Patent Application
20100028892
1. Field of the Invention
The disclosure generally relates to components and methods of using a high throughput screening (HTS) systems for intracellular proteases, using Caspases as a prototype. Disclosed are yeast-based cellular systems that permit facile expression of proteases and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor.
2. Background Information
Endopeptidases (“proteases”) play critical roles in many biological processes and are often excellent targets for drug discovery. Development of high throughput screening (HTS) assays using purified proteases can be relatively straightforward or it can be quite challenging, particularly when multi-component systems are required to achieve protease activation. Also, due to similarity of the active sites of some groups of proteases, selectivity of chemical inhibitors can be difficult if not impossible to achieve, highlighting the need for alternative screening methods for identifying compounds that target upstream activators of proteases rather than directly inhibiting the protease of interest.
Caspases represent an excellent example of a family of intracellular endopeptidases for which novel HTS tools are desired. Caspases are cysteine proteases that are conserved throughout the animal kingdom. The human genome contains at least 10 genes encoding Caspases. These proteases often collaborate in complex proteolytic networks that encompass upstream initiators and downstream effectors, where upstream members of the Caspase family cleave and activate downstream members. Upstream initiator Caspases become activated through protein interactions involving assembly of multi-protein complexes that are difficult to reconstitute in vitro. The substrates cleaved by active Caspases are responsible for either apoptotic cell death or for cytokine-mediated inflammation, thus making these proteases attractive targets for drug discovery for a wide variety of degenerative diseases, ischemic disease, autoimmunity, inflammatory conditions, and some host-pathogen interactions.
Currently, there is no assay technology for high-throughput screening which reconstitutes the use and economy as those facilitated by the present disclosure.
Herein are disclosed genetic systems for monitoring exogenous caspase activation pathways in the yeast, Saccharomyces cerevisiae. These systems rely, singly or in concert, on exogenous recombinant caspases and exogenous upstream activators of caspases to cleave a chimeric protein giving rise to a transcription factor which induces the expression of the LacZ and LEU2 genes. The activities of these genes result in blue cultures and impart the ability of the yeast to grow in leucine deficient media. The blue color is due to the cleavage of X-gal by the LacZ gene product, β-galactosidase. The intensity of the blue color is measured by colorimetry and quantified with OD units. The OD units are directly proportional to the activity of the caspase in the system. The method of quantification is referred to as the “readout”.
In a “Single Component” system an exogenous caspase is stably over-expressed and autoactivated in a yeast strain containing the transmembrane receptor, CD95/Fas, (“chimera”). The chimera is expressed such that most of its cytosolic domain is replaced by a chimeric transcription factor comprised of the DNA-binding domain of LexA and the transactivation domain of B42. Between Fas and the chimeric transcription factor (LexA-B42) domains are various numbers of tetrapeptide sequences known to be recognized and cleaved by various members of the Caspase family. Each genetic system of this disclosure contains an appropriately matched set of the appropriate caspase to the appropriate chimera. The activity of the entire system is monitored by the readout.
An embodiment of this disclosure provides for a method using a single-component system exemplified above for drug screening assays wherein the activities of Caspase 1, 2, 3, 4, 5, 6, 7, and 9 are measured and compounds that inhibit Caspase 1, 2, 3, 4, 5, 6, 7, and 9 are identified.
Furthermore, an embodiment of this disclosure provides for a method using a single-component system exemplified above for drug screening assays which may utilize any other substrate of the LacZ gene, beta-galactosidase, for quantification.
In a “Two-Component” system an exogenous caspase is expressed at a low level to maintain its inactive pro-caspase form along with its exogenous cogent upstream activator. The level of expression is engineered to prevent autoactivation such that the interaction of the upstream activator with the pro-caspase results in the activated caspase and the readout. The level of expression is engineered to impact assay performance by manipulating several variables of vector construction, including promoter strength, promoter inducibility, vector copy number, and number of operators (transcription factor binding sites) in a reporter gene.
An embodiment of this disclosure provides for a method using a two-component system exemplified above for drug screening assays where the activities of ASC, RAIDD, FADD, Apaf, and active caspase-9 are measured and compounds that inhibit the interaction or activity of ASC with pro-caspase-1, RAIDD with pro-caspase-2, FADD with pro-caspase-8 or pro-caspase-10, an active mutant of Apaf with pro-caspase-9, active caspase-9 with pro-caspase-3 or procaspase-7 are identified.
An embodiment of this disclosure provides for a method using a two-component system exemplified above for drug screening assays where the activities are used to identify or discover other or novel upstream activators of pro-caspase-1, pro-caspase-2, pro-caspase-3, pro-caspase-4, pro-caspase-5, pro-caspase-6, pro-caspase-7, pro-caspase-8, pro-caspase-9, and pro-caspase-10 via the introduction of a cDNA library or other expression conveyance to screen for biomolecular activators of the pro-caspases.
Furthermore, an embodiment this disclosure provides for a method using a two-component system exemplified above for drug screening assays which may utilize any other substrate of the LacZ gene, beta-galactosidase, for quantification
In a “Plural-Component” system an exogenous caspase is expressed in its inactive pro-caspase form along with its exogenous cogent activation pathway components consisting of two or more additional upstream proteins required for activation of the caspase and the subsequent readout. The levels of expression of the components are engineered to prevent caspase activation unless all of the components are present.
An embodiment of this disclosure provides for a method using a plural-component system exemplified above for drug screening assays where the activities of FAS, FADD, DR5, ASC, or NALP1 are measured and compounds that inhibit the interaction or activity of FAS with FADD, DR5 with FADD, FADD with pro-caspase-8 or pro-caspase-10, ASC with NALP1, or NALP1 with pro-caspase-1.
An embodiment of this disclosure provides for a method using a plural-component system exemplified above for drug screening assays where the activities are used to identify or discover other or novel upstream activators of FADD and NALP1 via the introduction of a cDNA library or other expression conveyance to screen for biomolecular activators of FADD or NALP1.
Furthermore, an embodiment this disclosure provides for a method using a plural-component system exemplified above for drug screening assays which may utilize any other substrate of the LacZ gene, beta-galactosidase, for quantification.
Another embodiment of the present disclosure incorporates the use of two-component and/or plural-component systems and by extension their specific examples to identify caspase activation protease networks in yeast, and to isolate and study an exogenous protease network in a cellular context.
An embodiment disclosed includes methods of performing chemical library screens to validate the performance of the assays and to further identify caspase activation protease networks in yeast, and to isolate and study an exogenous protease network in a cellular context. The screens are conducted such that all assay components may be added in automated fashion using integrated robotic liquid handling systems, moving the plates initially into carousels that hold ˜180 plates at room temperature, and then manually applying seals (breathable sealing film from Axygen Scientific) to reduce evaporation, and moving the bar-coded plates to a 30° C. incubator for the required time, generally 2-4 days. Each assay plate will contain a row of positive (min) and a row of negative (max) controls that do not receive compounds but that receive DMSO in volumes equivalent to the amount of DMSO in which compounds will be supplied. At the conclusion of the 72 hrs incubation, plates are robotically delivered to one of the integrated multi-purpose plate readers for reading at OD620 nm. Programmable robotic workstations sequence the additions of reagents, minimizing variations in incubation times. Data from primary screens are uploaded directly from plate readers into computers with customized Microsoftexcel software, set up to calculate Z′ factor for each plate, and with hit determinations set at 50% of the mean value for the negative control values.
An embodiment disclosed utilizes library screens which may be performed at several different concentrations (eg., 20, 10, and 5 μM) to compare the hit rates, and to empirically determine an acceptable concentration for conducting large-scale library screens. It is often best to employ as high a concentration of compounds as possible to maximize the chance of identifying active compounds (avoiding false-negatives), but balance that against an excessive frequency of hits (avoiding false-positives). A general rule of thumb is to empirically adjust compound screening concentrations to achieve a hit rate of 0.1-0.5%. Thus, before undertaking starting a large library screen, pilot experiments may be utilized to optimize results such as empirically determining the effects of DMSO on assay performance, through pilot experiments where increasing concentrations of DMSO (from 1-10% volume) are added to the positive and negative controls and the impact on assay signal and stability is determined. Second, when progressing with larger libraries (e.g., 50K Chembridge), it is useful to determine the stability of the positive and negative controls from plate to plate, assessing the assay performance as time is extended from minutes to hours, and calculating Z′ for each plate as the quality of the assay's performance is assessed in true screening mode.
For HTS assays we have measured β-galactosidase produced by yeast carrying the caspase-cleavable reporter proteins, and assayed the calorimetric product (OD620 nm) derived from X-gal substrate in 384 well plates, as a measure of the lac Z reporter gene activity.
An embodiment of the present disclosure comprises a high throughput screening of lac-Z reporter gene activity by measuring β-galactosidase produced by yeast carrying caspase-cleavable reporter proteins; and assaying the calorimetric product derived from x-gal substrate in 384 well plates, at an optical density of 620 nm.
Proteolytic processing of proteins is an irreversible post-translational modification of importance for a wide-variety of biological processes, including cell division, cell death, cell differentiation, innate immunity, host-pathogen interactions, and intracellular protein sorting and trafficking (Lopez-Otin C, and Overall CM. Protease degradomics: a new challenge for proteomics. Nat Rev Mol Cell Biol 2002; 3(7): 509-19). Consequently, proteases have emerged as promising targets for drug discovery for a wide variety of human diseases, including cancer, neurodegeneration, ischemic diseases, inflammation, and infectious diseases.
Proteases can be found in either intracellular or extracellular (or cell surface) locations, where they encounter their specific substrates. Among the intracellular families of proteases are Caspases, Calpains, Deubiquitinating enzymes and their homologs, and Separase. Still other proteases may have dual intracellular and extracellular lives. For example, Cathepsins are normally stored in lysosomes undergoing vesicular recycling between extracellular and intracellular compartments, but also are released into the cytosol under various pathological circumstances, promoting cell death (Cirman T, et al. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 2004; 279: 3578-87).
From the standpoint of high-throughput screening (HTS) for identifying chemical inhibitors of proteases, many endopeptidases (or at least their catalytic domains) can be readily produced in large quantities by recombinant DNA technology, purified, and formatted for HTS using convenient fluorigenic or colorimetric substrates. However, such assay formats have several limitations. First, standard HTS configurations for proteases are typically amenable only to screens for inhibitors, not activators of proteases. Second, assays requiring large amounts of purified, active protease can be hampered by difficulties in producing by recombinant methods or purifying from endogenous sources sufficient amounts of material, as well as by instability problems where purified proteases lose activity in vitro. Third, because one is often limited to using only the catalytic domain due to difficulties of expressing and purifying intact full-length proteases, opportunities to discover allosteric modulators of proteases are limited, with most of the standard assays limited to identification of compounds that target the active site in a competitive fashion. Fourth, when using single target systems consisting of purified protease or catalytic domain of protease, opportunities for identification of compounds that target other proteins involved in protease activation are lost. Fifth, several classes of intracellular endopeptidases exists as large families of closely related enzymes with structurally very similar active sites, making it difficult to achieve selective inhibitors in the absence of more context-dependent, multi-component systems that would provide the opportunity for selective inhibition. Sixth, some proteases are not active in purified form, requiring necessary cofactors (e.g. Separins involved in chromosome segregation and cell division) or requiring membranes (e.g. Presenilins (such as γ-secretase) involved in Amyloid-beta-peptide processing).
Caspases are intracellular cysteine proteinases with specificity for aspartic acid residues in the P1 position of substrates. These proteases are well conserved throughout animal evolution, where they play essential roles in programmed cell death (apoptosis). In Caenorhabiditis elegans, for example, all programmed cell deaths that occur during the development of this simple animal depend upon the presence of an intact CED-3 gene, which encodes a Caspase (Yuan J, et al., The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. CELL 1993; 75: 641-52). In humans, 12 genes encoding Caspases or Caspase-like proteins have been identified, 10 of which have been clearly and convincingly shown to possess protease activity and all showing absolute dependence on aspartic acid at the P1 position of substrates, including Caspases-1-10. Another member of the family, Caspase-12, is produced only in ˜1% of African populations, due to a polymorphism that produce a nonsense mutation that truncates the protein in most humans, and its proteolytic activity is questionable (Fischer H, et al., Human caspase-12 has acquired deleterious mutations. Biochem Biophys Res Comm 2002; 293: 722-6; and Saleh M, et al., Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice. Nature 2006; 440: 1064-8). Protease activity for the remaining member, Caspase-14, has not been demonstrated to date (Kuechle M K, et al., Caspase-14, a keratinocyte specific caspase: mRNA splice variants and expression pattern in embryonic and adult mouse. Cell Death Differ 2001; 8: 868-70). In addition to apoptosis, other biological roles for Caspases have been elucidated, the most studied of which is inflammation. Of the 10 established human Caspases, 3 of them cleave and activate pro-inflammatory cytokines (particularly pro-IL-1b, pro-IL-18, pro-IL-32), having major roles in host-defense and inflammation (Reed J C. Caspases and cytokines: roles in inflammation and autoimmunity. Adv Immunol 1999; 73 :265-99).
Caspases exist as inactive zymogens in all animal cells but can be activated by proteolytic cleavage of their proforms at conserved aspartic acid residues, thus generating the subunits of the enzymatically active proteases which consist of heterotetramers comprised of two large and two small subunits. Because caspases both cleave substrates at Asp residues and are themselves activated by cleavage at Asp residues, the potential for proteolytic cascades exists and indeed has been documented (reviewed in (Salvesen G S, et al., Intracellular signaling by proteolysis. Cell 1997; 91 :443-6; and Thornberry N A, and Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-6). The concept of upstream initiator and downstream effector caspases that operate within a proteolytic cascade thus has emerged (Salvesen G S, et al., Cell 1997; 91: 443-6; and Alnemri E S, et al. Human ICE/CED-3 Protease Nomenclature. Cell 1996; 87: 171).
The 3D-structure of the active sites of most human Caspases accommodates tetrapeptide substrates, in which Aspartic acid at the P1 position is invariant. In Drosophila, Glutamic acid can also be tolerated for some of the Caspases (Hawkins C J, et al., The drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID; and GRIM. J Biol Chem 2000; 275: 27084-93). Differences in the geometry and side-chains of amino acids surrounding the active site dictate preferences among Caspases for different tetrapeptide substrates. Surveys of peptide libraries have elaborated activity profiles for most of the human Caspases (Talanian R, et al. Substrate specificities of caspase family proteases. J Biol Chem 1997; 272: 9677-82), as summarized in Table 1. These peptidyl substrate preferences match the known cleavage sites of endogenous proteins that are hydrolyzed by specific Caspases. For example, the WEHD sequence preferred by the inflammatory Caspase is found in pro-IL-1b and pro-IL-18, while the DEVD sequence preferred by downstream apoptotic proteases Caspases-3 and -7 is found in enzymes responsible for the characteristic chromatin condensation and DNA fragmentation associated commonly with apoptosis (Timmer J C, and Salvesen G S. Caspase substrates. Cell Death Differ 2007; 14(1): 66-72).
One of the major pathways for Caspase activation involves the TNF family of cytokine receptors (reviewed in Wallach D, et al., Cell death induction by receptors of the TNF family: towards a molecular understanding. FEBS Lett 1997; 410: 96-106, and Ashkenazi A, and Dixit V M. Death receptors: signaling and modulation. Science 1998; 281: 1305-8). Several TNF family receptors are known to transduce signals that result in apoptosis, including TNF-R1(CD120a), Fas (CD95), DR3 (Wsl-1; Tramp); DR4 (Trail-R1); DR5 (Trail-R2); and CAR-1. These death receptors contain a conserved cytosolic domain known as a Death Domain (DD) that is responsible for recruiting adapter proteins such as Fadd/Mort-1 to the receptor complex after binding of ligand. The Fadd/Mort-1 protein contains both a DD domain and an additional protein-interaction domain called a Death Effector Domain (DED). The DED of Fadd/Mort-1 binds certain caspases which contain homologous DEDs within their prodomains, caspases-8 and -10. The oligomerization of caspases within the death receptor complex results in trans-processing of the zymogens (Muzio M, et al., An induced proximity model for caspase-8 activation. J Biol Chem 1998; 273: 2926-30; and Yang X, et al., Autoproteolytic activation of pro-caspases by oligomerization. Mol Cell 1998; 1: 319-25), which contain low levels of proteolytic activity even before undergoing processing to the fully active protease. Processing of caspase-8 removes the DED-containing prodomain, thus releasing the activated protease into the cytosol where it can cleave and activate other downstream pro-caspases such as caspase-3 (Stennicke H R, et al. Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem 1998; 273: 27084-90).
The pathway for apoptosis induction employed by the TNF family death receptors is sometimes referred to as the “Extrinsic Pathway.” This pathway for caspase activation plays a critical role in the mechanisms used by immune cells to induce apoptosis in target cells. Cytolytic T-cells, for example, employ death ligands, particularly Fas-Ligand (Fas-L), as a weapon for inducing apoptosis in target cells (Nagata S, and Golstein P. The Fas death factor. Science 1995; 267(5203): 1449-56). Several anti-apoptotic proteins that contain Death Effector Domains have also been described that operate as suppressers of apoptosis induced by TNF-family death receptors. These proteins contain DEDs, which allow them to interact with other DED-proteins, such as Fadd, pro-caspase-8, and pro-caspase-10, thereby interfering with assembly of the multiprotein complexes required for death receptor-mediated activation of caspases (Irmler M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997; 388: 190-5; and Tschopp J, et al., Inhibition of Fas death signals by FLIPs. Curr Opin Immunol 1998; 10: 552-8). For example, the cellular protein Flip (reviewed in Wallach D. Placing death under control. Nature 1997; 388: 123-6) is such an anti-apoptotic protein, representing a homologue of caspases-8 and -10 that contains DED domains but lacks proteolytic activity.
Standing apart from the Death Receptors is another mechanism for achieving initiator Caspase activation that relies on nucleotide-binding oligomerization domains in Caspase-binding proteins to assemble multi-protein complexes, sometimes referred to as “apoptosome” or “inflammasomes”, depending on whether the Caspases activated are involved in cell death versus inflammation. The prototype for this Caspase activation mechanism comes from pioneering work in Caenorhabiditis elegans, where the only documented mechanism of inducing caspase activation depends on CED-4. The CED-4 protein is an ATP-binding protein and putative ATPase, which binds to pro-CED-3, an initiator caspase (Seshagiri S, and Miller L K. Caenorhabditis elegans CED4 stimulates CED-3 processing and CED-3-induced apoptosis. Curr Biol 1997; 7: 455-60; and Chinnaiyan A, et al., Role of CED-4 in the activation of CED-3. Nature 1997; 388: 728-9). Binding of ATP to CED4 induces conformational changes that result in oligomerization of CED-4 proteins, followed by binding to pro-CED-3 (Yang X, et al., Essential role of CED-4 oligomerization in CED-3 activation and apoptosis. Science 1998; 281: 1355-7). The oligomerization of associated pro-CED-3 proteins is thought to bring these zymogens into close proximity, allowing them to dimerize and become active, typically cleaving themselves or trans-processing each other, thereby generating the fully active autonomous proteases. The first mammalian homologue of CED-4 identified was Apaf-1 (Apoptotic Protease Activating Factor-1). The human Apaf-1 protein shares in common with CED-4 the presence of a nucleotide binding NB-ARC domain and a caspase-binding CARD (CAspase Recruitment Domain) domain. However, Apaf-1 is structurally more complex than the worm CED-4 (Zou H, et al., Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997; 90: 405-13), in that it also contains 12-14 copies of a W) domain at its C-terminus. The WD domains function as negative-regulatory region, rendering Apaf-1 inactive until bound by cytochrome c from mitochondria (Li P, el al. Cytochrome c and dATP-dependent formation of Apaf-1/Caspase-9 complex initiates an apoptotic protease cascade. Cell 1997; 91: 479-89). The presence of this negative-regulatory domain thus defines a fundamental difference between the human Apaf-1 and worm CED-4 proteins. The human protein requires an activation step to interact with caspases and induce cell death, whereas the worm CED-4 protein has constitutive caspase-binding and death-inducing activity (reviewed in Reed J C. Cytochrome C: Can't live with it; Can't live without it. Cell 1997; 91 :559-62). The only known mechanism for activating Apaf-1 is cytochrome c, which binds to Apaf-1 apparently relieving the repression applied by the WD domains (Li P, et al. Cell 1997; 91: 479-89). Cytochrome c is normally sequestered inside mitochondria, between the inner and outer membranes of these organelles. However, it becomes released into the cytosol following exposure of cells to a variety of pro-apoptotic stimuli, including chemotherapeutic drugs (Liu X, et al., Induction of apoptotic program in cell-free extracts: requirement for dATP and Cytochrome C. Cell 1996; 86: 147-57; Kluck R M, et al., The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 1997; 275 :1132-6; and Yang J, et al., Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 1997; 275 :1129-32). The apical caspase in the cytochrome c/mitochondrial pathway is pro-caspase-9. Pro-caspase-9 possesses an N-terminal CARD domain that allows it to bind the CARD domain of Apaf-1. Oligomerization of Apaf-1 induced by cytochrome c and ATP (or dATP) brings associated pro-caspase-9 zymogens into close proximity, allowing them to dimerize and become active (Zou H, et al., An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 1999; 274: 11549-56; and Saleh A, et al., Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J Biol Chem 1999; 274: 17941-5). The caspase-activation pathway mediated by Apaf-1 as a result of cytochrome c release from mitochondria is sometimes referred to as the “Intrinsic Pathway.” The complex of Apaf1/cytochrome c/Caspase-9 is known as the “apoptosome” a donut-like structure with two stacked rings, each ring having stoichiometry of 7:14:7, based on high resolution cryo-electron microscopy imaging studies and other methods (Salvesen G S, and Renatus M. Apoptosome: The seven-spoked death machine. Develop Cell 2002; 2: 256-7).
An analogous system for achieving initiator Caspase activation has been described for inflammatory caspases, in which members of the NLR family (“NACHT+LRR” proteins, also known as “NOD-Like Receptors”) oligomerize to form a platform for recruitment and activation of inflammatory caspases such as Caspase-1, 4, and 5 in humans (reviewed in Martinon F, et al., The Inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-b. Mol Cell 2002; 10: 417-26). NLRs consist of a central nucleotide-binding domain called NACHT, flanked on the C-terminus by Leucine Rich Repeats (LRRs) and on the N-terminus by either CARDs or PYRIN Domains (PYDs). The LRRs of NLRs keep these proteins in an inactive monomeric state, until bound by pathogen-derived products such as components of bacterial peptidoglycan. In vitro reconstitution studies by our laboratory have shown that upon binding pathogen-derive ligands, a conformation change occurs in NLRs that renders the NACHT domain competent to bind nucleotide triphosphates (NTPs) and undergo NACHT-dependent oligomerization (Faustin B, et al. Reconstituted NALP1 inflammasome reveals two-step mechanism of Caspase-1 activation. Molecular Cell 2007; 25(5): 713-24). The N-terminal CARD and PYD domains of NLRs bind directly to the CARD of inflammatory caspases or indirectly link to them via the bipartite adapter protein ASC, which contains both a CARD and PYD (Stehlik C, and Reed J C. The PYRIN connection: novel players in innate immunity and inflammation. J Exp Med 2004; 200: 551-8). The complex of NLR/ASC/Caspase-1 is called the “inflammasome” (Martinon F, and Tschopp J. NLRs join TLRs as innate sensors of pathogens. Trends Immunol 2005; 26: 447-54). Cryo-EM imaging studies suggest that the NALP1 (NLRP1) inflammasome is a donut-like structure, akin to the apoptosome (Faustin B, et al. Molecular Cell 2007; 25(5): 713-24).
Other mechanisms of mammalian Caspase activation have also been described, including the PIDDosome, a multiprotein complex involving the p53-inducible protein PIDD, the adapter protein RAIDD, and Caspase-2 (Tinel A, and Tschopp J. The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress. Science 2004; 304: 843-6). PIDD contains an oligomerization domain and Death Domain (DD). The DD of PIDD binds a complementary DD found in the bipartite adapter protein RAIDD, which contains a DD and CARD. The CARD of RAIDD binds the CARD of pro-Caspase-2, thus providing the necessary link to the protease.
The phenotypes of animal Caspases expressed in yeast have been explored. (Kamada S, et al. A cloning method for caspase substrates that uses the yeast two-hybrid system: Cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 1998; 95: 8532-7; Hawkins C J, et al., A cloning method to identify caspases and their regulators in yeast: Identification of Drosophila DIAP1 as an inhibitor of the Drosophila caspase DCP-1. Proc Natl Acad Sci USA 1999; 96: 2885-90; Kang J, et al. Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae. J Biol Chem 1999; 274: 3189-98; Zhang H, and Reed J C. Studies of apoptosis proteins in yeast. In: Schwartz L, Ashwell, editors. Methods in Cell Biology. 2nd ed: Academic Press; 2001. p. 453-68; and Jin C, and Reed J C. Yeast and apoptosis. Nature Rev Mol Cell Biol 2002; 3: 453-9). Several lessons have been learned from these studies. First, over-expression of some zymogen forms of Caspases in yeast is sufficient to result in their activation, particularly the initiator Caspases, where it is likely that their CARD or DED-containing prodomains mediate dimerization when proteins are over-expressed. Second, some animal Caspases kill yeast, whereas others do not. Third, for those non-lethal Caspases, it is possible to devise cleavable reporter systems, particularly transcription factors that activate reporter genes only when cleaved by Caspases (Hawkins C J, et al., Proc Natl Acad Sci USA 1999; 96: 2885-90).
The following examples are intended to illustrate but not limit the invention.
A reporter gene system for monitoring protease activity in living yeast was devised. For this purpose, a Type I transmembrane receptor (CD95; Fas) 4, most of its cytosolic domain was replaced by a chimeric transcription factor comprised of the DNA-binding domain of LexA and the transactivation domain of B42. Between Fas and the chimeric transcription factor (LexA-B42) was cloned various numbers of tetrapeptide sequences known to be recognized and cleaved by various members of the Caspase family. As a control, constructs were prepared in which the sessile aspartic acid within the tetrapeptide sequence was replaced with glycine (
The demonstration of a genetic system for monitoring Caspase-1 activity in yeast cells is shown in
Caspases are initially produced as inactive zymogens, which typically undergo proteolytic processing to produce active enzymes composed in most cases of tetrameric assemblies with two large (˜20 kDa) and two small (˜10 kDa) subunits. To produce the active form of Caspases in yeast, each full-length Caspase construct was expressed (for Caspases-1, 2, 3, 4, 5, 6, 7, and 9) at high levels using plasmids with strong promoters, which results in ‘spontaneous” activation of these over-expressed proteases, generating the active, proteolytically processed forms of these proteases consisting large and small catalytic subunits. As for Caspases-8 and 10, however, a small amount of each full-length Caspase construct was expressed with a large amount of adaptor protein FADD to make the active forms, because large amounts of active Caspases-8 or -10 inhibited the yeast cell growth significantly. These active Caspases were then expressed in ΔLeu yeast with cleavable reporter constructs containing various tetrapeptide target sequences. Variables such as the strengths of the yeast promoters driving expression of the Fas-LexA-B42 cleavable fusion proteins to optimize signal:noise ratio were empirically adjusted, so that background (spontaneous) activation of the LEU2 and lacZ reporter genes was minimal (not shown), while titrating the number of LexA operators in the reporter genes to ensure a signal well above background, empirically determining that 2 LexA operators for lacZ and 6 LexA operators for LEU2 produced satisfactory results (not shown).
When plated on complete medium, all ΔLeu transformants grew, as expected (
Similarly, yeast containing the DEVD linker in the Fas-LexA-B42 fusion protein grew on leucine-deficient media and showed β-galactosidase-positivity when co-expressed with active Caspase-3 or Caspase-7, but not Caspases-1, 4, 5, 6, 8, 9, or 10, showing striking preferentiality among Caspases for cleavage of this reporter protein. Caspase-2 also cleaved the DEVD-containing reporter protein, which differs only slightly from the reported optimal substrate sequence of DEHD (Thornberry N A, et al. B. J Biol Chem 1997; 272: 17907-11). DEHD produced results very similar to DEVD, consistent with prior substrate specificity experiments suggesting that the P2 position where H was placed is the most flexible for Caspases-3 and -7 (Thornberry N A, et al. B. J Biol Chem 1997; 272: 17907-11). The other tetrapeptide sequences optimized for Caspase-6 (TEVD), Caspases-8/10 (LETD) and Caspase-9 (LEHD) resulted in less specific patterns of reporter protein activation, but nevertheless showed selectivity. For example, the “inflammatory” Caspases (Caspases-1, 4, and 5) did not activate the Caspase-6 substrate (TEVD). Also, the downstream effector Caspases (Caspases-3, 6, 7) did not activate the reporter proteins containing tetratpeptide sequences optimized for upstream initiator Caspases (LETD, Caspases-8/10; LEHD, Caspase-9).
As shown in
Taken together, these data demonstrate the performance of a one-component cleavable reporter gene system for measuring activity of the human Caspases in yeast. This one-component system was further used for screening human cDNA libraries expressed in yeast plasmids, where the cleavable Fas-LexA-B42 fusion protein was expressed without a Caspase, screening cDNA libraries for proteases that activate the reporter gene. Using the WEHD-containing reporter protein, eleven cDNAs that confirmed positive on repeated testing were obtained, including five that encoded Caspase-1 and six encoding Caspase-4. Using the DEVD-containing reporter, twelve clones that confirmed positive were obtained, including three that encoded Caspase-3 and nine encoding Caspase-7. These cDNA library screening results further validated the cleavable reporter system.
Two-component systems for assaying Caspase activity in yeast, in which the inactive proforms of the Caspases were co-expressed with various activator proteins were also created.
The schematic representation of a two-component system for caspase I activators in yeast cells is shown in
Among the two-component systems interrogated were combinations of upstream initiator Caspases co-expressed with known activators, including: (1) pro-Caspase-1 plus ASC; (2) pro-Caspase-2 plus RAIDD; (3) pro-Caspase-9 plus a gain-of-function mutant of Apaf1; (4) pro-Caspase-8 plus FADD; and (5) pro-Caspase-10 plus FADD. In each of these cases, the upstream initiator Caspase, contains a N-terminal pro-domain (either CARD or DED) that binds a compatible CARD or DED in the activator protein. A two-component systems involving an upstream and downstream protease was also tested. For example, Caspase-9 was previously known as a direct upstream activator of downstream proteases, Caspases-3 and -7 (Slee E, et al. Ordering the cytochrome c-initiated caspase cascade: Hierarchic activation of caspases-2,-3,-6,-7,-8 and -10 in a caspase-9-dependent manner. J Cell Biol 1999; 144: 281-92). Active Caspase-9 was over-expressed in combination with the inactive proforms of Caspase-3 or -7, expressed at low levels. In each case, an appropriate cleavable reporter protein was co-expressed with the two-component systems, in yeast containing LEU2 and lacZ reporter genes. For all pair-wise combinations of pro-Caspase and upstream activator, the pro-Caspase was expressed in a relatively small amount using a low-copy plasmid containing CEN/ARS (1 to 2 copies per cell), and the upstream activator in a large amount using 2μ origin-containing plasmids (20 to 50 copies per cell) with relatively strong promoters, TEF, or ADH. Each pair of pro-Caspase and upstream activators were empirically adjusted by manipulating the number of LexA operators driving the LEU2 or lacZ reporter genes to achieve an acceptable signal:noise ratio. For example, for the combination of pro-Caspase-1 and ASC, it was determined that 2 LexA operators for LEU2 were superior to 4 or 6 operators (
Successful activation of the cleavable reporter protein was achieved for each two-component system (
As shown in
An increase in the complexity of reconstituted Caspase-activating systems in yeast, was created utilizing a plural component network comprising three or more components. Two different mammalian Caspase-activating networks were explored. In the first, the proximal portion of the extrinsic pathway was recapitulated, expressing (1) death domain (DD)-containing TNF-family death receptor, Fas [CD95]; (2) bipartite adapter protein FADD, which contains DD and DED; and (3) the proform of a DED-containing protease, either Caspase-8 or Caspase-10, along with (4) a cleavable reporter protein containing the Caspase-8/10 substrate tetrapeptide LETD. Two strategies for reconstituting the Fas/FADD/pro-Caspase-8 network in yeast were compared. First, we expressed the bridging adapter protein FADD at low levels using a constitutive but weak promoter such that the amount of FADD was inadequate to achieve pro-Caspase-8 activation in the absence of Fas. For that system, Fas was expressed at high levels so that it could oligomerize with itself without requiring Fas Ligand confirm (
Additionally a 4-component system was reconstituted in yeast for γ-secretase activity, comprised of Presenlin-1, Nicastrin, APH-1, and PEN-2, using lacZ (β-galactosidase) reporter gene activation by a membrane-tethered cleavable transcription factor (Amyloid β-protein precursor [APP] fused to GAL1 transcription factor) as an endpoint for assessing activity of this transmembrane protease complex.
The schematic representation of a plural-component system for caspase-8 activators in yeast cells is shown in
By empirically comparing different strength promoters for driving expression of the three components and adjusting the number of LexA operators in the promoters of the LEU2 and lacZ reporter genes, the determination of conditions that produced the desired result for the approach was reached whereby low levels of constitutive FADD were complemented by expression of Fas confirm(
The results of testing yeast-based, plural-component systems for activating caspases-8 or -10 are shown in
Another plural-component Caspase-activating network was similarly created, using variations on the same approach. Specifically, the pro-inflammatory plural-component system was reconstituted in yeast and comprised: (1) the CARD-containing protease, pro-Caspase-1; (2) the bipartite adapter ASC, which contains CARD and PYD domains; and (3) the NLR-family protein NALP1, using a gain-of-function mutant of NALP1 that is constitutively active without requiring a bacterial ligand to induce its oligomerization (Faustin B, et al. Reconstituted NALP1 inflammasome reveals two-step mechanism of Caspase-1 activation. Molecular Cell 2007; 25(5): 713-24). For this system, the reporter protein contained the Caspase-1 cleavage sequence, WEHD (
The results of a yeast assay for NALP1 are shown in
The results of a yeast assay for NLRs are shown in
When converting the yeast-based protease reporter system to HTS assay, conditions from agar plates to liquid medium in microtiter wells must be revised. This revision comprises, utilizing the measured β-galactosidase produced by yeast carrying the Caspase-cleavable reporter proteins, and assaying the colorimetric product (OD620nm) derived from X-gal substrate in 384 well plates, as a measure of the lacZ reporter gene activity. We compared β-galactosidase activity produced in the presence or absence of the broad-spectrum caspase inhibitory compound, zVAD-fmk (benzoyl-Valinyl-Alaninyl-Aspartyl-fluoromethylketone). Among the variables that were initially interrogated were initial seeding cell density, time of culture, concentration of X-gal substrate, and supporting carbohydrate (raffinose vs mannose). Note that it was determined that it is not advisible to employ glucose in yeast media because it represses the GAL1 promoter used in some plasmids. An example of data obtained using the plural-component system of Fas/FADD/Caspase-8 is presented. All variables tested altered the signal:noise ratio of the assay. For the Fas/FADD/Caspase-8 plural-component assay, the best results were achieved with ˜2×105 cells per well starting density cultured for ˜3 days in raffinose-containing media with 4× concentration of X-gal substrate (
Optimization of Signal:noise Ratio in Microtiter Plates.
As shown in
The 384 well assay format was further validated, using zVAD-fmk, a peptidyl inhibitor with broad-spectrum activity against animal Caspases. For some experiments, a Calpain inhibitor was employed in side-by-side experiments as a control. The broad-spectrum Caspase inhibitor zVAD-fmk inhibited β-galactosidase activity in a dose-dependent fashion, with the concentration required for achieving 50% inhibition ranging from ˜1-10 μM in these assays (
The validation results of a yeast-based Caspase assay using a pharmacological inhibitor of Caspases are shown in
To assess the reproducibility of the 384 well microplate assay, multiple replicate wells in 384 well plates were prepared representing the negative (assay max) and positive (assay min) controls for the assays. For the plural-component system of NALP1ΔLRR/ASC/Caspase-1, the maximum signal was produced by cells grown with X-gal substrate, while the minimum was set by cells grown without X-gal substrate. As described above, for this assay, NALP1ΔLRR-mediated activation of Caspase-1 resulted in activation of the cleavable transcription factor, producing β-galactosidase, which was measured using a colorimetric substrate. The magnitude of the readout of the Z′ factor was optimized by empirically adjusting yeast cell density, incubation time, and other variables in ordere to employ a 384 well assay format. The Z′ factor for the assay was determined by reading multiple replicates of the assay max and min, and determined to be >0.6, and thus suitable for HTS (
The results of a HTS of a yeast-based NALP1 inflammasome assay are shown in
Determination of IC50 Values for zVAD-fmk Inhibition of Caspases in Yeast.
Yeast expressing various Caspases alone (at high levels) or in combination with upstream activators (at low levels) and cleavable substrates containing appropriate tetrapeptides reorganized by these proteases were used in 384 well β-galactosidase activity assays to assess inhibition by zVAD-fmk. The compound was titrated into assays at various concentrations and percentage inhibition was determined. IC50 values were determined using PRIZM software for analysis with the results displayed in Table 2 below.
As shown in
Flow Chart for cDNA Library Screening Using a Reporter Gene Strategy Based on Cleavable Transcription Factor.
The example shown in
Flow Chart for cDNA Library Screening Using Reporter Gene Strategy Based on Cleavable Transcription Factor.
The example shown in
Use of One-component Yeast-based Caspase Activity Assay for cDNA Library Screening.
As shown in
Note that the large amount of Caspase-9 (expressed from TEF promoter) activated the cleavable S3(DEVD) substrate when co-expressed with pro-Caspase-3 or pro-Caspase-7, but not in their absence, thus constituting a 2-component system. No lacZ reporter gene activity was detected when the non-cleavable substrate (DEVG) was employed (G3). In contrast, expressing a small amount of Caspase-9 (from the CYC promoter) or the catalytically-defective Caspase9 (C287→A287) did not activate Caspase-3 or Caspase-7. However, co-expressing active Apaf-1* with a small amount of pro-Caspase-9 activated the lacZ reporter gene in yeast expressing pro-Caspase-3 or -7 (but not in the absence of these downstream effector Caspases), thus constituting a 3-component system.
As shown in
Note that the lacZ reporter gene was activated only when the combination of an initiator Caspase and upstream activator was co-expressed, along with a cleavable substrate.
Transformants. The transformed yeast cell clones are: (A) EGY191-2op-LEU2/2oplacZ/TEF-Fas-d-S1 (WEHD)-TA/ΔTEF1-Caspase1-FLAG (S1, C1(WT)), EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-G1 (WEHG)-TA/ΔTEF1-Caspase1-FLAG (G1, C1(WT)), or EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-S1(WEHD)-TA/ΔTEF1-Caspase1 (C285→G285)-FLAG, (S1, C1(C285→G285)), were transformed with the plasmids encoding the activator Asc, or the empty vector (−); (B) EGY48-6op-LEU2/2op-lacZ/ΔTEF1-Fas-d-S2(DEHD)-TA/ΔGPD1-HA-Caspase2-FLAG(S2, C2(WT)), EGY48-6op-LEU2/2op-lacZ/ΔTEF1-Fasd-G2(DEHG)-TA/ΔGPD1-HA-Caspase2-FLAG (G2, C2(WT)), or EGY48-6op-LEU2/2op-lacZ/ΔTEF1-Fas-d-S2(DEHD)-TA/ΔGPD1-HA-Caspase2 (C320→A320)-FLAG (S2, C2(C320→A320)), were transformed with plasmids encoding the activator RAIDD, or the empty vector (−); (C) EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HACaspase9 (S9, C9(WT)), EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-G9(LEHG)-TA/TEF-HA-Caspase9 (G9, C9(WT)), or EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase9(C287→A287) (S9, C9(C287→A287)), were transformed with plasmids encoding an active form of Apaf-1 (Apaf*), or the empty vector (−); (D) EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADH-Caspase10-FLAG (S8, C10(WT)), EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8(LETG)-TA/ADH-Caspase10-FLAG (G8, C10(WT)), or EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/ADHCaspase10(C358→A358)-FLAG (C10(C358→A358)), were transformed with the plasmids encoding the activator FADD, or the empty vector (−). The large amount of FADD is enough to activate Caspases-10 by itself.
Specificity of Upstream Activators of Initiator Caspases—Tested by 2-component Systems.
As shown in
Note that results obtained were as predicted, with (1) Apaf-1* activating pro-Caspase-9, but not other initiator Caspases; (2) RAIDD activating pro-Caspase-2, but not other Caspases; (3) FADD activating pro-Caspases-8 and 10, but not other Caspases, and (4) Asc activating pro-Caspase-1 and 8. Note that while Asc contains a CARD that pairs with a complementary CARD in pro-Caspase-1 and would not be necessarily predicted to activate the DED-containing protease Caspase-8, it has previously been reported that Asc is an activator of Caspase-8 (Hasegawa M. et. al. J Biol Chem 280: 15122-30 (2005); Masumoto, J. et. al., Biochem. Biophys. Res. Commun 303: 69-73 (2003).
Transformants: Transformed yeast clones were as follows: S1,C1:EGY191-2op-LEU2/2op-lacZ/TEF-Fas-d-S1 (WEHD)-TA/PΔTEF1-Caspase1-FLAG; S2, C2: EGY48-6op-LEU2/2op-lacZ/PΔTEF1-Fas-d-S2(DEHD)-TA/PΔGPD1-HA-Caspase2-FLAG; S8, C8: EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)-TA/CYC1-Caspase8-HA; S9, C9: EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S9(LEHD)-TA/TEF-HA-Caspase-9; S8, C10: EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)-TA/ADH-Caspase10-FLAG. These cells were transformed with the plasmids encoding the activators (Asc, RAIDD, FADD, and Apaf*). For controls (−), the “empty” version of the plasmids were introduced.
Validation of 3-component Yeast-based Caspase Assay Reconstituting DISC.
As shown in
Transformants: (A) Yeast transformants included EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S8 (LETD)-TA/CYC1-Caspase-8-HA (S8, C8) EGY48-6op-LEU2/2op-lacZ/TEFFas-d-G8(LETG)-TA/CYC1-Caspase8-HA (G8, C8), without (−) or with Fas, and without (−) or with FADD, which were expressed from either ADH and ΔADH or CYC1 promoters, respectively, to achieve high expression of Fas and low expression of FADD. (B) Yeast transformants included: EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG (S8, C10); EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8(LETG)-TA/CYC1-Caspase10-FLAG (G8, C10); and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8 (LETD)-TA/CYC1-Caspase10(C358→A358)-FLAG (S8, C10(C358→A358)) each without (−) or with Fas, and without (−) or with FADD-expressing vector or the corresponding empty vectors.
Flow Chart for cDNA Library Screening Using 3 Component System—Application to Death Receptor Cloning—Example Screening Strategy #1.
Flow Chart for cDNA Library Screening Using 3 Component System to Clone Death Receptors—Example Screening Strategy #2.
Flow Chart for cDNA Library Screening Using 3-component System to to Clone Death Receptors—Example Screening Strategy #3.
Schematic Representation of 3-component System Used for Cloning Adapter Protein that Links Fas to Pro-Caspase-10.
As shown in
Transformants: (A) Yeast cell transformants included: EGY48-6op-LEU2/2op-lacZ/TEFFas-d-S8 (LETD)-TA/CYC1-Caspase8-HA (S8, C8) and EGY48-6op-LEU2/2oplacZ/TEF-Fas-d-G8 (LETG)-TA/CYC1-Caspase8-HA (G8, C8), with empty vector (−) or with plasmids encoding FADD (expressed from CYC1 promoter) or Fas (expressed from ADH promoter). (B) Yeast cell transformants included EGY48-6op-LEU2/2oplacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG (S8, C10), EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-G8 (LETG)-TA/CYC1-Caspase10-FLAG (G8, C10), and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8 (LETD)-TA/CYC1-Caspase10(C358→A358)-FLAG (S8, C10(C358→A358)), each with empty vector (−) or with plasmids encoding FADD or Fas as above.
Flow Chart for cDNA Library Screening Using 3 Component System to Clone Adapters.
As shown in
Examples of cDNA Cloning Results.
As shown in
Optimization of Signal:noise Ratio in Microtiter Plates: Cell Density.
As shown in
Optimization of Signal:noise Ratio in Microtiter Plates: Time and X-gal Concentration.
As shown in
Plasmids for Expression of Upstream Activators of Caspases and lacZ Reporter Gene in Yeast.
As shown in
Yeast Expression Plasmids for Functional Screening of cDNA Libraries and Expression of Upstream Activators of Caspases.
As shown in
Chemical Library Screens of Multi-component Yeast-based Protease Assay Systems were Performed to Define Hit-rates and Test Reliability.
Chemical library screens to validate the performance of the assays were completed. The screens were conducted such that all assay components were added in automated fashion using integrated robotic liquid handling systems, moving the plates initially into carousels that hold 180 plates at room temperature, and then manually applying seals (breathable sealing film from Axygen Scientific) to reduce evaporation, and moving the bar-coded plates to a 30° C. incubator for the required time (generally culturing for 2-4 days or less). Each assay plate contained a row of positive (min) and a row of negative (max) controls that did not receive compounds but that received DMSO in volumes equivalent to the amount of DMSO in which compounds will be supplied. At the conclusion of the 72 hrs incubation, plates were robotically delivered to one of the integrated multi-purpose plate readers for reading at OD620 nm. The programmable robotic workstations sequenced the additions of reagents, minimizing variations in incubation times. Data from primary screens were uploaded directly from plate readers into computers with customized Microsoft excel software, set up to calculate Z′ factor for each plate, and with hit determinations set at 50% of the mean value for the negative control values.
A screening of a LOPAC was performed at several different concentrations (typically 20, 10, and 5 μM) to compare the hit rates, and to empirically determine an acceptable concentration for conducting large-scale library screens. The empirically adjusted compound screening concentrations allowed for the employment of the highest screening concentration tolerable without causing artifacts or non-specific effects. A hit rate of 0.1-0.5% was achieved which was consistent with the general expectations for the screening results. Before undertaking this study, it was empirically determined what the effects of DMSO on assay performance would be, through pilot experiments where increasing concentrations of DMSO (from 1-10% volume) were added to the positive and negative controls and the impact on assay signal and stability was determined. Second, a progression was made to larger libraries (eg., 50K Chembridge library), it was determined which factors improve the stability of the positive and negative controls from plate to plate, assessing the assay performance as time is extended from minutes to hours, and calculating Z′ for each plate as the quality of the assay's performance is assessed in true screening mode.
For HTS assays the β-galactosidase produced by yeast carrying the caspase-cleavable reporter proteins was measured, by assaying the calorimetric product (OD620 nm) derived from X-gal substrate in 384 well plates, as a measure of the lac Z reporter gene activity.
In summary disclosed are HTS systems for intracellular proteases, using Caspases as a prototype. For this purpose, yeast-based cellular systems that permit facile expression of proteases and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes were devised. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor. In summary we disclose:
1. Multi-component systems that reconstitute mammalian protease activation pathways in yeast.
2. Optimized systems with adjusted expression levels, reporter gene sensitivity, and other parameters to achieve satisfactory signal:noise results and mechanisms for optimizing future systems.
3. Defined the key variables that require optimization for achieving HTS quality assay performance.
4. Performed chemical library screens of multi-component yeast-based protease assay systems to define hit-rates and test reliability.
The reporter, Fas-d-S1-TA for Caspase-1 and related proteases (called “S1”), was generated by using PCR and standard recombinant DNA techniques. This protein consists of, from N to C termini, amino acids M1-L224 of a type 1 transmembrane protein, human Fas 34 in which Fas is devoid of the cytosolic death domain (Fas-d), a linker containing the sequence GWEHDG between a Xhol and EagI site, and finally a transcriptional activator (TA) containing the LexA DNA binding domain and the B42 activation domain, taken from plasmids pRS305(Δwbpl-Cub-PLV) 35 and pJG4-5 (Invitrogen) with PCR. Other reporters were made by substituting the linker with oligonucleotides designed to encode in-frame the sequences GWEHGG (“G1”), GDEHDG (“S2”), GDEHGG (“G2”), GDEVDG (“S3”), GDEVGG (“G3”), GTEVDG (“S6”), GTEVGG (“G6”), GLETDG (“S8”), GLETGG (“G8”), GLEHDG (“S9”), or GLEHGG (“G9”), after digestion with Xhol and EagI.
Plasmids for expression of Caspases in yeast were derived from pRS series vectors 36-38 and Pesc series vectors (Stratagene [Agilent]). Expression levels were adjusted by using constitutive promoters of different strengths (CYC1, ADH, TEF, and GPD) 39 or using the inducible GAL1 promoter, in conjunction with different strength reporter genes carrying variable numbers of lexA operators 6, and using plasmids with different replication origins for low or high copy number replication (2u and CEN/ARS). To further control expression levels, several attenuated forms of the promoters were made by PCR-assisted deletional mutagenesis. Using a standard indicator gene, the approximate relative strength of the promoters was determined to be: ΔCYC4<ΔCYC2<CYC1<ΔGPD2<ΔGPD1<ΔADH1<ADH<ΔTEF3<ΔTEF2<ΔTEF1<TEF. However, the relative locations of the promoters in complex plasmids somewhat affects their strength, especially when two or three genes are contained in one plasmid. In this regard, two or three genes within one plasmid were sometimes co-expressed by placing the genes flanked by the above promoters and inserting transcription termination elements between them, because the selectable marker genes available are limited (URA3, HIS3, TRP1, and LEU2). In addition, many of the expressed Caspases and upstream activators were cloned with N-terminal HA or C-terminal HA or FLAG epitope tags for convenience of detection of the protein products by immunoblotting. Examples of complex plasmids are (a) p426-2op-lacZ/CYC1-FADD, which consists of 2μ origin, URA3 marker, lacZ gene under the control of two lexA operators, and a FADD gene with expression driven by a short form of the ADH promoter (ΔADH1), as in
Diagrams of complex plasmids containing 3 or more genes are provided in
Construction of Expression cDNA Libraries.
Oligo(dT)-primed or random heptamerprimed cDNA libraries were made in modified p424-GAL1, p424-GAL1-HA, p424-ADH, or p424-ADH-HA plasmids (carrying a TRP1 marker) using mRNAs derived from HEK 293 cells, HepG2 cells, HeLa cells, human liver, or human placenta, as in
Plasmids were introduced into yeast by lithium acetate transformation. The yeast strain EGY48, which carries 6 lexA operators upstream of LEU2 gene (6op-LEU2), was transformed with pJK103 6, which carries two lexA operators upstream of lacZ gene (p426-2op-lacZ), and subsequently with reporter plasmids (p413-TEF-Fas-d-S1-TA, p413-TEF-Fas-d-G1-TA, p413-TEF-Fas-d-S2-TA, p413-TEF-Fas-d-S3-TA, p413-TEF-Fas-d-S6-TA, p413-TEF-Fas-d-S8-TA, or p413-TEF-Fas-d-S9-TA). Caspase (all full-length zymogen proforms) expression plasmids (p424-ADH-Caspasel-FLAG, p424-ADH-HA-Caspase2, p424-ADH-Caspase3, p424-TEF-Caspase4, p424-ADH-Caspase5, p424-TEF-HA-Caspase6, p424-ADH-Caspase7, and p424-ADH-HA-Caspase9-FLAG) or empty vector (p424-ADH), were introduced into these backgrounds. As for assays with Caspase-8 and -10, small amounts of pro-Caspase-8 and -10 were expressed with a large amount of FADD by transforming yeast with the dual gene plasmids p424-CYC1-Caspase8-HA/TEF-HA-FADD and p424-CYC1-Caspase10-FLAG/TEF-HA-FADD, because expression of large amounts of human Caspase-8 or Caspase-10 significantly inhibited the cell growth. The transformants (two independent colonies for each transformation) were streaked on growth plates (minimum synthetic dropout (SD) medium containing 2% glucose and 50 μg/ml leucine) or on selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 μg/ml X-gal). Yeast growth and blue color development were monitored for four to six days at 30° C.
Reporter Gene Assays in Liquid Media Using 384-well Plates.
Assays were performed in a total volume of 40 ηl in triplicate. First, 20 μl of liquid selection media containing X-gal and a series of concentrations of reagents such as z-VAD was dispensed into each well of 384-well plates. Next, confluent yeast cells expressing various Caspases and cleavable substrates were collected, washed with water, and suspended in selection media of the same volume as the culture media. The yeast suspension was diluted to 1:5-10 (v:v) with the selection media, and 20 μl aliquots were added to the 384-well plates. Absorbance at 620 nm was measured 2-3 days after culture at 30° C.
For the Fas/FADD/Caspase-8 and the NLRP1/Asc/Caspase-1 assays, EGY48 yeast containing the desired plasmids were streaked onto SD plates (6.8 g Yeast Nitrogen Base w/o amino acids, 20 mg arginine, 50 mg threonine, 30 mg isoleucine, 60 mg phenylalanine, 20 mg valine) containing agar (1.7%), supplemented with 2% α-D-glucose and 50 μg/mL leucine. The plates were incubated at 30° C. for 48 hrs and a colony was picked and transferred to a 14 ml polypropylene tube containing 2 ml of SD media broth supplemented with α-D-glucose and leucine as above and grown at 30° C. for 16-24 hrs with shaking. Then, 1 ml of the overnight culture was transferred into 20 mls of growth media in a 500 ml flask and shaken at 30° C. for 16-24 hrs. The cells were collected by centrifugation at 1000×g for 5 minutes at room temperature, and washed with 20 mls of sterile water, then resuspended in 20 mls of SD broth supplemented with 1% galactose and 0.2% raffinose. The HTS Assay was performed at a final compound concentration of 10 μM (1% DMSO), with cell densities of 2×105 cells/ml (2×105 cells) in a volume of 40 μl. The assay was assembled by the addition of 4 μl of ≈100 μM compounds (final concentration of 10 μM) in 10% DMSO (1% final DMSO) to clear polystyrene 384-well microplates using a Beckman-Coulter Biomek FX, then 18 μl of cell suspension (1.1×107 cells/ml) (2×105 cells/well) and 18 μl of X-gal suspended in Selection Media (for a final concentration of 400 μg/mL) were added to the wells using a Matrix WellMate bulk reagents dispenser. Remaining solutions were added by using the WellMate from Matrix Technologies Corp. Controls were included with each plate, corresponding to cells treated with DMSO (without compounds) and cells cultured with and without X-gal. The plates were sealed with breathable sealing film (from Axygen Scientific) to reduce evaporation and transferred to 30° C. incubators. After 48 hours, the plates were brought to room temperature, the breathable film removed and replaced with the transparent polyester tape seal, and the plates were mixed, pelleted briefly and read using a Beckman DTX 880, recording absorbance at 620 nm.
EGY48 yeast inducibly (GAL1 promoter) expressing the LexA/B42 transcription factor or containing the empty vector were used to detect compounds that directly inhibit the lacZ reporter gene or that alter β-galactosidase activity. The plasmids employed were p424-GAL1 and p424-GAL1-TA (transcriptional activator) with transformed cells selected on tryptophan-deficient plates. Yeast expressing Caspase-1 activated by expression of high levels of Asc (ΔTEF3-Caspase-1-FLAG/TEF-HA-Asc) were used to detect compounds that cross-react with hits from the Fas/FADD/Caspase-8 screen. The Asc/Caspase-1 yeast cells were cultured and assayed under identical conditions to those for the primary NLRP1 HTS assay (see above), using cells expressing 6op-LEU2/2op-lacZ reporter genes and TEFFas-d-S1-TA substrate.
To clone cDNAs encoding proteins that activate Caspase-1, the yeast strain EGY191 containing 2op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units of TEF-Fas-d-S1-TA (substrate) and ΔTEF3-Caspase1-FLAG (pro-Caspase-1), as illustrated in
To clone cDNAs encoding proteins that activate Caspase-2, the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units of ΔTEF2-Fs-d-S2-TA (substrate) and ΔGPD1-HA-Caspase2-FLAG (pro-Caspase-2) and selected on histidine-deficient plates. As controls, cells were subsequently transformed with the RAIDD-expressing plasmid p424-TEF-HA-RAIDD or empty vector and selected an tryptophan-deficient plates.
To clone cDNAs encoding proteins that activate Caspase-3, the yeast strain EGY191 containing 2op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units for ΔTEF3-Fas-d-S3-TA and ΔCYC2-Caspase-3 and selected on histidine-deficient plates. As controls, cells were transformed with the Caspase-9-expressing plasmid p424-ADH-HA-Caspase9-FLAG or empty vector and selected on tryptophan-deficient plates.
To clone cDNAs encoding proteins that activate Caspase-4, the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units for GPD-Fas-d-S1-TA and ΔTEF2-Caspase4. As controls, cells were transformed with a plasmid expressing high levels of (Caspase-7 (p424-ADHCaspase-7), which results in active Caspase-7 did but does not cut the S1 site.
To clone molecules that activate Caspase-7, the yeast strain EGY48 expressing 6op-LEU2/2op-lacZ/TEF-Fas-d-S3-TA/CYC1-Caspase-7 was created. The small amount of Caspase-7 exists as inactive zymogen, and does not cut the S3 site. As a positive control, pro-Caspase-7 was activated by expressing Caspase-9 at high levels (p424-ADH-HA-Caspase9-FLAG). Active Caspase-9 did not cut the S3 site.
To clone cDNAs encoding proteins that activate Caspase-8, the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units for GPD-Fas-d-S8-TA and CYC1-Caspase8-HA and selected on histidine-deficient plates. As controls, cells were transformed with FADD-expression plasmid p424-TEF-HA-FADD or empty vector and selected on tryptophan-deficient plates.
To clone cDNAs that activate Caspase-9, the yeast strain EGY48 containing 6op-LEU2/2op-lacZ was transformed with plasmids containing transcriptional units for TEF-Fas-d-S9-TA and TEF-HA-Caspase9, on a low-copy plasmid p413 (CENLARS). The small amount of pro-Caspase-9 exists as inactive zymogen, and does not cut the S9 site. As controls, cells were transformed with the Apaf-1*-expressing plasmid p424-TEF-HA-Apaf* or empty vector and selected on tryptophan-deficient plates.
To clone cDNAs encoding proteins that activate Caspase-10, the yeast strain EGY48 containing 6op-LEU212op-lacZ was transformed with plasmids containing transcriptional units for GPD-Fas-d-S8-TA and ADH-Caspase10-FLAG and selected on histiodine-deficient plates. As controls, cells were transformed with the FADD expressing plasmid p424-TEF-HA-FADD or empty vector and selected on tryptophan deficient plates.
Procedures for cDNA Library Screening.
Screens for cDNAs encoding proteases (Caspases). To screen for cDNAs encoding proteases capable of cleaving the S1 site (WEHD), the yeast strain EGY48 containing 6op-LEU212op-lacZ was transformed with p413-TEF-Fas-d-S1-TA followed selection on histiodine deficient plates, then these cells were subsequently transformed with a HEK293 cDNA library (contains TRP1 marker) or a human placenta cDNA library (contains TRYP1 marker). The transformants were seeded on tryptophan-deficient growth plates (SD medium containing 2% glucose and 50 ug/ml leucine). Independent colonies of 5.1×105 appeared from the HEK293 cDNA library in 48 hours. The colonies were harvested and pooled, and a portion of the recovered cells (3.6×106 cells) was seeded onto leucine-deficient selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 ug/ml X-gal). Bluecolored colonies appeared within a week and were subjected to plasmid DNA extraction. The extracted plasmid DNAs were introduced into KC8 E. coli cells by electroporation to efficiently recover the cDNAs plasmids. The candidate cDNAs were again introduced into the yeast cells containing 6op-LEU2/2op-lacZ/TEF-Fas-d-S1-TA or 6op-LEU2/2op-lacZ/TEF-Fas-d-G1-TA to confirm whether they cleave S1 specifically.
To screen for cDNAs encoding proteases capable of cleaving the S3 site (DEVD), the yeast strain EGY48 containing 6op-LEU2/2op-lacZ, was transformed with p413-TEF-Fas-d-S3-TA, followed by selection on histiodine-deficient plates, and subsequently transformed with a HEK293 cell cDNA library, then processed as above.
Screening for cDNAs Encoding Proteins That Activate Caspase-8 in a FADD-dependent Manner.
The yeast strain EGY48 (6op-LEU2) was transformed with plasmids p426-2op-lacZ/ΔADH1-FADD and p413-TEF-Fas-d-S8-TA/CYC1-Caspase8, then subsequently transformed with a HepG2 cDNA library, a human liver cDNA library, or a HEK293 cDNA library. The transformants were seeded on growth plates (SD medium containing 2% glucose and 50 ug/ml leucine). Independent colonies of 1.7×106 appeared from the HepG2 library in 48 hours. They were harvested, and pooled, and a portion of the cells (2.4×107 cells) was seeded on selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 ug/ml X-gal). Blue-colored colonies appeared within a week, were subjected to plasmid DNA extraction. The extracted plasmid DNAs were introduced into KC8 E. coli cells by electroporation to recover the cDNAs plasmids. The candidate cDNAs were again introduced into the yeast cells containing 6op-LEU2/2op-lacZ and plasmids containing transcriptional units for ΔADH1-FADD, TEF-Fas-d-S8-TA, and CYC1-Caspase8; or TEF-Fas-d-S8-TA and CYC1-Caspase8; or TEF-Fas-d-S8-TA to confirm whether they activate Caspase-8 in a FADD-dependent manner.
Screening for cDNA Encoding Adapter Proteins That Link Fas or DR5 to Caspases-8 and -10.
The yeast strain EGY48 (6op-LEU2) was transformed with p426-2op-lacZ/ADHDR5-FLAG and p413-TEF-Fas-d-S8-TA/CYC1-Caspase8, then subsequently transformed with a HeLa cell cDNA library or a HEK293 cDNA library. The transformants were seeded on growth plates (SD medium containing 2% glucose and 50 ug/ml leucine). Independent colonies of 1.0×106 appeared from the HeLa cell cDNA library within 48 hours. They were harvested, pooled, and a portion of the cells (2.4×107 cells) was seeded on selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 ug/ml X-gal). Blue-colored colonies appeared within a week and were subjected to plasmid DNA extraction. The extracted plasmid DNAs were introduced into KC8 E. coli cells by electroporation to recover the cDNA plasmids. The candidate cDNAs were again introduced into the yeast cells expressing 6op-LEU2/2oplacZ/ADH-DR5-FLAG/TEF-Fas-d-S8-TA/CYC1-Caspase8, 6op-LEU2/2op-lacZ/TEFFas-d-S8-TA/CYC1-Caspase8, or 6op-LEU2/2op-lacZ/TEF-Fas-d-S8-TA to confirm whether they activate Caspase-8 in a DR5-dependent manner.
The yeast strain EGY48 (6op-LEU2) was transformed with p426-2op-lacZ/ADH-Fas and p413-GPD-Fas-d-S8-TA/CYC1-Caspase10, then subsequently transformed with a HEK293 cell cDNA library. The transformants were seeded on growth plates (SD medium containing 2% glucose and 50 ug/ml leucine). Independent colonies of 2.2×106 appeared within 48 hours, and were harvested, and pooled. A portion (3.2×107 cells) of the cells seeded on selection plates (SD medium containing 1% galactose, 0.2% raffinose, BU salts, and 80 ug/ml X-gal). Blue-colored colonies appeared in a week and were subjected to plasmid DNA extraction. The extracted plasmid DNAs were introduced into KC8 E. coli cells by electroporation to recover the cDNA plasmids. The candidate cDNAs were again introduced into the yeast cells expressing 6op-LEU2/2oplacZ/ADH-Fas/GPD-Fas-d-S8-TA/CYC1-Caspase10, 6op-LEU2/2op-lacZ/GPD-Fas-d-S8-TA/CYC1-Caspase10, or 6op-LEU2/2op-lacZ/GPD-Fas-d-S8-TA to confirm whether they activate Caspase-10 in a Fas-dependent manner.
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All references cited herein are hereby incorporated by reference in their entireties, whether previously specifically incorporated or not. As used herein, the terms “a”, “an”, and “any” are each intended to include both the singular and plural forms.
Having now fully described the disclosed subject matter, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the disclosure and without undue experimentation. While this disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the subject matter following, in general, the principles of the disclosure and including such departures from the disclosure as come within known or customary practice within the art to which the subject matter pertains and as may be applied to the essential features hereinbefore set forth.
Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Ser. No. 61/081,363, filed Jul. 16, 2008, the entire content of which is incorporated herein by reference.
This invention was made in part with government support under Grant No. GM085255 awarded by the NIH. The United States government may have certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61081363 | Jul 2008 | US |
