcl A yeast strains whose growth under non- permissive conditions is dependent upon co-expression of G ⁇ q subunits could be screened for loss of complementation when contacted with compounds.
- This screen could in theory detect compounds that act either by inhibiting PLC-isoforms directly or by blocking the interaction between PLC- ⁇ isoforms and G ⁇ q subunits or by blocking the activation of G ⁇ q subunits.
- functional complementation in screening it may be possible to construct yeast strains in which the activation or inhibition of signaling components downstream of PLC- ⁇ can be scored appropriately configured strains.
- strains expressing both mammalian PLC- ⁇ isoforms and PKC may be used to screen for compounds that modulate the activity of either PLC- ⁇ isoforms, PKC isoforms, or both.
- CDK2 X61622 1.3e-137 cdc28 P00546 Elled ⁇ e et al. (1991 ) CDK3 X66357 1.3e-137 cdc28 P00546 Meyerson et al. (1992)
- VDAC1 L06132 9.8e-15 porl P04840 Blachlv-Dvson E. et al. (1993)
- VDAC2 L06328 2.8e-21 porl P04840 Blachlv-Dvson E. et al. (1993)
- ligating a polynucleotide coding sequence into a gene construct such as an expression vector
- hosts either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells)
- eukaryotic yeast, avian, insect or mammalian
- prokaryotic bacterial cells
- Similar procedures, or modifications thereof, can be employed to prepare recombinant reagent cells of the present invention by tissue-culture technology in accord with the subject invention.
- the vector be capable of replication in the host cell. It may be a DNA which is integrated into the host genome, and thereafter is replicated as a part of the chromosomal DNA, or it may be DNA which replicates autonomously, as in the case of a plasmid. In the latter case, the vector will include an origin of replication which is functional in the host. In the case of an integrating vector, the vector may include sequences which facilitate integration, e.g., sequences homologous to host sequences, or encoding integrases.
- YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein).
- These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
- drug resistance markers such as ampicillin can be used.
- Suitable promoters include the promoters for metallothionein, 3 -phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Req. 7, 149 (1968); and Holland et al.
- Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
- promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and glyceraldehyde-3 -phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose utilization.
- promoters that are active in only one of the two haploid mating types may be appropriate in certain circumstances.
- the pheromone promoters MFal and MF ⁇ l are of particular interest.
- Phospholipases As set out above, one aspect of the present invention encompasses the functional expression of a heterologous phospholipase in a yeast cell.
- a preferred phospholipase is phospholipase C.
- Preferred PLCs for carrying out the subject screening assays include any isotype of any of the four classes of phospholipases C (e.g. ⁇ , ⁇ .4, ⁇ 1&2 > or ⁇ l-3)-
- the phospholipases may be recombinant forms of vertebrate (e.g. mammalian, such as bovine, porcine, caprine, laprine, feline, canine, rodent, human or non-human primate), invertebrate (e.g.
- PLC Genbank Accession number XI 4034
- PLCl Genebank Accession number M372308
- PLC ⁇ 2 Genebank Accession number M95678; see also Park et al. J. Biol. Chem. 1992. 267:16048
- PLC ⁇ 3 Genebank Accession number Z37566
- the heterologous phospholipase need not be a naturally occurring protein, rather, it may be a mutant form of a phospholipase.
- the mutant is substantially homologous to a naturally occurring phospholipase, or a mutant known to be functional.
- the structural gene encoding the phospholipase may be the wild-type mammalian gene, or a modified gene. "Silent" modifications may be made to improve expression, by, e.g., (1) eliminating secondary structures in the corresponding mRNA, or (2) substituting codons preferred by yeast for codons that are not so preferred, or to facilitate cloning, e.g., by introducing, deleting or modifying restriction sites.
- the gene may also be modified so that a mutant phospholipase is encoded.
- Analysis of yeast codon usage indicates that there exists a preferred codon set consisting of the most abundant isoaccepting tRNAs present in yeast and that this preferred set (25 out of the 61 possible coding triplets) is the same for all yeast proteins (Bennetzen and Hall (1981) J. Biol. Chem. 257, 3026-3031).
- the rapid translation rate required for abundant proteins is believed to provide the selective pressure for the existence of the preferred set of codons.
- As the extent of biased codon usage in specific genes correlates directly with the level of gene expression Hoekma et al. (1987) Mol. Cell. Biol.
- the yeast cells of the present invention can be further engineered to express one or more regulatory proteins which regulate an exogenous phospholipase.
- Preferred regulatory proteins are G-protein coupled receptors, G proteins, receptor tyrosine kinases, and/or non-receptor kinases.
- Examples of other regulatory proteins are proteins which bind to phospholipase substrates and influence the activity of a phospholipase, such as profillin. Phospholipase regulatory proteins are described in detail below.
- G proteins One family of signal transduction cascades found in eukaryotic cells utilizes heterotrimeric "G proteins.” Transduction of growth signals by G protein-coupled receptors has been demonstrated to include phospholipase-dependent pathways. For example, ⁇ -type PLC isoforms are activated by the heterotrimeric G protein subfamily Gq. The ⁇ -subunits of Gq / l l/ 16' t0 further illustrate, specifically regulate PLC- ⁇ l and PLC- ⁇ 3, and the ⁇ / ⁇ -subunits of the Gi subfamily interact with PLC- ⁇ 2. Agonist interaction with specific G protein- coupled receptors causes the dissociation of Gq proteins into G ⁇ and G ⁇ / ⁇ subunits and the exchange of GDP bound to G ⁇ for GTP.
- G protein signaling systems include three components: a receptor , a GTP-binding protein (G protein), and an intracellular target protein.
- the G proteins which consist of alpha ( ⁇ ), beta ( ⁇ ) and gamma ( ⁇ ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors.
- GDP nucleotide guanosine diphosphate
- the receptor changes conformation and this alters its interaction with the G protein. This spurs the ⁇ subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP), replaces it, thus activating the G protein.
- GTP nucleotide guanosine triphosphate
- the activated state lasts until the GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the ⁇ subunit.
- the enzyme in turn converts an inactive precursor molecule into an active "second messenger," which may diffuse through the cytoplasm, triggering a metabolic cascade.
- the G ⁇ converts the GTP to GDP, thereby inactivating itself.
- the inactivated G ⁇ may then reassociate with the G ⁇ complex.
- Hundreds, if not thousands, of receptors transduce signals through heterotrimeric G proteins, of which at least 17 distinct forms have been isolated.
- G protein coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven transmembrane receptors (STRs).
- transmembrane domain receptors that have been reported in the literature to be coupled to phosphoinositide metabolism in cells and accordingly suitable for generating the subject reagent cell: an ⁇ lA-adrenergic receptor, an ⁇ lB-adrenergic receptor, an ⁇ lC-adrenergic receptor, an M j ACh receptor, an M3 ACh receptor, an M5 ACh receptor, a D 2 dopamine receptor, a D 3 dopamine receptor, an Al adenosine receptor, a 5HTl-like receptor, a 5HTld- like receptor, a 5HTld beta receptor, a substance K (neurokinin A) receptor, a f-Met-Leu-Phe (FMLP) receptor, an angiotensin II type 1 receptor, a mas proto-oncogene receptor, an endothelin ETA receptor, an endothelin ETB receptor, a thrombin receptor, a growth hormone-
- yeast Specific human G protein-coupled receptors for which genes have been isolated and for which expression vectors could be constructed include those listed herein and others known in the art.
- the gene would be operably linked to a promoter functional in the cell to be engineered and to a signal sequence that also functions in the cell.
- suitable promoters include those derived from Ste2, Ste3 and gall genes.
- Suitable signal sequences include those of Ste2, Ste3 and of other genes which encode proteins secreted by yeast cells.
- the yeast genome is preferably modified so that it is unable to produce the yeast analog of the recombinant receptor in functional form.
- PLC- ⁇ enzymes are activated in response to ligand binding to G-protein coupled receptors by hormones, neurotransmitters, small molecules and other agonists. This occurs via a pertussis toxin insensitive mechanism mediated by members of the Gq family of G ⁇ subunits as well as via a pertussis toxin sensitive mechanism mediated by G-protein complexes containing G ⁇ subunits of the Gi or Go family.
- Gq family members all members of the Gq family (q, 11, 14, 15, and 16) have been demonstrated both in vitro and in cell culture systems to directly stimulate the catalytic activity of PLC- ⁇ 1, - ⁇ 2, and - ⁇ 3 and - ⁇ 4 enzymes (A.J.
- PLC- ⁇ isoforms are sensitive to stimulation directly by GjV subunits.
- G ⁇ subunits derived from G ⁇ i or G ⁇ O-containing heterotrimers could contribute to the pertussis toxin sensitivity of PLC- ⁇ stimulation observed in whole cell systems in the past.
- Certain of the regions of PLC- ⁇ isozymes that are required for activation by G ⁇ or G ⁇ ⁇ subunits have been characterized.
- deletion mutations have shown that residues at the carboxyl terminus of PLC- ⁇ 1 (Wu et al, 1993) are crucial for interaction with G ⁇ subunits, while residues at the amino terminus appear to be essential for interaction with G ⁇ subunits, perhaps through binding of G ⁇ subunits to a presumptive pleckstrin homology present in the amino terminal 150 residues of PLC- ⁇ 2 (Inglese et al. 1995, and references therein).
- the G ⁇ subunit may be modified to improve coupling. These modifications often will take the form of mutations which, relative to interaction with the receptor or G ⁇ complex, increase the resemblance of the G ⁇ subunit to the yeast G ⁇ while decreasing its resemblance to the receptor-associated G ⁇ . For example, a residue may be changed so as to become identical to the corresponding yeast G ⁇ residue, or to at least belong to the same exchange group of that residue. After modification, the modified G ⁇ subunit might or might not be "substantially homologous" to the foreign and/or the yeast G ⁇ subunit.
- the modifications are preferably concentrated in regions of the G ⁇ which are likely to be involved in one or both of receptor binding and G ⁇ binding.
- the modifications will take the form of replacing one or more segments of the receptor-associated G ⁇ with the corresponding yeast G ⁇ segment(s), thereby forming a chimeric G ⁇ subunit.
- segment refers to three or more consecutive amino acids.
- point mutations may be sufficient.
- This chimeric G ⁇ subunit will interact with the endogenous receptor and the yeast
- G ⁇ complex thereby permitting signal transduction to occur via a pathway which includes the heterologous phospholipase. While use of the endogenous yeast G ⁇ is preferred, if a foreign or chimeric G ⁇ capable of transducing the signal to the recombinant phospholipase may be used instead.
- Certain phospholipase such as PLC- ⁇ enzymes, are activated directly in response to phosphorylation of key tyrosine residues by both receptor and non-receptor tyrosine kinases (Summarized in Cook and Wakelam, 1992b).
- receptors which mediate PLC ⁇ activation include those for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), and erbB2 which have intrinsic tyriosine kinase activity (Kumjian et al. (1991) J Biol. Chem.
- MIRRs multisubunit immune recognition receptors
- membrane immunoglobulin M and CD40 in B lymphocytes
- T-cell antigen receptor Park et al. 1991. Proc. Natl. Acad. Sci. USA 88:5453; Weiss and Liftman. 1994. Cell 76:253
- high-affinity immunoglobulin E receptor in basophilic leukemia cells
- PLC- ⁇ isozymes bind to specific autophosphorylated tyrosine residues within the kinase domains of these PTKs via the SH2 domains present in the spacer region between the PLC X and Y regions and the PLCs themselves become substrates for tyrosine phosphorylation.
- Phosphorylation of specific tyrosine residues (Tyr783 and Tyr 1254 on PLC- ⁇ l and Tyr753 and Tyr759 on PLC- ⁇ 2) by the receptor associated tyrosine kinase activities in turn increases the catalytic activity of PLC- ⁇ isozymes which results in increased production of 1,4,5-IP3 and *yw-l,2-diacylglycerol (DAG).
- profilin-bound PIP 2 was found to be resistant to cleavage by PLC- ⁇ l except when the PLC- ⁇ l had been activated by tyrosine phosphorylation.
- Other exemplary molecules which may regulate a phospholipase include profilin, gelsonin, cofilin, alpha-actin, and villin. The binding sites for PtdIns(4,5)P 2 on gelsolin and villin have recently been identified (Janmey et al.,(1992) J. Biol Chem 267:11818).
- PLC- ⁇ regulation may be regulated by a novel class of regulatory proteins (pl22-RhoGAP) that show similarity to the GTPase activating protein homology region of bcr, display GAP activity towards RhoA, and bind to and directly stimulate PLC- ⁇ 1 (Homma, Y., and Y. Emori, 1995)
- pl22-RhoGAP novel class of regulatory proteins
- Other reports suggest that rho can regulate PtdIns(4,5)P 2 levels (Chong et al. (1994) Cell 79:507).
- PLCs such as PLC ⁇ l (Bernstein and Ross) have a GAP-like activity for certain Gq family members. This activity serves to down regulate the activity of both the Gq and the PLC itself.
- IP3 receptors include: IP3 receptors, calcium channels, DAG protein kinase C (PKC), PKA3 (cAMP dependent protein kinase catalytic subunit), SOK1 (suppressor of PKA overexpression), CMD1 (calmodulin, CMK1 (Calcium/calmodulin dependent protein kinase), and CMK2 (calmodulin dependent protein kinase).
- PKC DAG protein kinase C
- PKA3 cAMP dependent protein kinase catalytic subunit
- SOK1 uppressor of PKA overexpression
- CMD1 calmodulin
- CMK1 Calcium/calmodulin dependent protein kinase
- CMK2 calmodulin dependent protein kinase
- the assay is characterized by the use of recombinant cells to sample a variegated compound library for phospholipase agonists and/or antagonists.
- the reagent cells express a mammalian phospholipase capable of producing a detectable signal in the reagent cell.
- the reagent cell also produces the test compound which is being screened.
- the reagent cell can produce a test polypeptide, a test nucleic acid and/or a test carbohydrate which is screened for its ability to modulate the mammalian phospholipase activity.
- a culture of such reagent cells will collectively provide a variegated library of potential phospholipase effectors and those members of the library which either agonize or antagonize the phospholipase function can be selected and identified.
- the reagent cell can be used to detect agents which directly alter the activity of the mammalian phospholipase, or which act on some target upstream or downstream of the mammalian phospholipase.
- the test compound is exogenously added.
- the test compound is contacted with the reagent cell.
- Exemplary compounds which can be screened for activity include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries. In such embodiments, both compounds which agonize or antagonize the phospholipase function can be selected and identified.
- the reagent cell can be used to detect agents which directly alter the activity of the mammalian phospholipase, or which act on some target upstream or downstream of the mammalian phospholipase.
- test compound is produced by cells which are cocultured with the reagent cells expressing a mammalian phospholipase.
- intracellular second messenger generation can be measured directly.
- Exemplary assays for the direct measurement of phospholipase generated second messengers are provided below; other assays for the measurement of second messengers generated in a signal transduction pathway involving a phospholipase will be apparent to those of skill in the art and may be substituted for the assays described below.
- the phospholipase enzymatic activity can be measured in yeast cell extracts. In other embodiments measurements of a detecting a phospholipase activity are made using intact yeast cells.
- Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. DAG can also be measured using thin-layer chromatography. Water soluble derivatives of all three inositol lipids (IPj, IP 2 , IP3) can also be quantitated using radiolabelling techniques or HPLC.
- the mobilization of intracellular calcium or the influx of calcium from outside the cell can be measured using standard techniques.
- the choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca ++ -sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56).
- Ca ++ detection cells could be loaded with the Ca ⁇ sensitive fluorescent dye fura-2 or indo-1, using standard methods, and any change in Ca "1-1" measured using a fluorometer.
- the other product of PIP2 breakdown, DAG can also be produced from phosphatidyl choline. The breakdown of this phospholipid in response to receptor-mediated signaling can also be measured using a variety of radiolabelling techniques.
- the activation of phospholipase A2 can easily be quantitated using known techniques, including, for example, the generation of arachadonate in the cell.
- the assay it may be desirable to screen for changes in cellular phosphorylation.
- the ability of compounds to modulate phospholipase activation could be screened using colony immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) using anti-phosphotyrosine.
- Reagents for performing such assays are commercially available, for example, phosphotyrosine specific antibodies which measure increases in tyrosine phosphorylation and phospho-specific antibodies can be purchased (New England Biolabs, Beverly, MA).
- Tests for phosphorylation could be useful to measure the phosphorylation of proteins in response to the modulation of a phospholipase activity; it is noted that protein phosphorylation need not be directly affected by the modulation in phospholipase activity, but rather may amplify signals generated by the phospholipase.
- Multi-kinase cascades allow not only signal amplification but also signal divergence to multiple effectors that are often cell-type specific, allowing a growth factor to stimulate mitosis of one cell and differentiation of another.
- MAP kinase pathway that appears to mediate both mitogenic, differentiation and stress responses in different cell types. Stimulation of growth factor receptors results in Ras activation followed by the sequential activation of c-Raf, MEK, and p44 and p42 MAP kinases (ERK1 and ERK2). Activated MAP kinase then phosphorylates many key regulatory proteins, including p90RSK and Elk-1 that are phosphorylated when MAP kinase translocates to the nucleus. Homologous pathways exist in mammalian and yeast cells. For instance, an essential part of the S.
- cerevisiae pheromone signaling pathway is comprised of a protein kinase cascade composed of the products of the STE11, STE7, and FUS3/KSS1 genes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to assay phospholipase modulation.
- Reporter gene based assays of this invention measure the end stage of the above described cascade of events, e.g., transcriptional modulation.
- a reporter gene construct is inserted into the reagent cell in order to generate a detection signal dependent on phospholipase signaling.
- the reporter gene construct will include a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to phospholipase signal modulation, with the level of expression of the reporter gene providing the receptor-dependent detection signal. If only one transcriptional regulatory element is included it must be a regulatable promoter.
- At least one the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the heterologous phospholipase whereby activity of the phospholipase can be monitored via transcription of the reporter genes.
- Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art.
- a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable.
- the reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.
- reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.
- CAT chloramphenicol acetyl transferase
- Transcriptional control elements which may be included in a reporter gene construct include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, after modulation of a phospholipase.
- genes include those which are responsive to the second messengers generated upon phospholipase activation, such as Ca """1" , cAMP, and others.
- One such gene is c-fos, an immediate early gene (see, Sheng et al. (1990) Neuron 4: 477-485).
- Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein.
- the characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.
- the amount of transcription from the reporter gene may be measured using any method known to those of skill in the art to be suitable.
- specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain or an intrinsic activity.
- the gene product of the reporter is detected by an intrinsic activity associated with that product.
- the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detection signal based on color, fluorescence, or luminescence.
- the amount of expression from the reporter gene is then compared to the amount of expression in either the same cell in the absence of the test compound or it may be compared with the amount of transcription in a substantially identical cell that lacks heterologuos DNA, such as the phospholipase. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the activity of the phospholipase.
- the reporter or marker gene provides a selection method such that cells in which the peptide is a ligand for the receptor have a growth advantage. For example the reporter could enhance cell viability, e.g., by relieving a cell nutritional requirement, and/or provide resistance to a drug.
- the reporter gene could encode a gene product which confers the ability to grow in the presence of a selective agent, e.g., canavanine.
- a selective agent e.g., canavanine.
- use of the S. cerevisiae plcl A allele as an endogenous mutant phospholipase C gene provides a readout system, whereby functional complementation (i.e. detection of the second detectable phenotype rather than the first detectable phenotype) can be detected or quantitated by cell growth.
- functional complementation i.e. detection of the second detectable phenotype rather than the first detectable phenotype
- growth systems suffer from their "quantal" nature.
- a transcriptionally based readout may be preferred because it is rapid (i.e. hours instead of days) and affords greater flexiblity both in terms of types of readout and dynamic range of the readout.
- a transcriptionally based readout may be preferred because it is rapid (i.e. hours instead of days) and affords greater flexiblity both in terms of types of readout and dynamic range of the readout.
- by placing the bacterial gene encoding lacZ under the control of the promoter sensitive to signals generated upon activation of a phospholipase pathway can be detected in less than an hour by monitoring the ability of permeabilized yeast to produce color from a chromogenic substrate. The rapidity of such a readout would, in itself, be advantageous. And such a readout would be necessary to monitor phospholipase activity under conditions where the yeast do not grow.
- a transcriptional readout can be developed by identifying genes/promoters that are positively or negatively regulated by the PLCl locus (i.e. promoters that are up or down- regulated in PLCl vs. plcl A strains or in plcl A strains expressing human PLC- ⁇ 2 plus G ⁇ 16). These promoters can then be linked to a reporter gene. Alternatively, the reporter gene can be expressed in response to a downstream phospholipase C signal molecule, such as IP3, DAG, increased intracellular calcium levels or increased PKC activity.
- a transcriptional based readout can be constructed using yeast expressing mammalian PLC enzymes in the following manner.
- CREB is a transcription factor whose activity is regulated by phosphorylation at a particular serine (SI 33).
- SI 33 serine
- phospho-CREB binds to a recognition sequence known as a CRE (cAMP Responsive Element) found to the 5' of promotors known to be responsive to elevated cAMP levels.
- CRE cAMP Responsive Element
- a transcriptional-based readout can be constructed in plcl ⁇ yeast strains expressing a mammalian PLC enzyme, regulatory proteins (G ⁇ and/or G ⁇ ), if necessary, and/or a reporter gene (i.e.
- lacZ whose expression is driven by a basal promoter containing one or more CREs in the following manner.
- Changes in the intracellular concentration of Ca + ⁇ (a result of alterations in the activity of the recombinant PLC) will result in changes in the level of expression of the reporter gene if: a) CREB is also co-expressed in the cell, and b) either the endogenous yeast CaM kinase will phosphorylate CREB in response to increases in calcium or if an exogenously expressed CaM kinase IV is present in the same cell.
- the promoter is activated upon activation of the phospholipase, in which case, for selection, the expression of the marker gene should result in a benefit to the cell.
- a preferred marker gene is the imidazoleglycerol phosphate dehydratase gene (HIS3). If a phospholipase responsive promoter is operably linked to a beneficial gene, the cells will be useful in screening or selecting for phospholipase activators. If it is linked to a deleterious gene, the cells will be useful in screening or selecting for inhibitors.
- the promoter may be one which is repressed by phospholipase, thereby preventing expression of a product that is deleterious to the cell.
- a phospholipase- repressed promoter one screens for agonists by linking the promoter to a deleterious gene, and for antagonists, by linking it to a beneficial gene.
- Repression may be achieved by operably linking a phospholipase-induced promoter to a gene encoding mRNA that is antisense to at least a portion of the mRNA encoded by the marker gene (whether in the coding or flanking regions), so as to inhibit translation of that mRNA.
- Repression may also be obtained by linking a phospholipase induced promoter to a gene encoding a DNA-binding repressor protein, and incorporating a suitable operator site into the promoter or other suitable region of the marker gene.
- suitable positively selectable (beneficial) genes include the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1,2,3, 4,5, 7,8; ARGl, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2, 5; THR1, 4; TRP2, 3, 4, 5; LEW, 4; MET2,3,4,8,9,14,16,19; URA1, 2,4,5, 10; H0M3,6; ASP3; CHOI; ARO 2, 7; CYS3; OLE1; IN01,2,4; PR01,3 Countless other genes are potential selective markers. The above are involved in well-characterized biosynthetic pathways.
- the imidazoleglycerol phosphate dehydratase (IGP dehydratase) gene (HIS3) is preferred because it is both quite sensitive and can be selected over a broad range of expression levels.
- the cell is auxotrophic for histidine (requires histidine for growth) in the absence of activation. Activation leads to synthesis of the enzyme and the cell becomes prototrophic for histidine (does not require histidine). Thus the selection is for growth in the absence of histidine. Since only a few molecules per cell of IGP dehydratase are required for histidine prototrophy, the assay is very sensitive.
- Suitable genes include: URA3 (orotidine-5'-phosphate decarboxylase; inhibits growth on 5-fluoroorotic acid), LYS2 (2-aminoadipate reductase; inhibits growth on ⁇ -aminoadipate as sole nitrogen source), CYH2 (encodes ribosomal protein L29; cycloheximide-sensitive allele is dominant to resistant allele), CAN1 (encodes arginine permease; null allele confers resistance to the arginine analog canavanin), and other recessive drug-resistant markers.
- the marker gene may also be a screenable gene.
- the screened characteristic may be a change in cell morphology, metabolism or other screenable features.
- Suitable markers include beta-galactosidase (Xgal, C12FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkaline phosphatase, horseradish peroxidase, exo-glucanase (product of yeast exbl gene; nonessential, secreted); luciferase; bacterial green fluorescent protein; (human placental) secreted alkaline phosphatase (SEAP); and chloramphenicol transferase (CAT).
- a preferred screenable marker gene is beta-galactosidase; yeast cells expressing the enzyme convert the colorless substrate Xgal into a blue pigment. Again, the promoter may be receptor-induced or receptor-inhibited.
- the present invention also facilitates the detection of inhibitors or activators of proteins other than phospholipase provided that the yeast cell expresses or is engineered to express the protein of interest in such a manner that it is functionally "coupled", directly or indirectly, to the phospholipase.
- yeast cells can also be used in more general readout systems for modulation of the activity of a variety of different cellular signalling components which regulate phospholipase activation, such as receptors, which are known to couple to phospholipases, either directly, (e.g. the PDGF receptor) or via a regulatory protein (e.g.
- a G- protein coupled receptor or tyrosine kinase coupled receptor a G-protein heterotrimer
- G ⁇ subunit a G ⁇ subunit
- G ⁇ subunit another protein known to interact functionally with a known phospholipase substrate (e.g., profilin, gelsolin, cofilin, and alpha-actin); and other effectors whose activity is dependent on phospholipase C activity (i.e., IP3 receptors/calcium channels, other potential IP3 sensitive calcium channels, and other calcium sensitive enzyme activities such as PKC or CaM kinase).
- phospholipase substrate e.g., profilin, gelsolin, cofilin, and alpha-actin
- effectors whose activity is dependent on phospholipase C activity (i.e., IP3 receptors/calcium channels, other potential IP3 sensitive calcium channels, and other calcium sensitive enzyme activities such as PKC or CaM kinase).
- the yeast cells of the present invention may be used to identify drugs which modulate the activity of a mammalian G protein-coupled receptor.
- the yeast cell is engineered to express a mammalian G protein-coupled receptor.
- Exemplary compounds which can be screened for activity include peptides, nucleic acids, carbohydrates, small organic molecules, and natural product extract libraries.
- peptide class One class of potential modulators of particular interest is the peptide class.
- the term "peptide” is used herein to refer to a chain of two or more amino acids, with adjacent amino acids joined by peptide (-NHCO-) bonds.
- the peptides of the present invention include oligopeptides, polypeptides, and proteins.
- the peptides of the present invention are 2 to 200, more preferably 5 to 50, amino acids in length.
- the minimum peptide length is chiefly dictated by the need to obtain sufficient potency as an activator or inhibitor.
- the maximum peptide length is only a function of synthetic convenience once an active peptide is identified.
- Synthetic peptides are also of interest.
- peptides based on the calmodulin-binding domain of calmodulin-dependent phospholipases could serve as modulators of phospholipase activity.
- peptides or molecules of any structure which inhibit the interaction between the phospholipase and known endogenous modulators of phospholipase activity are of interest.
- Known endogenous phospholipase modulators include Ca2+, Ca2+/calmodulin, protein kinase C, protein kinase A, Gas, Gai, G ⁇ , and adenosine.
- the yeast cells When peptide drugs are being assayed, the yeast cells may be engineered to express the peptides, rather than being exposed to the peptides simply by adding the peptides to the culture medium.
- Peptide libraries are systems which simultaneously display, in a form which permits interaction with a target, a highly diverse and numerous collection of peptides. These peptides may be presented in solution (Houghten 1991), or on beads (Lam 1991), chips (Fodor 1991), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull 1992) or on phage (Scott, Devlin, Cwirla, Felici, Ladner '409).
- the screening is for binding in vitro to an artificially presented target, not for activation or inhibition of a cellular signal transduction pathway in a living cell. While a cell surface receptor may be used as a target, the screening will not reveal whether the binding of the peptide caused an allosteric change in the conformation of the receptor.
- Ladner, USP 5,096,815 describes a method of identifying novel proteins or polypeptides with a desired DNA binding activity.
- Semi-random (“variegated") DNA encoding a large number of different potential binding proteins is introduced, in expressible form, into suitable host cells.
- the target DNA sequence is incorporated into a genetically engineered operon such that the binding of the protein or polypeptide will prevent expression of a gene product that is deleterious to the cell under selective conditions. Cells which survive the selective conditions are thus cells which express a protein which binds the target DNA. While it is taught that yeast cells may be used for testing, bacterial cells are preferred.
- the interactions between the protein and the target DNA occur only in the cell, not in the periplasm, and the target is a nucleic acid, not a protein.
- Yeast have been engineered to express foreign polypeptide variants to be tested as potential antagonists of mammalian receptors.
- Libraries encoding mutant glucagon molecules were generated through random misincorporation of nucleotides during synthesis of oligonucleotides containing the coding sequence of mammalian glucagon. These libraries were expressed in yeast and culture broths from transformed cells were used in testing for antagonist activity on glucagon receptors present in rat hepatocyte membranes (Smith et al. 1993).
- the yeast cells are engineered to express a peptide library.
- a "peptide library” is a collection of peptides of many different sequences (typically more than 1000 different sequences), which are prepared essentially simgrowthultaneously, in such a way that, if tested simultaneously for some activity, it is possible to characterize the "positive" peptides.
- the peptide library of the present invention takes the form of a yeast cell culture, in which essentially each cell expresses one, and usually only one, peptide of the library. While the diversity of the library is maximized if each cell produces a peptide of a different sequence, it is usually prudent to construct the library so there is some redundancy. Moreover, each sequence should be produced at assayable levels.
- the peptides of the library are encoded by a mixture of DNA molecules of different sequence. Each peptide-encoding DNA molecule is ligated with a vector DNA molecule and the resulting recombinant DNA molecule is introduced into a yeast cell. Since it is a matter of chance which peptide-encoding DNA molecule is introduced into a particular cell, it is not predictable which peptide that cell will produce. However, based on a knowledge of the manner in which the mixture was prepared, one may make certain statistical predictions about the mixture of peptides in the peptide library.
- the peptides of the library will have at least one, and usually more than one, variable residue.
- a variable residue may vary among any of two to any of all twenty of the genetically encoded amino acids; the range of possibilities may be different, if desired, for each of the variable residues of the peptide.
- the frequency of occurrence of the allowed amino acids at particular residue positions may be the same or different.
- the peptide may also have one or more constant residues.
- the DNAs are synthesized a base at a time.
- a suitable mixture of nucleotides is reacted with the nascent DNA, rather than the pure nucleotide reagent of conventional polynucleotide synthesis.
- the second method provides more exact control over the amino acid variation.
- trinucleotide reagents are prepared, each trinucleotide being a codon of one (and only one) of the amino acids to be featured in the peptide library.
- a mixture is made of the appropriate trinucleotides and reacted with the nascent DNA.
- the cytoplasm of the yeast cell is bounded by a lipid bilayer called the plasma membrane. Between this plasma membrane and the cell wall is the periplasmic space.
- Peptides secreted by yeast cells cross the plasma membrane through a variety of mechanisms and thereby enter the periplasmic space.
- the secreted peptides are then free to interact with other molecules that are present in the periplasm or displayed on the outer surface of the plasma membrane.
- the peptides then either undergo re-uptake into the cell, diffuse through the cell wall into the medium, or become degraded within the periplasmic space.
- the peptide library may be secreted into the periplasm by one of two distinct mechanisms, depending on the nature of the expression system to which they are linked.
- the peptide may be structurally linked to a yeast signal sequence, such as that present in the ⁇ -factor precursor, which directs secretion through the endoplasmic reticulum and Golgi apparatus. Since this is the same route that the receptor protein follows in its journey to the plasma membrane, opportunity exists in cells expressing both the receptor and the peptide library for a specific peptide to interact with the receptor during transit through the secretory pathway. This has been postulated to occur in mammalian cells exhibiting autocrine activation.
- Such interaction would likely yield activation of the linked pheromone response pathway during transit, which would still allow identification of those cells expressing a peptide agonist.
- An alternative mechanism for delivering peptides to the periplasmic space is to use the ATP-dependent transporters of the STE6/MDR1 class. This transport pathway and the signals that direct a protein or peptide to this pathway are not as well characterized as is the endoplasmic reticulum-based secretory pathway. Nonetheless, these transporters apparently can efficiently export certain peptides directly across the plasma membrane, without the peptides having to transit the ER/Golgi pathway.
- the present invention does not require periplasmic secretion, or, if such secretion is provided, any particular secretion signal or transport pathway.
- agents identified in the subject assay can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.
- the subject compounds selected in the subject, or a pharmaceutically acceptable salt thereof may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
- a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
- a biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art.
- Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on the above, such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the compound in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and isosmotic with physiological fluids. In preferred embodiment, the compound can be disposed in a sterile preparation for topical and/or systemic administration.
- excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH.
- Similar solutions may also be used for the pharmaceutical compositions of compounds in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).
- All expression plasmids are based upon the shuttle vectors originally described by Sikorski and Hieter (R.S. Sikorski and P.Hieter (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27).
- the expression plasmids with the GPAl promoter (Cadus 1127), the PGK promoter (Cadus 1447) and the CUPl promoter (Cadus 1725 and 1728) have been described elsewhere (Cadus published Patent Application WO 94/23025 entitled "Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor).
- G -subunits Construction of Low Copy Expression Plasmids
- the following full length G-protein encoding inserts G ⁇ S, G ⁇ i2, G ⁇ i3, G ⁇ q, G ⁇ l 1,
- G ⁇ l6, G ⁇ OA, G ⁇ OB, G ⁇ l 2, GNAZ, and activated alleles (GTPase-deficient) of G ⁇ S, G ⁇ i2, G ⁇ q, and G ⁇ l 6 were amplified by polymerase chain reaction using VENT polymerase, with cloning sites added to the 5' and 3' ends of the coding regions. In all cases, the restriction site added to the 5' end of the gene encoded a novel Ncol compatible overhang, while the restriction site added to the 3' end of the gene encoded a novel Xhol compatible overhang.
- Cadus 1127 is a low copy plasmid (CEN6 ARS4) in which heterologous gene expression is driven by the GPAl promoter (-1400 to +1 of the native GPAl locus).
- CEN6 ARS4 low copy plasmid
- a novel Ncol site has been engineered into this vector such that the initiator methionine (at position +1) is embedded in this site (CCATGG). All G-protein expression cassettes were constructed as described below for G ⁇ l 6.
- Template 100 ng
- primers 1 uM final concentrations
- lOx reaction buffer 2 mM MgSO4.
- Template was denatured at 94°C for 30 seconds in the presence of primers followed byaddition of lul per lOOul reaction volume of VENT polymerase.
- the amplified product was purified by gel electrophoresis, cut with BsmBI and Xhol, and ligated to Ncol / Xhol cut and phosphatase treated Cadus 1127 vector DNA. Recombinant clones were identified by colony PCR using primers flanking the inserted G ⁇ l 6 sequences, confirmed by restriction digestion, and validated by dideoxynucleotide sequencing of the PCR product.
- G ⁇ q and Gal l expressing plasmids were prepared in a similar fashion using the following oligonucleotide primer pairs for amplification:
- G -subunits - High Copy Expression Plasmids Full length G-protein encoding inserts were amplified by polymerase chain reaction using VENT polymerase as described above, with cloning sites added to the 5' and 3' ends of the coding regions. In all cases, the restriction site added to the 5' end of the gene encoded a novel Ncol compatible overhang, while the restriction site added to the 3' end of the gene encoded a novel Xhol compatible overhang. PCR products were gel purified, restricted with the appropriate endonucleases, and cloned into Ncol/Xhol cut Cadus 1447.
- Cadus 1447 is a high copy plasmid (2 micron origin of replication) in which heterologous gene expression is driven by the phosphoglycerol kinase (PGK) promoter.
- PGK phosphoglycerol kinase
- a novel Ncol site is present at the initiator methionine, and a unique Xhol site is downstream from it.
- G-proteins cloned in this manner include G ⁇ S, G ⁇ q, Gal l, Gal6, GaOA, GaOB, Gal2, GNAZ, and activated alleles (GTPase-deficient) of G ⁇ S, G ⁇ i2, G ⁇ q, and G ⁇ l 6.
- Rat PLC- ⁇ 1 (Suh et al., (1988)) was cloned into Cadus 1725 (CUPp LEU2 CEN ARS AmpR) and Cadus 1728 (CUPp LEU2 2 micron origin AmpR).
- CUPp LEU2 CEN ARS AmpR Cadus 1725
- Cadus 1728 Cadus 1728
- ATG initiator methionine codon
- the resulting plasmid, Cadus 1642 (Rat PLC- ⁇ 1), was cleaved with ApaLI and ligated to a duplex adapter encoding the amino terminal seven residues of rat PLC- ⁇ 1 with yeast codon bias.
- the ligation mixture was treated with polynucleotide kinase, cleaved with BamHI and a 3.8 kb fragment with the entire coding region of rat PLC- ⁇ 1 was isolated by gel electrophoresis. This 3.8 kb PLC- ⁇ 1 fragment was then ligated to Ncol - BamHI cut Cadus 1725 and Cadus 1728. Plasmids containing inserts in the correct orientation were identified by colony PCR and verified by restriction analysis and double-stranded DNA sequencing using Sequenase V2.0 (U.S. Biochemicals).
- Oligo D was treated with polynucleotide kinase prior to annealing to oligo C and ligation to EcoRI-blunt/Eco47III cut Cadus 1680. Following ligation, the ligation mixture was treated with polynucleotide kinase to phosphorylate the Ncol overhang at the 5' end of the gene and the 3.8 kb fragment with the entire coding region of human PLC- ⁇ 2 was purified by gel electrophoresis. This 3.8 kb fragment has Ncol compatible overhangs at both termini, and was ligated to Ncol cut/ shrimp alkaline phosphatase treated Cadus 1725 and 1728. Plasmids containing insert in the correct orientation were identified by colony PCR and verified by restriction analysis and double-stranded DNA sequencing using Sequenase V2.0.
- S.cerevisiae strain CY 2954 (plcl*l::HIS3 ade2-101 his3*200 leu2*l lys2-801 trpl *l ura3-52 [CUP RAT PLCbetal LEU2 2mu-ori REP3 AmpR], GPAlp TRPl CEN6 ARS4 AmpR] was deposited with the American Type Culture Collection (Rockville, Maryland) on June 5, 1995 and has been designated with the ATCC Accession Number: 74343.
- GAL1 promoter driven PLC- 1 does not complement the plcl mutant phenotype
- Phospholipase-C ⁇ dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form inositol 1,4,5, trisphosphate (IP3) and diacylglycerol (DAG) is stimulated by both G ⁇ and G ⁇ subunits of heterotrimeric G-proteins. Stimulation of PLC- ⁇ activity by G ⁇ subunits is restricted to members of the Gq family that includes Gq, -11, -14, -15, and -16. G ⁇ subunits belonging to other families (-s, -i, and -12/13) do not stimulate PLC- ⁇ dependent hydrolysis of PIP2 .
- agonist dependent stimulation of IP3 and calcium mobilization have been shown to be partially sensitive to pertussis toxin. This observation initially lead to speculation that Gi or Go could stimulate PLC- ⁇ activity. More recently the pertussis toxin sensitive component of agonist dependent IP3 production has been shown to be due to the ability of G ⁇ subunits to stimulate PLC- ⁇ isoforms directly.
- Cadus yeast strains CY1901, CY1903 and CY1904 (all CY1630 derivatives containing the plcl ⁇ l::HIS3 allele) contain, respectively, Cadus plasmids 1443 (no PLC), 1637 (GALlp-rat PLC- ⁇ l) or 1639 (GALlp-PLCl)(See Strain List, Table 2).
- CY1904 While all three strains grow at 30°C on both synthetic complete and rich media, CY1904, harboring a GALlp-PLCl expression cassette on a 2 micron plasmid, grows better than either CY1901 (empty vector) or CY1903 (harboring a GALlp-rat PLC- ⁇ l expression cassette on a 2 micron plasmid). At non-permissive temperatures (>35°C), CY1901 and CY1903 failed to form colonies after 3 days in culture, while CY1904 forms large colonies.
- CY1901, CY1903, and CY1904 (all haploid strains) were transformed by lithium acetate transformation procedures (Clontech, as per manufacturer's recommendations) with a panel of low-copy number (CEN ARS) G ⁇ subunit expression plasmids coding for the following genes under the transcriptional control of the GPAl upstream promoter: Cadus plasmid 1127 (empty vector), 1179 (GPAl), 1181 (G ⁇ S), 1606 (G ⁇ i2), 1617 (G ⁇ l6), 1753 (G ⁇ l6-Q209L GTPase " activated allele), 1685 (G ⁇ q), and 1749 (G ⁇ q-Q209L GTPase " activated allele)(See Strain List, Table 1).
- a second potential difficulty presented with the GALl promoter relates to the inability of plcl strains to utilitize galactose as a carbon source. If galactose utilization is compromised in these strains, then induction of galactose sensitive promoters like GALlp and GALl Op would also be compromised. To circumvent both of these potential problems, the mammalian PLC- ⁇ isozymes were expressed from an inducible promoter whose activation is not dependent on carbon sources other than glucose. Unlike other high level constitutive promoters (i.e.
- PGK or ADH1 has not been associated with cellular toxicity. This sort of toxicity has, in fact been reported in the literature by Flick and Thorner (1993) with respect to overexpression of the yeast PLCl gene from the GALl promoter in the presence of galactose. Flick and Thorner report that the growth of wildtype strains of yeast that contained the PLCl gene under the control of the GALl promoter was compromised when the GALl promoter was induced on galactose but not when it was repressed on glucose-containing media.
- Yeast Strain CY1630 was transformed with each of these plasmids, as well as other PLC constructs in which PLC isozymes are driven by the GALlp or ADH1 promoters, in the presence or absence of plasmids expressing various G ⁇ subunits.
- this particular hybrid subunit is functionally expressed in yeast (i.e. it couples to yeast G ⁇ subunits and supresses activation of the pheromone response pathway in gpal- strains).
- yeast G ⁇ subunits couples to yeast G ⁇ subunits and supresses activation of the pheromone response pathway in gpal- strains.
- the structural determinants involved in effector activation by G ⁇ subunits are thought to map to residues in the carboxyl terminal portions of G ⁇ subunits (Berlot and Bourne (1992); Rarick et al. (1992); Artemeyer et al.(1993)).
- Y1630 ( plcl) derivatives See Strain List, Table 2) harboring combinations of mammalian PLC- ⁇ isozymes and G ⁇ subunits were first colony purified and then duplicate sibs of each co-transformation assayed for growth at 30oC and 37oC in the presence or absence of lOOuM CuSO4.
- all plcl strains including those containing plasmids encoding rat PLC- ⁇ l, grew at 30oC, albeit less well than strains CY1633 (PLCl) or CY1904 (plcl ⁇ l::HIS3 [GALlp-PLCl]).
- CY1630 derivatives co-transformed with a low copy (CEN ARS) plasmid encoding CUP-human PLC- ⁇ 2 and a low copy (CEN ARS) plasmid encoding the G ⁇ l6-Bam allele grew at both 30oC and at 37oC.
- growth of CY1630 derivatives at 37oC in this assay was not due to expression of the G ⁇ l6-Bam allele alone as strains containing the G ⁇ subunit variant alone in the absence of rat or human PLC- ⁇ isozymes did not support growth at 37oC.
- strains expressing human PLC- ⁇ 2 and activated forms of G ⁇ q (Q209L) or G ⁇ l 6 (Q212L) grew robustly at both 30oC and 37oC while strains harboring human PLC- ⁇ 2 and an activated form of G ⁇ S (Q227L) failed to support growth at 37oC. 3
- Human PLC- ⁇ 3 has been found to complement the temperature-sensitive and NaCl sensitive phenotypes associated with the plcl genotype in yeast in a G-protein independent fashion. These conclusions are based on the finding that expression of the human PLC- ⁇ 3 in yeast strains with the plcl background under the control of the CUP promoter on either high or low copy plasmids, results in growth on 0.5M NaCl or at elevated temperatures, independent of the co-expression of G ⁇ subunits of the Gq family (i.e. q, 11, 14, 15, or 16).
- the characteristics of the human PLC- ⁇ 3 isoform expressed in yeast resemble those of rat PLC- ⁇ l in exhibiting G-protein independent growth under non-permissive conditions, as distinct from the G-protein dependent growth phenotype of the human PLC- ⁇ 2 isoform.
- Epidermal growth factor increases sn-1,2,- diacylglycerol levels and activates phospholipase D-catalysed phosphatidylcholine breakdown in Swiss 3T3 cells in the absence of inositol-lipid hydrolysis. Biochem. J., 285, 247-253.
- Lysophosphatidate-induced cell proliferation identification and dissection of signalling pathways mediated by G proteins.
- Emori Y., Homma, Y., Sorimachi, H., Kawasaki, H., Nakanishi, O., Suzuki, K., & Takenawa, T. (1989).
- Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma pituicytes by inactivating the inhibitory GTP-binding protein G j . Eur. J. Biochem, 183, 397-406.
- Dog and rat pancreatic phospholipases A-2 Complete amino acid sequences deduced from complementary DNAs. J. Biochem., 99, 733-739. Park, D., Jhon, D.-Y., Kriz, R., Knopf, J., & Rhee, S. G. (1992). Cloning, Sequencing,
- Pancreatic phospholipase A2 Isolation of the human gene and cDNAs from porcine pancreas and human lung. DNA, 5(6), 519-527.
- NK-1 Human substance P receptor
- cDNA for the human ⁇ 2 ⁇ adrenergic receptor A protein with multiple membrane spanning domains and a chromosomal location shared with the PDGF receptor gene. Proc. Natl. Acad. Sci. USA 84,
- Neote K. DiGregorio, D., Mak, J.Y., Horuk, R., and Schall, T. J. (1993) Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72, 415-425. Ogawa-Y. Nakao-K. Arai-H. Nakagawa-O. Hosoda-K. Suga-S.
- MOLECULE TYPE cDNA
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Endocrinology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP50214497A JP2001507201A (en) | 1995-06-07 | 1996-06-07 | Expression of functional vertebrate phospholipase in yeast |
AU61108/96A AU6110896A (en) | 1995-06-07 | 1996-06-07 | Expression of functional vertebrate phospholipases in yeast |
EP96918448A EP0832248A2 (en) | 1995-06-07 | 1996-06-07 | Expression of functional vertebrate phospholipases in yeast |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US48163295A | 1995-06-07 | 1995-06-07 | |
US08/481,632 | 1995-06-07 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1996040939A2 true WO1996040939A2 (en) | 1996-12-19 |
WO1996040939A3 WO1996040939A3 (en) | 1997-01-23 |
Family
ID=23912754
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/010002 WO1996040939A2 (en) | 1995-06-07 | 1996-06-07 | Expression of functional vertebrate phospholipases in yeast |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0832248A2 (en) |
JP (1) | JP2001507201A (en) |
AU (1) | AU6110896A (en) |
CA (1) | CA2220459A1 (en) |
WO (1) | WO1996040939A2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998055654A2 (en) * | 1997-06-06 | 1998-12-10 | University Of Iowa Research Foundation | CANDIDA ALBICANS TWO-COMPONENT HYBRID KINASE GENE, CaNik1, AND USE THEREOF |
JP2003180386A (en) * | 2000-02-08 | 2003-07-02 | Sanagamo Biosciences Inc | Cell for drug discovery |
CN111004787A (en) * | 2020-01-06 | 2020-04-14 | 江南大学 | Streptomyces phospholipase D mutant, transformation method and application thereof |
WO2020076697A1 (en) * | 2018-10-08 | 2020-04-16 | Novozymes A/S | Enzyme-expressing yeast for ethanol production |
CN114525266A (en) * | 2022-02-22 | 2022-05-24 | 华南理工大学 | Phospholipase D mutant from Antarctic bacteria and application thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115161303B (en) * | 2019-11-21 | 2024-02-13 | 天津科技大学 | Phospholipase mutant and method for synthesizing glycerophospholipids by using phospholipase mutant |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992005244A1 (en) * | 1990-09-13 | 1992-04-02 | Duke University | Expression of g protein coupled receptors in yeast |
DE4138621A1 (en) * | 1991-11-25 | 1993-06-17 | Boehringer Ingelheim Int | METHOD FOR SCREENING SUBSTANCES WITH MODULATING EFFECT ON A RECEPTACLE-RELATED CELLULAR SIGNAL TRANSMISSION PATH |
WO1994023025A1 (en) * | 1993-03-31 | 1994-10-13 | Cadus Pharmaceuticals, Inc. | Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor |
WO1995008624A1 (en) * | 1993-09-24 | 1995-03-30 | Shionogi & Co., Ltd. | HUMAN PHOSPHOLIPASE C-α AND DNA SEQUENCE CODING FOR THE SAME |
WO1995021925A1 (en) * | 1994-02-14 | 1995-08-17 | American Cyanamid Company | Heterologous g protein coupled receptors expressed in yeast, their fusion with g proteins and use thereof in bioassay |
WO1995030012A1 (en) * | 1994-04-26 | 1995-11-09 | Cadus Pharmaceutical Corporation | Functional expression of mammalian adenylyl cyclase in yeast |
-
1996
- 1996-06-07 CA CA 2220459 patent/CA2220459A1/en not_active Abandoned
- 1996-06-07 EP EP96918448A patent/EP0832248A2/en not_active Withdrawn
- 1996-06-07 AU AU61108/96A patent/AU6110896A/en not_active Abandoned
- 1996-06-07 WO PCT/US1996/010002 patent/WO1996040939A2/en not_active Application Discontinuation
- 1996-06-07 JP JP50214497A patent/JP2001507201A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992005244A1 (en) * | 1990-09-13 | 1992-04-02 | Duke University | Expression of g protein coupled receptors in yeast |
DE4138621A1 (en) * | 1991-11-25 | 1993-06-17 | Boehringer Ingelheim Int | METHOD FOR SCREENING SUBSTANCES WITH MODULATING EFFECT ON A RECEPTACLE-RELATED CELLULAR SIGNAL TRANSMISSION PATH |
WO1994023025A1 (en) * | 1993-03-31 | 1994-10-13 | Cadus Pharmaceuticals, Inc. | Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor |
WO1995008624A1 (en) * | 1993-09-24 | 1995-03-30 | Shionogi & Co., Ltd. | HUMAN PHOSPHOLIPASE C-α AND DNA SEQUENCE CODING FOR THE SAME |
WO1995021925A1 (en) * | 1994-02-14 | 1995-08-17 | American Cyanamid Company | Heterologous g protein coupled receptors expressed in yeast, their fusion with g proteins and use thereof in bioassay |
WO1995030012A1 (en) * | 1994-04-26 | 1995-11-09 | Cadus Pharmaceutical Corporation | Functional expression of mammalian adenylyl cyclase in yeast |
Non-Patent Citations (7)
Title |
---|
BIOCHIMICA AND BIOPHYSICA ACTA, vol. 1092, 1991, pages 49-71, XP000611209 MELDRUM, E. ET AL.: "The PtdIns-PLC Superfamily and Signal Transduction" cited in the application * |
FEBS LETTERS, vol. 364, May 1995, pages 45-50, XP002018653 ARKINSTALL, S. ET AL.: "Mapping Regions of Galphaq Interacting with PLCbeta1 Using Multiple Overlapping Synthetic Peptides" * |
FEBS LETTERS, vol. 375, November 1995, pages 183-187, XP002018654 ARKINSTALL, S. ET AL.: "Co-Expression of the Neurokinin NK2 Receptor and G-Protein Components in the Fission Yeast Schizosaccharomyces pombe" * |
MOLECULAR AND CELLULAR BIOLOGY, vol. 13, no. 9, September 1993, pages 5861-5876, XP000609195 FLICK, J. S. AND THORNER, J.: "Genetic and Biochemical Characterization of a Phosphatidylinositol-Specific Phospholipase C in Saccharomyces cerevisiae" cited in the application * |
MOLECULAR AND CELLULAR BIOLOGY, vol. 15, no. 3, March 1995, pages 1431-1438, XP000609196 ARKINSTALL, S. ET AL.: "Activation of Phospholipase Cgamma in Schizosaccharomyces pombe by Coexpression of Receptor or Nonreceptor Tyrosine Kinase" cited in the application * |
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 90, March 1993, pages 1804-1808, XP002018652 YOKO-O, T. ET AL.: "The Putative Phosphoinositide-Specific Phospholipase C Gene, PLC1, of the Yeast Saccharomyces cerevisiae is important for Cell Growth" cited in the application * |
TCM, vol. 4, no. 2, 1994, pages 88-95, XP000611204 BOYER, J. ET AL.: "G-Protein-Mediated Regulation of Phospholipase C - In volvement of beta / gamma Subunits" * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998055654A2 (en) * | 1997-06-06 | 1998-12-10 | University Of Iowa Research Foundation | CANDIDA ALBICANS TWO-COMPONENT HYBRID KINASE GENE, CaNik1, AND USE THEREOF |
WO1998055654A3 (en) * | 1997-06-06 | 1999-03-04 | Univ Iowa Res Found | Candida albicans two-component hybrid kinase gene, canik1, and use thereof |
US6803191B2 (en) | 1997-06-06 | 2004-10-12 | University Of Iowa Research Foundation | Candida albicans two-component hybrid kinase gene, CaNik1, and use thereof |
JP2003180386A (en) * | 2000-02-08 | 2003-07-02 | Sanagamo Biosciences Inc | Cell for drug discovery |
JP2011147448A (en) * | 2000-02-08 | 2011-08-04 | Sanagamo Biosciences Inc | Cell for drug discovery |
WO2020076697A1 (en) * | 2018-10-08 | 2020-04-16 | Novozymes A/S | Enzyme-expressing yeast for ethanol production |
CN113286889A (en) * | 2018-10-08 | 2021-08-20 | 诺维信公司 | Enzyme-expressing yeast for producing ethanol |
US11807889B2 (en) | 2018-10-08 | 2023-11-07 | Novozymes A/S | Yeast expressing a heterologous phospholipase for ethanol production |
CN111004787A (en) * | 2020-01-06 | 2020-04-14 | 江南大学 | Streptomyces phospholipase D mutant, transformation method and application thereof |
CN111004787B (en) * | 2020-01-06 | 2022-05-10 | 江南大学 | Streptomyces phospholipase D mutant, transformation method and application thereof |
CN114525266A (en) * | 2022-02-22 | 2022-05-24 | 华南理工大学 | Phospholipase D mutant from Antarctic bacteria and application thereof |
CN114525266B (en) * | 2022-02-22 | 2023-06-20 | 华南理工大学 | Phospholipase D mutant from Antarctic bacteria and application thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0832248A2 (en) | 1998-04-01 |
JP2001507201A (en) | 2001-06-05 |
CA2220459A1 (en) | 1996-12-19 |
AU6110896A (en) | 1996-12-30 |
WO1996040939A3 (en) | 1997-01-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2266543C (en) | Methods and compositions for identifying receptor effectors | |
CA2188950C (en) | Functional expression of mammalian adenylyl cyclase in yeast | |
Ghislain et al. | Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin‐mediated proteolysis in Saccharomyces cerevisiae. | |
Yan et al. | Two novel transcriptional regulators are essential for infection-related morphogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae | |
AU685103B2 (en) | Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor | |
US7319009B2 (en) | Methods and compositions for identifying receptor effectors | |
JP2009143932A (en) | Yeast cells expressing modified g protein and method of use thereof | |
Fischer et al. | Regulation of cell-to-cell communication and cell wall integrity by a network of MAP kinase pathways and transcription factors in Neurospora crassa | |
US20080268475A1 (en) | Yeast cells expressing modified g proteins and methods of use therefor | |
Noble et al. | Development of a yeast bioassay to characterize G protein-coupled receptor kinases: Identification of an NH2-terminal region essential for receptor phosphorylation | |
Mayrhofer et al. | Functional characterization of an α-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae | |
EP0832248A2 (en) | Expression of functional vertebrate phospholipases in yeast | |
US7273747B2 (en) | Cell having amplified signal transduction pathway responses and uses therefor | |
US20030009022A1 (en) | Methods and compositions for identifying receptor effectors | |
Yerra | Relationship Between Morphogenesis and Secretion in the Filamentous Fungus Aspergillus Nidulans | |
MXPA05000837A (en) | Method for identifying substances. | |
Weng | Characterization of the Saccharomyces cerevisiae gene KAP104: Its role in G (alpha)-mediated adaptation | |
Pateman | RGS proteins and G protein signalling | |
Zhou | Isolation and characterization of G (alpha) mutations that uncouple G (beta-gamma) binding from adaptive function in yeast | |
AU7169600A (en) | Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA |
|
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase in: |
Ref country code: CA Ref document number: 2220459 Kind code of ref document: A Format of ref document f/p: F Ref document number: 2220459 Country of ref document: CA |
|
ENP | Entry into the national phase in: |
Ref country code: JP Ref document number: 1997 502144 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1996918448 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1996918448 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1996918448 Country of ref document: EP |