Patent Application
20100160217
This invention relates to biotechnology, and more particularly to receptor chaperons and methods of using the same.
Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels that mediate fast excitatory neurotransmission in the central and peripheral nervous system. nAChRs are distinct from metabotrophic receptors, including the muscarinic acetylcholine receptor, in that they are pentameric integral membrane proteins that form a cation selective channel gated by acetylcholine (Changeux et al., 1984; Karlin and Akabas, 1995). These channels are tightly clustered at the postsynaptic region and modulate the postsynaptic membrane potential in response to presynaptic release of acetylcholine. Adult mammalian muscle type nAChRs are heteropentamers composed of two α1 subunits and one β1, γ and ε subunit. Each of the subunits have the same membrane topology and are composed of a large extracellular region, a transmembrane region composed of four membrane spanning domains and a cytoplamsic region formed by an intracellular loop between the third and fourth membrane spanning domains. (Corringer et al., 2000; Unwin, 2005). The subunits of mature pentameric nAChRs are arranged with a five-fold axis of symmetry with the second transmembrane spanning region lining the central pore of the ion channel (Akabas et al., 1994; Imoto et al., 1988). In addition to an invariant stoichometry, the circular arrangement of subunits is also fixed in the circular order α γ α β ε. The correct ordered assembly of nAChR subunits is functionally important because the acetylcholine binding site is formed at the interface between α/γ and α/ε subunits (Green and Wanamaker, 1998).
After translation, formation of mature nAChRs occurs in different cellular compartments and is a slow inefficient process (Merlie et al., 1983; Baker et al., 2004). Early steps in the formation of nAChRs occur co-translationally on each subunit and are common among transmembrane proteins of the plasma membrane. These early processing steps include signal peptide cleavage, insertion of transmembrane spanning regions into the membrane in the correct orientation, glycosylation, and disulfide bond formation. After or during processing the individual subunits are assembled into pentamers and assembly is regulated to ensure that the correct composition and arrangement of subunits is achieved. Finally, the mature assembled pentamer is trafficked to the plasma membrane. Trafficking of nAChRs is a regulated process; individual nAChR subunits are not trafficked, but remain in the ER until incorporated into pentamers. In contrast to the structural and functional studies of individual receptor subunits, little is known about the molecules and mechanism that govern formation of nAChRs.
nAChRs belong to a family of ligand gated ion channels that all contain a conserved extracellular cystiene loop (Connolly and Wafford, 2004). The Cys-loop is present on all subunits of the family and is formed by a disulfide bond between two cystine residues separated by 13 amino acids. The Cys-loop family includes receptors for common neurotransmitters including, GABA, glycine and serotonin, as well as uncommon ligands such as histidine and zinc (Davies et al., 2003; Le Novere and Changeux, 2001). The functional significance of this conserved structural feature is not well established. High-resolution structural determination of the nAChR, and the acetylcholine binding protein (AChBP), place the Cys-loop near the extracellular linker between the second and third transmembrane spanning region. In addition, mutation of residues within the Cys-loop of a glycine receptor results in a decrease in receptor activation, suggesting that the Cys-loop may have a functional role in gating. (Brejc et al., 2001; Schofield et al., 2003; Unwin, 2005). However, the presence of the Cys-loop in the AChBP, which does not contain a channel, argues against a functional gating role for the Cys-loop. Other groups assert that formation of the Cys-loop in nAChR subunits plays an essential role in nAChR formation, since mutation of the cysteine residues of the Cys-loop blocks assembly of nAChRs and prevents trafficking (Green and Wanamaker, 1997; Sumikawa and Gehle, 1992). Hence, the functional significance of the Cys-loop remains unclear.
C. elegans is an ideal genetic model system to identify genes whose protein product has a role in nAChR formation (Miller et al., 1996). Cholinergic neurotransmission at the worm neuromuscular junction is mediated by two genetically distinct classes of nAChR based on sensitivity to the anthlementic levamisole (Richmond and Jorgensen, 1999a). Levamisole causes activation of the levamisole sensitive nAChR, but does not affect the levamisole insensitive nAChR. Application of levamisole to nematodes causes chronic activation of levamisole sensitive nAChRs and results in hyper-contraction of body muscles, causing paralysis. Forward genetic screens to isolate mutants resistant to levamisole have led to the identification of four genes (unc-29, unc-38, unc-63, and lev-1) that encode subunits of the levamisole sensitive nAChR. In addition, other genes that do not encode nAChR subunits have been shown to confer levamisole resistance when mutated. The protein products of these genes may be involved in formation of the nAChR (Lewis et al., 1980a; Lewis et al., 1980b).
The present invention provides receptor chaperons and means for producing cells having increased and/or decreased expression of nAChR subunit combinations and/or nAChR subtypes, which provide useful compositions and tools for investigating pharmacological properties of the receptors and the regulation of the binding sites of nAChR subtypes. In addition, the invention provides compositions and methods for screening nicotinic compounds.
The invention relates to a receptor chaperon protein or a functional fragment thereof. For example, a polypeptide of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, or
The invention also relates to nucleic acid sequences, isolated and/or recombinant, encoding a receptor chaperon protein. The nucleic acid sequences may be in a vector, including an expression vector, and may be introduced into a host cell.
The invention also relates to a method of producing a heterologous receptor in a cell, the method comprising: providing a host cell; introducing a nucleic acid sequence encoding at least one subunit of a heterologous receptor that does not efficiently produce a functional receptor on the surface of the host cell; producing the at least one subunit of a receptor in the host; introducing a nucleic acid sequence encoding a receptor chaperon of the invention into the host cell; producing the receptor chaperon in the host cell; and increasing production of a functional receptor comprising the at least one subunit of a heterologous receptor on the surface of the host cell. The receptor subunit of the method may be a nicotinic Acetylcholine receptor subunit derived from a multicellular organism (a subject), such as a vertebrate, insect, Caenorhabditis or mammal, including a human, cow, or horse.
The invention also relates to a method of reducing or eliminating expression of a receptor on a cell surface by inhibiting a function of a receptor chaperon of the invention in a cell. Such inhibitors may be used to treat a subject, for example, the inhibitor may be made into a medicament for the treatment of a disease characterized by receptor hyperactivity. Alternatively, the inhibitor may be used as an insecticide, particularly, when the inhibitor has a high affinity for an insect receptor chaperon, relative to the affinity for a human and/or mammalian receptor chaperon.
The invention further relates to a method of producing a recombinant nematode nicotinic acetylcholine receptor, comprising culturing a host cell under conditions which permit the expression of UNC-74. The unc-74 gene may be coexpressed with one or more nAChr subunits, preferably the nAChR subunits derived from Caenorhabditis.
The invention further relates to a method of screening for anthelmintic compounds by introducing a receptor chaperon of the invention into a host cell; expressing the receptor chaperon in the host cell; contacting the host cell with a compound to be screened for antihehnintic activity; selecting a compound which interacts with said receptor chaperon; and characterizing the selected compound as an anthelmintic compound.
The invention further relates to a method of controlling parasitic nematode growth in a host, comprising: administering an effective amount of an anthelmintic compound identified by a method of the invention to a subject. The invention further relates to a method of controlling parasitic nematode growth in soil or a crop, comprising: administering an effective amount of an anthelmintic compound identified by a method of the invention to the soil or crop. For example, the invention also relates to methods of screening for neonicotinoids and their use for crop protection by screening for compounds that inhibit or prevent the production of functional nAChRs on the surface of insect cells. Preferably, the compound exhibits selective toxicity toward insects based, at least in part, on a higher affinity for an insect receptor chaperon.
T) causes a nonsense change at lysine 95; ox145 (ΔC1117-A1159) is a small 43 base pair deletion in exon 5 that causes a frameshift after glycine 213; x19 (C1319
T) results in a nonsense change at arginine 280; ox111 (G2096
A) disrupts the splice donor of intron 6 which results in a frameshift starting at S405.
The invention provides a greater understanding of ligand gated ion channel formation, thereby providing compositions and methods useful in the production of such channels. The molecules of the invention, which have not been identified through traditional biochemical analysis, are involved in nAChR formation.
The invention utilizes the cloning and characterization of unc-74, which encodes the worm homologue of TMX3, a transmembrane thioredoxin domain containing protein (Haugstetter et al., 2005), to describe receptor chaperon proteins involved in the formation of multimeric nAChRs.
The data presented herein describes the cloning and characterization of unc-74, which is believed to be an exemplary member of a class of proteins required for the formation of a specific acetylcholine-gated ion channels. Using a transposon-based mutagenesis approach described in International Patent Publication WO 00/73510 (Dec. 7, 2000), the unc-74 locus was cloned and shown to encode the worm homologue of TMX3, a transmembrane thioredoxin containing protein. Electrophysiological and genetic analysis of unc-74 mutant animals demonstrated that UNC-74 is required for the formation of the levamisole-sensitive nAChR. In the absence of unc-74 function, nAChR subunits are retained in the ER of both muscle and neurons. UNC-74 is expressed in muscle cells and localized to the ER, where it is believed to function in the trafficing of nAChRs to the plasma membrane. However, this function does not require the catalytic activity of its thioredoxin domain.
That the thioredoxin active site of UNC-74 is dispensable for function is discordant with the conservation of the UNC-74/TMX3 thioredoxin domain and active site. These data suggest that UNC-74 may have two functions: the promotion of acetylcholine-gated ion channel formation independently of redox, and a redox-dependent function that is not essential and does not produce a visible phenotype when perturbed. For example, UNC-74/TMX3 could provide a redundant contribution to redox homeostasis in the ER.
The protein structure and sequence conservation of UNC-74/TMX3 homologues suggests that this family of thioredoxins has a similar function in all metazoans. Although mammalian nAChRs are insensitive to levmamisole, they do express a variety of nAChRs that are distinct with respect to subunit composition and activity. For example, mammalian capsaicin-sensitive and -insensitive nAChRs are known. Therefore, mammalian UNC-74/TMX3 are believed to distinguish between different nAChR subtypes and is required for the formation of nAChR. Hence, the addition of UNC-74/TMX3 to a cell is believed to allow or facilitate the formation of receptor subtypes that are refractory to heterologous expression.
Three lines of evidence suggest that the UNC-74 transmembrane spanning domain has a function other than just membrane anchoring. First, there is a high level of sequence conservation within this domain compared to other regions of the protein (Table 1). In general, transmembrane domains do not exhibit high sequence identity between species, but rather are composed of similar hydrophobic residues, which anchor the transmembrane spanning region in the membrane. Signal peptides are analogous to transmembrane spanning regions in that they span the membrane and identical sequence conservation is not required for signal peptide function. Comparison of the identity and similarity between UNC-74 and TMX3 (the human homologue) within different regions of the protein indicates that the transmembrane domain is considerably more conserved than the signal peptide. Second, there is a conserved (G/A)XXX(G/A) motif present in the transmembrane domain. This motif is common within helices of both transmembrane and soluble proteins and is involved in helix-helix interactions (Gerber et al., 2004; Senes et al., 2004). The presence of the small side groups at each end of this motif allows the helix to adopt a conformation ideal for hydrogen bonding between two helices. This is suggestive that the transmembrane region of UNC is able to interact with other proteins via helix-helix interactions within the membrane. Finally, there are two conserved proline residues present in the transmembrane domain, suggesting that this region is arranged in a specific conformation. Together, these three lines of evidence indicate that the transmembrane domain of this class of receptor chaperons, e.g., UNC-74 or TMX3, is functionally significant for a function other than just membrane anchorage.
Many mammalian nAChR subunits contain a motif that is necessary and sufficient for ER retention (Wang et al., 2002; Wang et al., 1996). This motif PL(F/Y)(F/Y)XXN (a ER retention motif) is frequently present at the amino terminal end of the first transmembrane spanning region, however it is not clear whether this motif is within the lipid bi-layer of the membrane or located on the luminal side of the membrane (Unwin, 2005). While the precise location of the motif is not currently known, it is believed to play a role in retaining subunits in the ER until they are assembled into functional receptors, e.g., pentamer nAChRs. For example, once subunits of the nAChR are assembled into pentamers, the motif is believed to become buried and the mature pentamer is trafficked to the plasma membrane. This provides a means of regulation that ensures trafficking of only assembled subunits. The affects of mutation within this motif on the formation of functional receptors has not been fully determined, but alanine scanning mutagenesis indicated that perturbation of the motif results in the surface localization of individually expressed subunits that are normally retained in the ER. This motif is found in some, but not all nAChR subunits, e.g., levamisole sensitive nAChR subunits of C. elegans (Table 2). For example, UNC-39 contains this motif, but UNC-29, LEV-1, and UNC-63 have amino acid substitutions that disrupt this motif. UNC-29, LEV-1 and UNC-63 all have a threonine residue in place of the proline at the first position of this motif. In addition, both UNC-29 and LEV-1 contain a hydrophobic residue in place of the terminal asparagine. Similar amino acid changes are also found in mammalian α5 and β3 subunits. The absence of this motif in some, but not all, subunits of nAChRs suggest that some subunits are kept in the ER prior to assembly via their own ER retention motif, while the remaining subunits may require other factors to keep them in the ER prior to assembly. In the absence of such a factor, subunit that require such a factor, are trafficked to the cell surface prior to proper assembly into the receptor. Thereby, reducing or eliminating the function of the receptor.
The present observations suggest that receptor chaperons, e.g., UNC-74 or TMX3, function as an exogenous ER retention factor that retains specific nAChR subunits in the ER. Thereby keeping subunits lacking the ER retention motif in the ER, and allowing assembly with other subunits kept in the ER via endogenous the ER retention motifs (
Without wishing to be bound by theory, UNC-74 is believed to keep these subunits in the ER through direct interaction between the UNC-74 transmembrane helix and transmembrane spanning regions of nAChR subunits. This may be accomplished two different ways. First, UNC-74 may remain in the ER interacting with unassembled nAChR subunits, thus keeping them in the ER until assembly. Alternatively, UNC-74 may shuttle between the ER and cis-Golgi, retrieving subunits that have escaped the ER. This model makes three readily testable predictions, one or more of which are tested. First, a functional UNC-74 transmembrane domain and an intact ER retention motif should be required for UNC-74 function. Second, subunits that lack the ER retention motif should not form, or have a reduced ability to form, dimers or higher order oligomers with other subunits in the absence of UNC-74. Finally, nAChR subunits that do not contain the ER retention motif should not be retained in the ER of unc-74 mutants and/or addition of this ER retention motif to subunits that do not have it should bypass the requirement for UNC-74 function. These models can be tested using the guidance of the present invention, in combination with established genetic and biochemical techniques.
Previously published reports on TMX3, the human homologues of UNC-74, were limited to characterization of predicted motifs in the protein and cellular localization, but did not mention a function for TMX3. Northern blot data indicated that TMX3 transcripts were very broadly, if not ubiquitously, expressed, but enriched in skeletal cardiac muscle. The enrichment in skeletal muscle is now consistent with the present invention, where TMX3 is believed to play a role in the formation of muscle type nAChR. Heterologous expression of mammalian muscle type nAChRs in Xenopus oocytes was used to test whether mammalian UNC-74 can increase the formation of Xenopus muscle type nAChR.
While no increase in native Xenopus nAChR expression, measured by two-electrode voltage clamp, was observed by the addition of the mouse homologue of UNC-74, the ubiquitous expression of TMX3 is consistent with Xenopus homologues of unc-74 being expressed in Xenopus oocytes, allowing the formation of vertebrate nAChRs. Therefore, the fact that expression of a mammalian UNC-74 homologue does not appear to increase production of native nAChRs in Xenopus oocytes is consistent with the proposed mechanism of action. However, expression of heterologous nAChR subunits (homomultimers and heteromultimeric forms) has been shown to be limited and expression of a complementary receptor chaperon, for example, a receptor chaperon derived from the same species as the nAChR subunit, may overcome the limited expression.
The model presented herein suggests that unc-74 homologues function in the promotion of nAChR subunits that do not contain the ER retention motif, including α7, α5 and β3 subunits. α7 subunits are notoriously difficult to express in certain cell lines. The low level of α7 homopentamer expression in these cell lines may be due to low-level expression or the complete lack of a complimentary UNC-74 homologue. Therefore, expression of a mammalian homologue in these cells may increase production of homopentamers. In addition, α5 subunits require the presence of another α subunit to be incorporated into pentamers. Interestingly, both UNC-63 and UNC-38 encode α-like subunits and are likely to be incorporated into the same channel. Therefore, the present data is believed to define a class of unc-74 homologues (e.g., orthologous) that have a similar function in other systems.
A model presented herein predicts that the UNC-74 transmembrane spanning region and ER retention motifs are necessary, and possibly sufficient, for unc-74 function. This prediction could be tested by generating a set of deletions and point mutations that disrupt the unc-74 transmembrane spanning region and ER retention motif. It is expected that expression of mutant constructs will fail to rescue the unc-74 mutant phenotype. Alternatively, unc-74 mutants could be rescued with just the luminal portion of UNC-74. This might indicate that unc-74 has a processing or folding role by acting on the extracellular region of nAChR subunits. If the carboxy terminal end of UNC-74 is found to be dispensable for function, then a noncomplementation screen could be done to isolate other alleles of unc-74 that may identify functionally important regions of the protein. Because levamisole resistance is a robust phenotype it should be easy to obtain a large panel of alleles, some of which may be due to point mutations in essential regions of the protein.
The model presented herein also hypothesizes that pentamers are not formed in unc-74 mutants because subunits that make up the pentamer quickly leave the ER prior to assembly. Biochemical techniques could be used to see whether pentamers are formed. In addition, co-immunoprecipitation could be used to determine whether subunits that contain the motif are in a complex with other subunits. An alternative to the model is that UNC-74 has a role in trafficking of assembled pentamers out of the ER. Immunoprecipiation of pentamers in unc-74 mutants would provide support for this alternative model.
The localization of UNC-38:GFP in an unc-74 mutant background established that UNC-38 is retained in the ER. However, the localization of other levamisole sensitive nAChR subunits that do not contain the motif has not been determined. The model predicts that subunits that do not contain the ER retention motif will not be localized to the ER in unc-74 mutants. This prediction could be tested by examining the cellular localization of GFP fusion proteins. The model presented herein hypothesizes that the direct target of UNC-74 is subunits without the ER retention motif. If this is the case, then restoration of the motif to UNC-29, UNC-63 and LEV-1 should bypass the requirement for unc-74. This proposition can be tested by replacing the motif of UNC-29, UNC-63 and LEV-1 with the intact motif of UNC-38 and assaying whether expression of these subunits rescue unc-74 mutants. If the model is correct, restoration of the motif will keep these subunits in the ER, allowing them to assemble in the absence of unc-74. It is possible that ER retention motif has functional role and that perturbation of the ER retention motif disrupts function, but not formation of nAChRs. If this were the case, then the phenotype of ER retention motif mutants would be identical to unc-74 mutants making interpretation of a negative result impossible. To get around this issue, GFP tagged subunits could be used and examined for proper localization in the absence of unc-74.
One feature of UNC-74 is the presence of homologues in other systems. All metazoans in which it has been found contain nAChRs and those organisms in which it has been shown to be absent (i.e., fungi) do not have nAChRs. UNC-74 may have a similar role in the formation of mammalian nAChRs that it has in C. elegans. To date, there is no evidence other than protein conservation to indicate that homologues of unc-74 have a role in nAChR formation. Expression of Munc-74 (the mouse homologue) in body wall muscles of unc-74 mutants failed to rescue the unc-74 mutant phenotype (data not shown). This could be due to lack of interaction between Munc-74 and worm nAChRs subunits or other unidentified proteins that require unc-74 function, perhaps unc-50. Previously published reports on TMX3, the human homologues of UNC-74, were limited to characterization and cellular localization and did not mention a function for TMX3. Northern blot data indicated that TMX3 transcripts were very broadly, if not ubiquitously, expressed but enriched in skeletal cardiac muscle. The enrichment in skeletal muscle suggests that TMX3 may have a role in the formation of muscle type nAChR. Heterologous expression of mammalian muscle type nAChRs in Xenopus oocytes was used to test whether mammalian UNC-74 has a role in the formation of mammalian muscle type nAChR. No increase in nAChR expression, measured by two electrode voltage clamp, was observed by the addition of Munc-74. The ubiquitous nature of TMX3 expression suggests that homologues of unc-74 may be expressed in Xenopus oocytes, allowing the formation of vertebrate nAChRs.
Reduction of expression of a receptor chaperon may be used to treat diseases. For example, in humans, the epidermal growth factor receptor (Egfr) belongs to a family of ErbB receptors. Hyperactive receptor signaling of these receptors has been linked to human cancers, including, but not limited to, brain, breast, lung, colon, and epidermis. For example, ErbB1 is frequently amplified in about 85% of squamous cell carcinomas and ErbB2 is frequently amplified in breast, stomach, and ovarian cancer. Hence, reducing the number of ErbB receptors, hyperactive receptors, may provide a method of treating such diseases. For example, RNAi may be used to reduce the expression of a receptor chaperon, or compounds that inhibit a receptor chaperon, may be used to reduce the number of receptors that are trafficked to the cell surface, thereby treating the disease.
As used herein, “receptor chaperon” means a protein or polypeptide that functions in the formation of a multimeric receptor on the surface of a cell, the protein or polypeptide may function during subunit processing and folding, subunit assembly, or trafficking of mature receptors. The receptors are preferably pentamers. As will be recognized by a person of ordinary skill in the art, the current use of the phrase “receptor chaperon” is not be limited to the activities of known chaperone proteins, however, the reader will also recognize common attributes between the present class of receptor chaperons and more “classical” chaperones. Thus, the term is not intended to limit the invention, but rather to provide a convenient descriptive term.
Any of the methods for studying ligand-gated ion channels known in the art, for example, see U.S. Patent Publication 20040132187 to Groppi et al. (Jul. 8, 2004), may be used in the invention. For the sake of brevity, descriptions of these methodologies, which include such things as RNAi, mutagenesis, cell culture, preparation an effective amount of a pharmaceutical, and the like, are omitted and the reader being referred to alternative descriptions, including, but not limited to, U.S. Patent Publications 20040224910, 20030153519, 20030175772, 20040203132, 20030138911, 20040185468, 20040248189, 20050033522, 20040132187, Ausubel, F. M. et al. (eds) (1997) C
As used herein, “substantially pure” means a preparation which is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
As used herein, an “isolated nucleic acid” means a nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example, a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequences.
As used herein, a “substantially identical” polypeptide sequence means an amino acid sequence which differs from a reference sequence only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (for example, valine for glycine, arginine for lysine, etc.) or by one or more nonconservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the polypeptide (assayed, for example, as described herein). Preferably, such a sequence is at least 73%, more preferably at least 85%, and most preferably at least 95% substantially identical at the amino acid level to the sequence used for comparison. The invention encompasses polypeptide sequences being 73-99% substantially identical to the amino acid sequences set forth in any one of SEQ ID NO:9 through SEQ ID NO:27. Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis., 53705, or BLAST software available from the National Library of Medicine). Examples of useful software include the programs, PILE-UP™ and PRETTYBOX™. Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
As used herein, “an expression vector” means that the nucleic acid molecule of interest is operably linked to a sequence which directs transcription and/or translation of the nucleic acid molecule.
As used herein, “peptide,” “polypeptide” and “protein” are used interchangeably, no distinction, based on length, is intended between a peptide, a polypeptide or a protein.
Swimming assay. Well bottoms of a 96-well microtiter plate were layered with 50 μl of 2% agarose in M9. Individual young adult worms were suspended in 50 μl M9 in single wells. After a >5 minute recovery period, the number of complete body bends were counted for a minimum of 60 seconds. Each worm was assayed a minimum of three times and body bend frequency per worm is the average of all assays. Data presented is the mean +/− SEM of n=number of worms.
Pharmacological resistance assays. Plates were equilibrated to room temperature, weighed, and equal volumes of different concentrations of levamisole or aldicarb were added to make plates containing specific concentrations of drug. Once plates dried, they were seeded with a drop of OP50 and incubated overnight at room temperature. A minimum of 15 young adult animals (levamisole) or L4 animals (aldicarb) were placed on drug plates and maintained for two (levamisole) or eight (aldicarb) hours. The percentage of animals on each plate that were paralyzed in response to mild stimulation was determined. Data presented represents the mean +/− SEM of n=number of plates.
Locomotion assay. Unseeded plates were stained with 0.1% Bromophenol blue and seeded with a thin lawn of HB101. Four young adult animals of the same genotype were placed on plates and allowed to recover for 10 minutes. Digital movies were obtained of the entire plate, and the velocity each animal moved per one second frame was determined using Image J Worm Tracker 6. Overall mean velocity was calculated for each animal by averaging over a minimum of 500 frames. Data presented represents the mean +/− SEM for n=number of animals.
Sequencing unc-74 mutant alleles. The molecular lesion of unc-74 alleles was determined by genomic amplification of the unc-74 locus from different strains using oligonucleotides oDW05 (5′-CAGATCACATAATAAGCCCGGAACC-3′; SEQ ID NO:1, of the incorporated herein Sequence Listing) and oDW06 (5′ CCATTCCTTATCGACGAGCCTTTGG-3′; SEQ ID NO:2). For each allele sequenced, the entire 3491 bp amplicon was directly sequenced at the University of Utah Sequencing Facility.
pJL29[UNC-38::GFP] Generated by Jean-Louis Bessereau. Contains GFP in the intracellular loop between TM3 and TM4 of UNC-38 (Jean-Louis Bessereau, personal communication).
mCTRAM[TRAM::CFP] Gift from M. Rolls and T. Rappaport. (Rolls et al., 2002).
pDW14[Punc-74:GFP] A 924 bp BamHI to NsiI fragment of the unc-74 promoter region cloned into the BamHI—PstI sites of pPD95.69.
pDW61[unc-74 cDNA] A 1364 amplicion, generated with oDW73 (5′-TCACTCGAGCTGGGTCACTCAGCTTTTTCGT-3′; SEQ ID NO:3) and oDW86 (5′-CAGGCTATGCAAAAATATTTCTTATTACC-3′; SEQ ID NO:4) using a cDNA clone (Vidal ORFeome clone) as template, blunt cloned into pCR-Blunt.
pDW80[Pmyo-3:unc-74cDNA] A 1454 by fragment generated by digestion of pDW61[unc-74cDNA] with XbaI and Acc65I, inserted into the NheI—Acc65I sites of the myo-3 promoter vector pPD95.62, from Fire Vector Kit.
pDW84[Pmyo-3:unc-74cDNA::GFP] A 945 by fragment from XmaI digestion of pPD 102.33 (GFP exon protein fusion, Fire Vector Kit), inserted into the BspEI site of pDW80[Pmyo-3:unc-74cDNA::GFP].
pDW88[Prab-3:unc-74cDNA] A 1267 by fragment generated ZraI—NotI digest of Prab-3:pGEMT (M. Hammarlund and K. Schuske) subclone, inserted in the PmlI—NotI sites of pDW80[Pmyo-3:unc-74cDNA]. This replaced the myo-3 promoter with the rab-3 promoter to drive the expression of the unc-74 cDNA.
pUNC-49B:GFP[UNC-49:GFP] Generated by Bruce Bamber. Contains the coding region of GFP between TM3 and TM4 of UNC-49B (B. Bamber, personal communication).
UNC-74 Thioredoxin Active Site Mutations
pDW85[Pmyo-3:unc-74(SAHC)]
pDW86[Pmyo-3:unc-74(CAHS)]
pDW87[Pmyo-3:unc-74(SAHS)]
A mutated thioredoxin active site amplicon was generated by PCR amplification using oDW43[SAHC] (5′-TACGCTCCATGGAGTGCTCACTGCAAGCGC-3′; SEQ ID NO:5), oDW44[CAHS] (5′-TACGCTCCATGGTGTGCTCACAGCAAGCGC-3′; SEQ ID NO:6), or oDW45[SAHS] (5′-TACGCTCCATGGAGTGCTCACAGCAAGCGC-3′; SEQ ID NO:6) as the upstream primer and oDW46 (5′-TTCACCGTCATCACCGAAACGCGCGAGG-3′; SEQ ID NO:7) using pDW80[Pmyo-3:unc-74cDNA] as template. The amplicons were cut with NcoI and ClaI and inserted into the NcoI—ClaI sites of pDW80[Pmyo-3:unc-74cDNA]. Verification of active site mutations was determined by sequencing
Immunocytochemistry and Confocal Microscopy.
Antibodies against SNB-1 were a gift from M. Nonet (Nonet et al., 1998). For immunocytochemistry worms were fixed in cold 2% paraformaldehyde and then cut into ˜0.1 mm transverse sections. Sections were washed in PBS, then decorated with primary anti-SNB-1 as previously described (Nonet et al., 1997). After washing, worm sections were treated with secondary goat anti-rabbit conjugated Alexa 568 antibodies (Molecular Probes). Worm sections were mounted on agarose pads and examined using a Bio-Rad Radiance Laser 2000 laser-scanning confocal microscope.
Intact double transgenic animals expressing UNC-74:GFP and TRAM:CFP were mounted on agarose pads and immobilized in 2% phenoxypropanol. Images were collected on a Zeiss Laser Scanning Microscope 5 PASCAL; GFP was excited using a 488 nm laser and emissions collected from 505-600 nm and CFP was excited at 405 nm and emissions collected from 420-480 nm. Transgenic animals with only one transgene were examined to ensure no bleed through of one fluorescent protein in the other channel.
Electrophysiology
Electrophysiology was performed as previously described (Richmond and Jorgensen, 1999a).
unc-74 Mutants Lack Functional Levamisole Sensitive nAChRs
Mutations in unc-74 cause behavioral and pharmacological phenotypes that are characteristic of defects in cholinergic neurotransmission mediated by the levamisole sensitive nAChR (
To directly examine levamisole sensitive nAChR function, electrophysiological analysis was performed on wild type and unc-74 mutant animals (
nAChR subunits are retained in the ER.
The lack of functional levamisole sensitive nAChRs on the muscle cell surface could be due to a block in nAChR formation in unc-74 mutant animals. To examine this possibility further, the cellular localization of UNC-38, an α subunit of the levamisole sensitive nAChR, was determined (
The unc-74 locus encodes a product homologous to TMX3
To address the question of how unc-74 is promoting formation of nAChRs, the unc-74 locus was cloned using Mos1 mediated mutagenesis (WO 00/73510; Bessereau et al., 2001; Williams et al., 2005). The F2 progeny of mutagenized animals were screened for animals with cholinergic neurotransmission defects using the acetylcholinesterase inhibitor aldicarb (Miller et al., 1996; Nguyen et al., 1995). From this screen one aldicarb resistant mutant, ox167, was isolated that was also uncoordinated and resistant to levamisole. Complementation testing demonstrated that ox167 failed to complement other alleles of unc-74, suggesting that ox167 is an allele of unc-74. A single Mos1 insertion in the fifth exon of the predicted gene ZK973.11 was tightly linked to the uncoordinated and drug resistance of unc-74(ox167). Germ line transformation rescue with a polymerase chain reaction (PCR) derived genomic fragment containing only the ZK973.11 open reading frame rescued the uncoordinated and drug resistance of ox167, as well as other alleles of unc-74 (data not shown). Finally the molecular lesions of many unc-74 alleles were determined by sequencing ZK973.11 obtained by PCR amplification using mutant genomic DNA as a template. Each mutant allele was due to a sequence change that is expected to disrupt UNC-74 protein function (
Reverse-transcriptase PCR analysis and the sequence of expressed sequence tags were used to validate the predicted unc-74 gene structure (Reboul et al., 2001). The unc-74 coding region is 1344 by long and produces a mature protein product of 423 amino acids after cleavage of a 24 residue signal peptide. The amino terminal fourth of the protein is composed of a single thioredoxin domain. Thioredoxins and thioredoxin domains are prevalent in both prokaryotes and eukaryotes, where the primary biochemical activity is the formation and cleavage of disulfide bonds. This activity is dependent on a conserved C-X-Y-C active site that can be reversibly reduced or oxidized. The central UNC-74 region, comprising the majority of the protein, does not contain any identified functional domains, although there are two putative glycosylation sites. The carboxy terminal end contains a hydrophobic stretch that is predicted to be a transmembrane spanning region and a di-lysine like ER retention signal that is a hallmark of ER localized transmembrane protein. (Hardt and Bause, 2002). Based on these structural features, UNC-74 is believed to be a type I transmembrane protein with a thioredoxin domain, central domain, and to be retained in the ER lumen.
Thioredoxin domains are found in prokaryotes and eukaryotes, however, UNC-74 homologues make up a unique class of thioredoxin domain containing proteins. The central domain shares significant homology only with other proteins in this class. The central domain is not found in other thioredoxin domain containing proteins, for example, protein disulfide isomerases. UNC-74 homologues are predicted to have a signal peptide and transmembrane spanning region, as well as di-lysine ER retention signal. Homologues of UNC-74 have been found in other metazoans, but are conspicuously absent in fungi, which do not have a nervous system (no nAChRs). In addition to general protein organization and structural feature conservation, members of this unique class contain regions of sequence conservation identity outside of the thioredoxin domain. Intriguingly, the predicted transmembrane region contains significant sequence homology with polar resides, a conserved G-X-X-X-G motif, and multiple proline residues (Gerber et al., 2004; Niimura et al., 2005; Senes et al., 2004). Thus, the transmembrane region of UNC-74 is likely to have a function beyond just crossing the membrane. Recently a human homologue of UNC-74, called TMX3, was characterized and shown to be glycosylated and localized to the ER (Haugstetter et al., 2005). However, the cellular function of TMX3 was not established.
UNC-74 Functions in the ER of Muscle Cells.
Genes encoding subunits of the levamisole sensitive acetylcholine gated ion channel are expressed in both neurons and muscle cells (Culetto et al., 2004). However, expression of UNC-29 in muscle cells, expressed under the control of the myo-3 promoter, was sufficient to rescue unc-29 locomotion and levamisole resistance phenotypes (Fleming et al., 1997). This indicates that the levamisole resistance and uncoordinated phenotype is due to the lack of levamisole sensitive nAChRs in muscle cells. As shown above, UNC-38:GFP is retained in the ER of both neurons and muscle. To determine whether UNC-74 acts cell autonomously to promote formation of levamisole sensitive acetylcholine gated ion channels, the expression pattern of GFP under control of the unc-74 promoter was determined. In transgenic animals expressing this fusion protein, GFP signal was seen in neurons as well as body wall and head muscle cells (data not shown). This indicates that unc-74 is expressed in the same tissue types in which levamisole sensitive nAChR subunits are expressed and function. In addition, transgenes that express unc-74 in specific tissues were tested for phenotypic rescue (
UNC-74 is localized to the ER
Analysis of UNC-74 protein sequence predicts that the mature protein will be localized to the ER. This prediction was tested by examination of the sub-cellular localization of a UNC-74:GFP fusion protein by confocal microscopy. The coding region of GFP was inserted in-frame into the carboxy terminal end of UNC-74, between the transmembrane spanning region and the putative ER localization signal. Expression of this transgene was able to rescue the unc-74 locomotion and levamisole resistance phenotypes (data not shown). In animals expressing UNC-74:GFP, signal was concentrated around the nucleus and diffuse throughout the muscle cell (
UNC-74 Functions Independently of Redox Chemistry
A straightforward model for the mechanism by which UNC-74 promotes formation of nAChRs is that the thioredoxin domain of UNC-74 catalyzes formation of the Cys-loop on nAChRs subunits. Thus, the lack of unc-74 may be affecting nAChR formation by preventing formation of the disulfide bonds in a Cys-loop. This model would be consistent with the UNC-74 tissue and cellular localization data, which places the thioredoxin domain in the ER lumen, the site of Cys-loop formation. To test this model, unc-74 transgenes containing mutations in the thioredoxin active site were tested for in vivo function. Site-directed mutagenesis was used to mutate one or both of the unc-74 active site cysteine residues to serines. These constructs were introduced into unc-74 mutant animals and transgenic strains were assayed for rescue (
unc-74 is Specific for the Levamisole Sensitive nAChR
In addition to the levamisole sensitive nAChR, C. elegans expresses other Cys-loop channels. UNC-49 encodes a GABAA receptor that is expressed in body wall muscles and EXP-1 is a cationic GABA gated channel expressed in enteric muscle and is required for the expulsion step of the defecation motor program (Bamber et al., 1999; Beg and Jorgensen, 2003; Richmond and Jorgensen, 1999a). In addition, the acr-16 locus encodes a Cys-loop, levamisole insensitive, nAChR (M. Francis et al., in press). Therefore, to determine if UNC-74 has a similar function in the formation of these related Cys-loop ligand gated ion channels, additional experiments were conducted. Five lines of evidence demonstrate that UNC-74 is specific for the levamisole sensitive nAChR (
TMX3 Knockout in Human Cells
To examine the function of TMX3 on nAChR function in human cells, electrophysiological analysis is performed on differentiated PC12 cells transfected with an RNAi construct that inhibits expression of TMX3 (Meyer et al. (1998) Analysis of 3-(4-Hydroxy, 2-Methoxybenzylidene)Anabaseine Selectivity and Activity at Human and Rat Alpha-7 Nicotinic Receptors, J. Pharmacol. Exp. Ther. 287:918-925). After initial northern blot or western blot analysis to confirm the expression of TMX3, PC12 are exposed to either an RNAi construct specific to TMX3 or a negative control sequence. The cells are then voltage clamped and the amount of current elicited in response to various nAChR agonists and/or antagonists is measured. Pulses of a nAChR agonist or antagonist administered to the PC12 cells having the control RNA are found to produce an agonist dependent current or an antagonist dependent reduction in current. In contrast, the agonist induced current, or the antagonist dependent reduction, is decreased or abolished in PC12 cells where expression of TMX3 is reduced by the RNAi construct. These results will show that TMX3 functions in the production of functional nAChRs in PC12 cells.
Screening Compounds for Inhibition of a Receptor Chaperon
Differential expression of nAChR subunit genes from the AChR superfamily produces distinct receptor subtypes. Since each AChR subtype has a specific subunit composition, each subunit must contain some information leading to proper assembly. The neuronal AChR subunits α3 and α7 are presumably members of two different AChR subtypes. These subunits have different assembly behavior when expressed in heterologous expression systems: alpha 7 subunits are able to produce homomeric AChRs, whereas alpha 3 subunits require an additional factor(s) for functional expression of AChRs (Garcia-Guzman et al. (1994) Role of two acetylcholine receptor subunit domains in homomer formation and intersubunit recognition, as revealed by alpha 3 and alpha 7 subunit chimeras, Biochemistry 33(50):15198-203). This provides the ability to dissect the requirement for subunit interactions during AChR formation. Analysis of α7/α/3 chimeric constructs identified two regions essential to assembly and intersubunit recognition: an N-terminal extracellular region, a second domain within a region comprising the first putative transmembrane segment, M1, and the cytoplasmic loop coupling it to the pore-forming segment, M2, involved in the subsequent interaction and stabilization of the oligomeric complex (Id.).
Xenopus laevis oocytes are extracted from anesthetized females and placed in ND-96 medium (mM: NaCl 96, KCl 2, MgCl2 1, CaCl2·H2O 1.8, HEPES 5, Na-pyruvate 2.5, theophylline 0.5, and gentamicin, adjusted to pH 7.5). The oocyte clusters are incubated in 0.2% collagenase (type IA, Sigma-Aldrich) in ND-96 medium for defolliculation. Oocytes are agitated at 18.5° C. for 4 hours and then rinsed with Barth's medium (mM: NaCl88, KCl 1, NaHCO3 2.4, HEPES 15, pH 7.6). The oocytes are then left to recover for 24 h in oocyte medium, before injection of cDNA or RNA encoding TMX3. Appropriate amounts and ratios of cDNA or RNA are then injected into individual oocytes. The oocytes are then incubated at about 17° C. for about 1-2 days in ND-96 medium prior to injection of cDNA or RNA encoding nAChR subunits that are identified as requiring TMX3 for proper assembly. The oocytes may be precultured in the presence of potential inhibitors or the potential inhibitors may be added to the media after injection of the nucleic acid sequences encoding the nAChR subunits. After 1 to 2 days of incubation with the potential inhibitors, electrophysiology current recordings are made using whole oocytes. Recording electrodes preferably contain atropine to prevent muscarinic receptor stimulation and barium in place of calcium to avoid current amplification by calcium activated chloride currents (Coates, K. M. and Flood, P. (2001) Ketamine and its Preservative, Benzethonium Chloride, both Inhibit Human Recombinant 60 7 and α4β2 Neuronal Nicotinic Acetylcholine Receptors in Xenopus oocytes, Br. J. Pharmacol. 137:871-879). ACh is applied at a flow rate of approximately 4 ml min−1 in about two second bursts. Oocytes, and the respective inhibitor, showing significant reductions in ACh triggered currents are identified.
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All references, including publications, sequence identifiers, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
While this invention has been described in certain embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.
This application claims the benefit of the filing date of U.S. Provisional Patent Application Ser. No. 60/678,121, filed May 4, 2005, for “ACETYLYCHOLINE GATED ION CHANNEL CHAPERONS AND METHODS OF USING THE SAME”, the contents of which are incorporated by this reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2006/017430 | 5/4/2006 | WO | 00 | 3/4/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60678121 | May 2005 | US |
