WO1998038210A2 - Methodes et compositions de modulation de la secretion vesiculaire - Google Patents

Methodes et compositions de modulation de la secretion vesiculaire

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Publication number
WO1998038210A2
WO1998038210A2 PCT/US1998/003789 US9803789W WO9838210A2 WO 1998038210 A2 WO1998038210 A2 WO 1998038210A2 US 9803789 W US9803789 W US 9803789W WO 9838210 A2 WO9838210 A2 WO 9838210A2
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PCT/US1998/003789
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WO1998038210A3 (fr
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Andrew J. Bean
Richard H. Scheller
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to AU64407/98A priority Critical patent/AU6440798A/en
Publication of WO1998038210A2 publication Critical patent/WO1998038210A2/fr
Publication of WO1998038210A3 publication Critical patent/WO1998038210A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods and compositions useful for identifying compounds capable of modulating vesicular release.
  • the invention relates to (i) the Hrs-2 ATPase, and (ii) methods of identifying compounds capable of modulating vesicular release employing the Hrs- 2 ATPase.
  • Signal transmission between nerve cells typically involves the release of neurotransmitter from a presynaptic cell onto a postsynaptic cell.
  • the neurotransmitter in the presynaptic cell is contained in synaptic vesicles positioned above the release sites at the presynaptic membrane (active zones).
  • a release signal typically a local influx of calcium due to a depolarization of the presynaptic terminal
  • the vesicles undergo a series of mobilization steps culminating in the fusion of the vesicles with the presynaptic terminal membrane, and a dumping of vesicle contents into the synaptic cleft.
  • the neurotransmitter molecules diffuse across the synaptic cleft and bind to corresponding receptors in the postsynaptic membrane to communicate the appropriate signal (typically a depolarization or hyperpolarization of the postsynaptic membrane) to the postsynaptic cell.
  • Much of the neurotransmitter in the synapse is subsequently re-absorbed by the presynaptic cell through specific transmitter uptake mechanisms.
  • CNS central nervous system
  • PNS peripheral nervous system
  • phenoxybenzamine block specific post-synaptic receptors
  • others such as clonidine and diethylamide, stimulate such receptors
  • still others e.g., desipramine, imipramine
  • neurotransmitter synthesis e.g., ⁇ -Methyltyrosine, p-Chlorophenylalanine
  • degradation e.g., monoamine oxidase inhibitors, iproniazid, pargyline.
  • the present invention provides a tool for the screening and identification of drugs capable of affecting secretory processes, such as neurotransmitter release at the active zones of presynaptic membranes.
  • the present invention includes a substantially purified Hrs-2 polypeptide, such as a polypeptide encoded by a polynucleotide sequence derived from the genome of a mammal (e.g. , a rat or a human).
  • the polypeptide contains a region of at least 6, preferably at least 8, more preferably at least 10 or more consecutive amino acids corresponding to a region contained in SEQ ID NO:4 or SEQ ID NO:6.
  • An rat Hrs-2 polypeptide contains the sequence represented as SEQ ID NO:4.
  • An exemplary human Hrs-2 polypeptide contains the sequence represented as SEQ ID NO:6.
  • the invention includes a substantially purified Hrs-2 polynucleotide, such as a polynucleotide having a sequence derived from the genome of a mammal (e.g. , a rat or a human).
  • a substantially purified Hrs-2 polynucleotide such as a polynucleotide having a sequence derived from the genome of a mammal (e.g. , a rat or a human).
  • the polynucleotide contains a region of at least 12, preferably at least 16, more preferably at least 20 or more consecutive nucleotides corresponding to a region contained in
  • SEQ ID NO: 3 SEQ ID NO:5.
  • An exemplary rat Hrs-2 polynucleotide contains the sequence represented as SEQ ID NO: 3.
  • An exemplary human Hrs-2 polynucleotide contains the sequence represented as SEQ ID NO:5.
  • the invention also includes a method of identifying a compound capable of modulating (e.g. , potentiating or inhibiting) calcium-regulated secretion of secretory vesicles (e.g. , release of neurotransmitter-containing synaptic vesicles).
  • the method includes the steps of: (i) contacting a SNAP-25 polypeptide (e.g. , a SNAP-25 polypeptide having the sequence of SEQ ID NO: 12) with an Hrs-2 polypeptide (e.g.
  • an Hrs-2 polypeptide having a sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:6) in the presence and absence of a test compound, (ii) measuring the effect of the test compound on the extent of binding between the SNAP-25 and Hrs-2 polypeptides, and (iii) identifying the compound as effective if its measured effect on the extent of binding is above a threshold level.
  • the test compound is capable of inhibiting the binding between the SNAP-25 and Hrs-2 polypeptides.
  • the test compound is capable of potentiating the binding between the SNAP-25 and Hrs-2 polypeptides.
  • the contacting may include contacting an Hrs-2 that is immobilized on a solid support, or it may include contacting a SNAP-25 polypeptide that is immobilized on a solid support.
  • the method may be used, for example, to assay small molecule test compounds contained in a small molecule combinatorial library, peptide test compounds (e.g. , from a combinatorial peptide library), a library of toxins, such as conotoxins, and the like.
  • Fig. 1 is a schematic illustration of the domain structure of Hrs-2.
  • Fig. 2 A shows Hrs-2 binding to GST-SNAP-25 immobilized on glutathione-agarose beads and inhibition of the binding by Zn 2+ .
  • Fig. 2A is plot of the binding as a function of Hrs-2 concentration.
  • Figs. 2B shows binding of Hrs-2 to immobilized GST-SNAP-25 in the absence and presence of increasing concentrations of free calcium.
  • Fig. 2B is plot of the binding as a function of free calcium concentration.
  • Fig. 3A is the raw data and plot showing that recombinant Hrs-2 elutes from a gel filtration column in two peaks.
  • Fig. 3B is a plot showing the NTPase activity of monomeric Hrs-2.
  • Fig. 3C is a plot showing the NTPase activity of oligomeric Hrs-2.
  • Fig. 3D is a plot showing the inhibition of the ATPase activity of monomeric Hrs-2 by ATP ⁇ S.
  • Fig. 3E is a plot showing the inhibition of the ATPase activity of monomeric Hrs-2 by ATP ⁇ S.
  • Figs. 4A-D are computer-generated image of immunohistochemical localization of Hrs-2 in cultured hippocampal neurons.
  • Fig. 5 is a plot showing dose-dependent and saturable inhibition of the release of 3 H- norepinephrine (NE) from permeabilized PC 12 cells by Hrs-2.
  • Figures 6 A and 6B show fragmentary plan views of control (Fig. 6 A) and experimental (Fig. 6B) multiwell plates used in a biochemical binding assay to identify compounds affecting binding of Hrs-2 to SNAP-25.
  • substantially purified refers to the at least partial purification of a selected polynucleotide, polypeptide, antibody or related compound away from unrelated or contaminating components (e.g. , serum cells, other proteins).
  • first polynucleotide fragment or polypeptide fragment When a first polynucleotide fragment or polypeptide fragment is said to "correspond to" a second polynucleotide fragment or polypeptide fragment, respectively, it means that the fragments or regions are essentially co-extensive with one another when the sequences representing the fragments are aligned using a sequence alignment program, such as "MACVECTOR” (IBI, New Haven, CT). "Corresponding" polynucleotide or polypeptide fragments typically contain a similar, if not identical, number of residues. It will be understood, however, that corresponding fragments may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences.
  • significant when used with reference to "significantly different”, “significantly inhibits” or “significantly stimulates”, refers to a difference in a quantifiable parameter between the two groups being compared that is statistically-significant using standard statistical tests.
  • the degree of binding in a protein binding assay may be quantified using standard methods, and the degree of binding under different conditions can be compared for statistically-significant differences.
  • An antibody or antibody composition (e.g. , polyclonal antibodies) is "specifically immunoreactive" with a selected protein when the antibody or antibody composition is not reactive with antigens typically present in normal sera, not exposed to the selected protein.
  • a polypeptide is "characterized by" a selected sequence when the polypeptide contains a sequence that is identical or substantially identical to the selected sequence.
  • SNAP-25 refers to synaptosomal-associated protein of 25 kDa (Pevsner, et al., 1994; Scheller, 1995).
  • ⁇ -SNAP refers to soluble NSF attachment protein (Pevsner, et al., 1994; Scheller, 1995).
  • NSF refers to N-ethylmaleimide-sensitive factor (Pevsner, et al., 1994; Scheller, 1995).
  • VAMP refers to vesicle-associated membrane protein (Pevsner, et al., 1994; Scheller, 1995).
  • VAMP refers to vesicle-associated membrane protein (Pevsner, et al., 1994; Scheller, 1995;
  • the present invention relates to methods and compositions effective to modulate neurotransmitter release.
  • the invention is based on the discovery, described herein, of Hrs-2 - a novel ATP-preferring nucleotidase that associates with SNAP-25, a component of the protein polypeptides thought to underlie vesicle docking and fusion.
  • Hrs-2 - a novel ATP-preferring nucleotidase that associates with SNAP-25, a component of the protein polypeptides thought to underlie vesicle docking and fusion.
  • Experiments performed in support of the invention demonstrate, among other things, that (i) the binding of recombinant Hrs-2 protein to SNAP- 25 is reduced by addition of calcium in the concentration range that supports neurotransmission (Smith and Augustine, 1988), and (ii) Hrs-2 dose-dependently inhibits calcium-triggered 3 H-NE release from permeabilized PC12 cells.
  • the results support a role for Hrs
  • eucaryotic cells possess transport vesicles, which continuously carry new plasma membrane components to the plasma membrane from the Golgi apparatus and incorporate such components into the plasma membrane through a process termed vesicle fusion.
  • Eucaryotic cells also typically secrete various types of molecules through a process termed exocytosis, whereby the contents of the transport vesicles are released outside of the cell during the vesicle fusion process.
  • exocytosis There are two basic types of exocytosis — constitutive and regulated.
  • Constitutive exocytosis refers to the process whereby the proteins and/or other components destined for secretion are packaged in the Golgi apparatus into transport vesicles, which are then promptly transported to and fused with the plasma membrane.
  • Regulated exocytosis refers to the type of exocytosis where the vesicles are stored in the cell and released only when triggered by some extrinsic event, such as the influx of calcium (as happens, e.g., at a neural synapse in the case of neurotransmitter release), or the binding of a ligand to a receptor (as happens, e.g., in the case of histamine release from mast cells).
  • some extrinsic event such as the influx of calcium (as happens, e.g., at a neural synapse in the case of neurotransmitter release), or the binding of a ligand to a receptor (as happens, e.g., in the case of histamine release from mast cells).
  • An exemplary system in which to study regulated exocytosis is chemical synaptic transmission in neurons.
  • Neurons communicate with target cells through regulated exocytosis of chemical messengers (reviews: Scheller, 1995; S ⁇ dhof, 1995).
  • neurotransmitter is packed into synaptic vesicles which are targeted to active zones at the nerve terminal plasma membrane.
  • Action potential-induced elevation of intracellular calcium increases the probability of fusion between synaptic vesicles and the plasma membrane lipid bilayer, resulting in the release of neurotransmitter into the synaptic cleft.
  • a vesicle to the appropriate acceptor membrane occurs through the formation of a 7S polypeptide, which is comprised of the two vesicle proteins, VAMP and synaptotagmin, along with the two target membrane proteins, SNAP-25 and syntaxin (Bennett and Scheller, 1994; Scheller, 1995; Sollner, et al. , 1993a, 1993b; Chapman, et al. , 1994; Sudhof, 1995; Pevsner, et al , 1994; Rothman, 1994; Oyler, et al , 1989).
  • the vesicle fusion process is then thought to progress with the addition of ⁇ SNAP to the 7S polypeptide, followed by the binding of NSF to form the 20S particle, consisting of syntaxin, VAMP, SNAP-25, ⁇ -SNAP and NSF.
  • NSF a group consisting of syntaxin, VAMP, SNAP-25, ⁇ -SNAP and NSF.
  • NSF non- hydrolyzable forms of ATP must be bound to NSF.
  • the 20S polypeptide Upon ATP hydrolysis by NSF, the 20S polypeptide dissociates into its component subunits and the vesicle fuses with the target membrane.
  • Hrs-2 a novel protein that interacts with SNAP-25 has been discovered.
  • the binding of Hrs-2 to SNAP-25 is inhibited by calcium in the physiological concentration range that supports synaptic transmission.
  • Hrs-2 binds and hydrolyzes nucleoside triphosphates with kinetics suggesting that ATP is the physiological substrate for this enzyme.
  • Hrs-2 is expressed broadly throughout the brain, is present in nerve terminals, and recombinant Hrs-2 inhibits calcium-triggered 3 H-norepinephrine release from permeabilized PC 12 cells.
  • Hrs-2 polynucleotide sequences encoding a novel protein termed Hrs-2.
  • the invention includes, in one aspect, such substantially isolated or substantially purified Hrs-2 polynucleotide sequences.
  • Hrs-2 sequences can be isolated, for example, from cDNA mammalian cDNA libraries, as described in Example 1.
  • Exemplary Hrs-2 sequences include the rat sequence (SEQ ID NO:3 and a partial human sequence (SEQ ID NO:5). The human sequence is missing the portion encoding the amino terminal 149 amino acids of the protein.
  • Full-length sequence information can be obtained using standard methods (e.g., Sambrook, et al. , 1989; Ausubel, et al. , 1988).
  • the sequences provided herein may be used to design forward and reverse polymerase chain reaction (PCR) primers, which can be employed to amplify longer cDNA fragments (Mullis, 1987; Mullis, et al. , 1987) for library screening or "rapid amplification of cDNA ends" (RACE) PCR (Chenchik, et al., 1995).
  • PCR polymerase chain reaction
  • DNA or cDNA libraries may be made according to established methods (Ausubel, et al. , 1988; Sambrook, et al.
  • a convenient method of performing PCR amplifications where the relative position of the peptide fragments in the protein is unknown is to use a cDNA library in a known vector as the template, employing one of the peptide specific primers in combination with a vector primer, such as a primer directed to a T3 or T7 sequences that flank the multiple cloning site in many vectors. Accordingly, if a clone containing one of the target sequences is present in the template mix, the portion between the sequence corresponding to the peptide specific primer and the end of the insert is amplified.
  • Additional sequences may also be obtained by hybridization screening a human DNA or cDNA library with a probe having all or a portion of the sequence represented as SEQ ID NO:5.
  • the probe preferably contains a portion of SEQ ID NO:5 near the 5' end, to increase the chance of obtaining a clone encoding the 149 N-terminal amino acids.
  • clones encoding full-length proteins may be identified in an expression library using antibodies raised against one or more of the peptides characterized by SEQ ID NOs:4 and SEQ ID NO:6.
  • Methods for the screening of expression libraries are well-known (e.g. , Unit 6.7 of Ausubel, et al. (1988), incorporated herein by reference).
  • Polynucleotide sequences such as described above can be used, for example, as probes to detect other Hrs-2 sequences.
  • Such probes typically contain at least 12, preferably at least 16, more preferably at least 20 or more consecutive nucleotides corresponding to a region contained in an Hrs-2 polynucleotide sequence.
  • Hrs-2 sequences may also be used to produce recombinant Hrs-2 proteins, which in turn are useful in the assays described below.
  • polynucleotide sequences encoding Hrs-2 proteins of the present invention may be cloned into an expression plasmid, such as p-GEX-KG, to produce corresponding polypeptides, as is described in the Examples below.
  • Recombinant pGEX-KG plasmids can be transformed into appropriate strains of E. coli and fusion protein production can be induced by the addition of IPTG (isopropyl-thio galactopyranoside). Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using glutathione agarose affinity chromatography according to standard methods (described below; Ausubel, et al., 1988).
  • affinity chromatography may also be employed for isolating fusion proteins consisting of, e.g. , glutathione-S-transferase (GST) and the recombinant protein.
  • GST glutathione-S-transferase
  • bacterial cell lysates are prepared and passed over agarose beads derivatized with glutathione as described below. This results in the attachment of the GST portions of the fusions to the glutathione on the agarose beads. The beads are then washed, and the recombinant protein is cleaved with thrombin and eluted for further analysis.
  • the "immobilized" protein may be left attached to the beads for use in the assay.
  • Isolated recombinant polypeptides produced as described above may be further purified by standard protein purification procedures. These procedures may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography.
  • Hrs-2 proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies (as described below). Further, Hrs-2 peptides may be chemically synthesized using methods known to those skilled in the art. It will be understood that polypeptides or protein used as the "free" protein in a partner capture assay are synthesized so that they remain soluble during the binding assay. Accordingly, if SNAP-25 is used as the free protein, it may be modified for increased solubility by, for example, expressing a truncated version of the proteins that is missing its membrane anchor — i.e. , expressing only the cytoplasmic domain.
  • the invention includes a method of identifying a compound capable of modulating (e.g. , potentiating or inhibiting) calcium-regulated secretion, such as the release of neurotransmitter- containing synaptic vesicles.
  • a SNAP-25 polypeptide is contacted with an Hrs-2 polypeptide, in the presence and absence of a test compound.
  • the effect of the test compound on the extent of binding between the SNAP-25 and the Hrs-2 polypeptides is measured, and a compound is identified as effective if its effect on the extent of binding is above a threshold level (e.g., a several- fold difference in binding level between control and experimental samples).
  • a threshold level e.g., a several- fold difference in binding level between control and experimental samples.
  • test compound may be effective to enhance (potentiate) or inhibit binding between the SNAP-25 and Hrs-2 polypeptides.
  • Compounds tested may include small molecules in a small molecule combinatorial library, peptides in a peptide combinatorial library, and the like.
  • the invention may be practiced using only a fragment of a full length Hrs-2, so long as that fragment retains the ability to bind to SNAP-25 at its normal binding site.
  • the experiments described in Examples 2, 3, and 5 were performed using the human Hrs-2 clone represented as SEQ ID NO:5. This clone is missing the N-terminal 149 amino acids of Hrs-2, yet is capable of effectively binding to SNAP-25. Accordingly, the SNAP-25 binding site (SBS) on Hrs-2 is formed by a region of Hrs-2 encoded by the partial sequence represented as SEQ ID NO: 5.
  • Compounds which affect the binding of the SNAP-25 and Hrs-2 polypeptides to one another, when applied to and internalized by target cells, are expected to modulate vesicular release by those cells.
  • the modulation may be an inhibition of release or stimulation of release, either when the compound is applied alone, or when the compound is applied in conjunction with another compound having an effect on vesicular release.
  • the assay is conducted using a co-immunoprecipitation method such as is described in the Materials and Methods and Example 2.
  • Anti-Hrs-2 antibodies are coupled to protein A beads at a final concentration of about 2 mg/ml using the cross-linker dimethylpimelimidate (Pevsner, et al. , 1994).
  • Rat brain membranes prepared as described herein are resuspended in 20 mM Tris, pH 8.0, 150 mM NaCl and solubilized with either 1 % CHAPS or Triton X-100 at a final protein concentration of —2 mg/ml.
  • solubilized sample is incubated at 4°C for 30 min before centrifuging at 20,000 x g for 20 min.
  • the solubilized brain membranes are pre-cleared by incubation with protein A beads (1 ml solubilized membranes per 200 ⁇ l protein A beads) for 30-60 min at 4 °C.
  • the pre-cleared supernatant is then incubated with (i) a selected concentration of the test compound and (ii) 20 ⁇ l protein A beads with either immobilized anti-Hrs-2 antibodies or immobilized control IgG for 4-16 hrs at 4 °C.
  • the beads are then washed three times with 300-500 ⁇ l 20 mM Tris, pH 8.0, 150 mM NaCl containing either 0.7% CHAPS or Triton X-100. Proteins bound to the beads are analyzed by Western blotting for the presence of SNAP-25 as described in the Materials and Methods and Examples below. If the test compound is effective to inhibit binding of a SNAP-25 polypeptide to the immobilized Hrs-2 polypeptide, the amount of SNAP-25 detected in the Western blot will be diminished. Conversely, if the test compound is effective to potentiate the binding of a SNAP-25 polypeptide to the immobilized Hrs-2 polypeptide, the amount of SNAP-25 detected in the Western blot will be increased.
  • the assay is typically conducted with both negative controls (e.g. , beads coated with control rabbit Ig) and positive controls (no test compound in assays with anti-Hrs-2 antibody).
  • co-immunoprecipitation assay may be practiced by using immobilized SNAP-25 and soluble Hrs-2 changing the which of using other antibodies which recognize and can immunoprecipitate the SNAP-25 polypeptide, such as anti-rsec ⁇ antibodies or antibodies directed against one or more of the other proteins in the polypeptide.
  • a variety of approaches may be employed to assay the binding of SNAP-25 with a corresponding SNAP-25 binding site (SBS) on Hrs-2, and/or the binding or Hrs-2 with a corresponding Hrs-2 binding site (HBS) on SNAP-25. and/or the binding of an SBS to an HBS. While the binding assays are described below using intact, full length proteins, it will be appreciated that such binding assays may be conducted using only those portions of Hrs-2 and SNAP-25 that are responsible for binding to the other.
  • SBS corresponding SNAP-25 binding site
  • HBS Hrs-2 binding site
  • Binding assays which may be used in the practice of the invention include, but are not limited to, biochemical approaches (such as immobilized GST fusion protein constructs), bioassays (such as the yeast two-hybrid system), and physical biosensor assays (such as surface plasmon resonance). Some of these assays are described in more detail below.
  • Hrs-2 to SNAP-25 a test compound is included in the solution (containing the "free" soluble protein; e.g., Hrs-2) that is contacted with the immobilized protein (e.g. , the GST-SNAP-25).
  • the amount of bound Hrs-2 is detected and compared to the amount bound under similar conditions in the absence of the test compound (control). If the compound has a significant effect on the binding of Hrs-2 to the GST-SNAP-25 (i. e.
  • the compound is identified as effective to affect or alter the binding of the Hrs-2 to SNAP-25.
  • Figs. 6 A and 6B Illustrations of results that may be obtained with such an assay in a multiwell plate are shown in Figs. 6 A and 6B. Both figures show fragmentary plan views of a multiwell plates.
  • the plate 22 in Fig. 6 A is the control plate (without test compounds). Detection of binding of the free protein to the immobilized protein is indicated by the stippled pattern in the wells 24.
  • the wells of the plate 26 in Fig. 6B each contain different test compounds, two of which inhibit the binding of the free protein to the immobilized protein. This inhibition is evidenced by a lighter stippled pattern in the wells 28 containing the effective compounds.
  • the roles of the proteins can be switched —that is, the Hrs-2 may be immobilized to the solid support and a solution containing SNAP-25 (or a polypeptide containing the Hrs-2 binding site of SNAP-25) may be contacted with the Hrs-2. Additionally, the immobilized protein or the free protein may be exposed to a test compound prior to the binding assay, and the effects of this pre-exposure may be assessed relative to controls. Compounds identified in this manner also inhibit the binding of Hrs-2 to SNAP-25 or vice versa. Alternatively, the test compound may be added subsequent to the mixing of Hrs-2 with SNAP-25. A compound effective to reduce the level of binding in such an assay displaces Hrs-2 from SNAP-25, or vice versa.
  • a partially-purified (e.g., by the GST methods above) SNAP-25 polypeptide may be attached to the bottoms of wells in a multiwell plate (e.g., 96- well plate) by introducing a solution containing the polypeptide into the plate and allowing the polypeptide to bind to the plastic. The excess peptide-containing solution is then washed out, and a blocking solution (containing, for example, bovine serum albumin (BSA)) is introduced to block nonspecific binding sites.
  • BSA bovine serum albumin
  • the plate is then washed several more times and a solution containing an Hrs-2 polypeptide and, in the case of experimental (vs. control) wells, a test compound added.
  • Different wells may contain different test compounds, different concentrations of the same test compound, or different concentrations of Hrs-2 or SNAP-25.
  • the wells of a multiwell plate may be coated with a polypeptide containing Hrs-2, rather than SNAP-25, and binding interactions assayed upon addition of free SNAP-25.
  • the wells may also be precoated with substance(s) that enhance attachment of the protein to be immobilized and/or decrease the level of non-specific binding.
  • the wells may be derivatized to contain glutathione and may be pre-coated with BSA, to promote attachment of the immobilized protein in a known orientation with the binding site(s) exposed.
  • Detection methods useful in such assays include antibody-based methods (i.e., an antibody directed against the "free” protein), direct detection of a reporter moiety incorporated into the "free” protein (such as a fluorescent label), and proximity energy transfer methods (such as a radioactive "free” protein resulting in fluorescence or scintillation of molecules incorporated into the immobilized protein or the solid support).
  • antibody-based methods i.e., an antibody directed against the "free” protein
  • direct detection of a reporter moiety incorporated into the "free” protein such as a fluorescent label
  • proximity energy transfer methods such as a radioactive "free” protein resulting in fluorescence or scintillation of molecules incorporated into the immobilized protein or the solid support.
  • multiwell plates e.g. , 96-well plates
  • a scintillating material in the wells available from, e.g. , Wallac, Gaithersburg, MD
  • a scintillating material in the wells available from, e.g. , Wallac, Gaithersburg, MD
  • Free protein that binds the immobilized protein is constrained within a few nanometers of the well surface, resulting in light emission from the scintillation material in the wells.
  • the signal can be quantitated using a plate reader or counter, such as the "MICROBETA PLUS" plate counter (Wallac), to generate standard binding plots.
  • Such plots may be used to determine the optimal concentrations of proteins used in the assay, and may be useful in identifying compounds with more subtle effects on Hrs-2/SNAP-25 binding that can be detected using some other methods.
  • yeast Two-hybrid protein interaction assay may also be employed to identify compounds that affect the binding of Hrs-2 to SNAP-25.
  • the assay is based on the finding that most eukaryotic transcription activators are modular (e.g, Brent, et al.,
  • the activators typically contain activation domains that activate transcription
  • DNA binding domains that localize the activator to the appropriate region of a DNA molecule.
  • a first fusion protein contains one of a pair of interacting proteins fused to a DNA binding domain
  • a second fusion protein contains the other of a pair of interacting proteins fused to a transcription activation domain.
  • the two fusion proteins are independently expressed in the same cell, and interaction between the "interacting protein" portions of the fusions reconstitute the function of the transcription activation factor, which is detected by activation of transcription of a reporter gene.
  • the yeast GAL4 two hybrid system (Fields and Song, 1989; Chien, et al., 1991; Durfee, et al., 1993; Bartel, et al., 1993) was developed to detect protein-protein interaction based on the reconstitution of function of GAL4, a transcriptional activator from yeast, by activation of a GAL1- lacZ reporter gene.
  • the GAL4 protein contains two distinct domains, a DNA binding domain and a transcription activation domain. Each domain can be independently expressed as a portion of a fusion protein composed of the domain, and a second, "bait" interacting protein. The two fusion proteins are then independently expressed together in a cell.
  • the reporter gene typically has a promoter containing GAL4 protein binding sites (GAL upstream activating sequences, UAS G ).
  • a two hybrid system such as is described above may be used to identify compounds effective to disrupt the binding of Hrs-2 to SNAP-25 as follows.
  • a polynucleotide encoding SNAP-25 is fused to the GAL4 DNA binding domain (G4BD) in a yeast expression vector (e.g., pG4BD-SNAP-25).
  • the vector is used to generate yeast cells harboring pG4BD-SNAP-25 and a GAL4-activated reporter gene (e.g., LacZ). These cells are then transformed with a vector carrying a fusion between the transcription activating domain of yeast GAL4 (G4AD) and Hrs-2 (e.g., pG4AD-Hrs-2).
  • Transformants are screened (e.g., using a j3-galactosidase (0-gal) assay on plates containing the chromogenic substrate X-gal) for expression of the reporter.
  • Reporter-expressing cells are selected, cloned, and used to screen test compounds.
  • Compounds which increase or decrease reporter expression relative to a user-defined threshold e.g., five-fold increase or five-fold decrease
  • a user-defined threshold e.g., five-fold increase or five-fold decrease
  • the extent of binding may be measured using densitometry of the Western blot image.
  • the densitometry values are typically normalized, and a threshold level is set based on the amount of variation in the signal between a series of "control" samples (samples not containing test compounds). The smaller the variation, the smaller the effect of a test compound that can be reliably detected.
  • the threshold is typically set at a several-fold difference, such as a 3-5 fold increase or decrease in binding affinity. If a multiwell plate screen is used, the output of a plate reader used to score the results of the experiment may be used as a measure of the effect on the extent of binding, and a threshold set as described above.
  • threshold level used in a particular application of the invention is determined in view of the specific requirements of that particular application. For example, if it is desired to isolate only compounds with a very high activity, the threshold is set to a relatively high value, such as a 10 to 100-fold difference in binding affinity. If, on the other hand, it is desired to isolate compounds having a subtle effect on the binding, a lower threshold level may employed.
  • the effect of a particular compound on the binding of a SNAP-25 polypeptide to an Hrs-2 polypeptide depends on the concentration of the compound. At relatively high compound concentrations the effect may be large, and be manifested as, e.g., a 50-fold difference in binding between control and experimental samples, whereas at lower compound concentrations, the effect may be smaller.
  • SNAP-25 polypeptide to an Hrs-2 polypeptide may be further evaluated for their ability to modulate vesicular release in vitro and in vivo.
  • the compounds may be tested using the PC 12 cell Dj3H vesicular release assay (Bennett, et al. , 1993), which detects a membrane-associated form of the enzyme dopamine ⁇ - hydroxylase (D3H) on the luminal side of catecholamine-containing granules.
  • D3H dopamine ⁇ - hydroxylase
  • the cells are depolarized in the presence of calcium, granule fusion with the plasma membrane results in the exposure of D/3H on the cell surface, where it can be quantitatively detected by immunofluorescence microscopy (Elferink, et al. , 1983).
  • a compound identified as affecting the binding of a SNAP-25 polypeptide to an Hrs-2 polypeptide By treating a sample of cells with a compound identified as affecting the binding of a SNAP-25 polypeptide to an Hrs-2 polypeptide, depolarizing the cells (e.g., with a pulse of KC1) in the presence of calcium, and comparing the response to that obtained with an untreated sample of cells, the effects of the compound on vesicle release in PC 12 cells may be assessed. Similar assays may be employed using freshly-isolated cells (e.g., in brain slices), or suitable animal models.
  • the compounds will likely need to be internalized by the target cells to have the desired effect on vesicle release.
  • Methods of promoting uptake of different types of compounds by cells are well known in the art. For example, certain classes of compounds, e.g. , lipophilic compounds and esters, can simply diffuse across the lipid bilayer.
  • Other types of compounds may utilize membrane transport proteins to be internalized, and still others can be internalized by endocytosis or liposome-mediated targeting.
  • a variety of different compounds may be screened using methods of the present invention. They include peptides, macromolecules, small molecules, chemical and/or biological mixtures, and fungal, bacterial, or algal extracts. Such compounds, or molecules, may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.
  • a set of potentially-effective test peptides can be generated from overlapping peptides spanning the entire sequence of each of the proteins involved in the SNAP-25 polypeptide/Hrs-2 polypeptide interaction. Such a set is likely to contain peptides which may be effective to disrupt the interactions of the SNAP-25 polypeptide with the Hrs-2 polypeptide.
  • an identified active compound is a peptide.
  • the peptide may be utilized to aid in the discovery of orally-active small molecule mimetics.
  • Inhibitory compounds isolated using methods of the present invention may be employed to inhibit or enhance vesicle-mediated secretion from cells.
  • compounds which enhance or potentiate the binding of an SNAP-25 polypeptide to an Hrs-2 polypeptide may be used to upregulate vesicle-mediated secretion.
  • the ability to modulate secretion processes has utility in a variety of areas, some of which are identified below.
  • CNS Disease Applications A number of disorders and/or conditions of the central nervous system (CNS) may be alleviated by selectively enhancing or inhibiting vesicular release in specific areas of the brain. They include affective disorders (e.g., depression), disorders of thought (e.g., schizophrenia) and degenerative disorders (e.g. , Parkinson's disease), as well as applications such as anesthesia. A variety of drugs are currently used to treat such disorders and/or conditions. Compounds identified by methods of the present invention may be used either alone, or in combination with currently used therapies to alleviate symptoms associated with the disorders.
  • Drugs used to treat affective disorders typically fall into three classes: (i) monoamine oxidase (MAO) inhibitors, such as phenelzine, (ii) tricyclic compounds, such as imipramine, and (iii) serotonin uptake blockers, such as fluoxetine and trazodone. All of these drugs work, at least in part, by increasing the concentration of either serotonin or biogenic amine neurotransmitters in CNS synapses of treated individuals. According to methods of the present invention, compounds which enhance the release of serotonin or biogenic amines at selected brain synapses may be similarly effective at treating depressive disorders. Such compounds may be identified by screening for compounds effective to enhance the binding of Hrs-2 to SNAP-25.
  • disorders of thought such as schizophrenia
  • antipsychotic drugs including phenothiazines, such as chlorpromazine, butyrphenones, such as haloperidol, xithioxanthenes, and newer drugs, such as clozapine
  • compounds identified as inhibitors of release of dopamine-containing vesicles particularly vesicles released from cells having their cell bodies in the arcuate nucleus of the hypothalamus, the substantia nigra, or the ventral tegmental area, may be employed to relieve symptoms of schizophrenia.
  • Parkinson's disease is due to degeneration of the nigrostriatal pathway, raphaei nuclei, locus ceruleus, and motor nucleus of vagus, which result in a reduction of dopamine, serotonin and norepinephrine levels.
  • the symptoms of Parkinson's may be alleviated by administering compounds identified according to the teachings presented herein as stimulating release of vesicles containing the above neurotransmitters.
  • compounds identified employing methods of the present invention may be used to therapeutically intervene in a variety of other systems. They include the endocrine system for treatment of hormonal imbalances, the immune system for intervention in antigen processing, secreted immunomodulators, and viral processing, as well as anti-tumor applications, such as regulation of membrane trafficking during rapid cell division.
  • [ 125 I]goat anti-rabbit secondary antisera and the Enhanced Chemo-Luminescence (ECL) system were obtained from Amersham Corp. (Arlington Heights, IL). Nitrocellulose paper was obtained from Schleicher and Schuell (Keene, NH). Materials for SDS-polyacrylamide gel electrophoresis (SDS- PAGE) were obtained from Bio-Rad Laboratories (Hercules, CA). Other chemicals were purchased from Sigma (St. Louis, MO) or United States Biochemical (Cleveland, OH). All protein purification procedures were carried out at 4°C unless otherwise noted.
  • PBS Phosphate-buffered saline
  • mouse SNAP-25b (Oyler, et al. , 1989) was amplified using the polymerase chain reaction using primers S25F (SEQ ID NO:l - forward: 5' CCG AAT TCA TGG CCG AGG ACG CAG ACA 3') and S25R (SEQ ID NO:2 - reverse: 5' CCG TCG ACT AAC CAC TTC CCA GCA TCT 3') containing internal EcoRI and Sail restriction sites. Amplification products were inserted in frame with the GAL4 binding domain (GAL4BD) in the pGBT9 vector (Fields and Song, 1989) creating PGBT9/SNAP-25. All constructions were verified by sequencing (Sequenase, USB).
  • a human brain cDNA library inserted downstream of the GAL4 activation domain (GAL4AD) in the pGADIO vector was purchased from Clontech (Palo Alto, CA).
  • Yeast HF7c strain, Clontech
  • pGBT9/SNAP-25 4 x 10 6 colonies
  • agar containing yeast nitrogen base 6. g/L, Difco Laboratories, Detroit, MI
  • adenine 0.054 mM
  • lysine (0.165 mM
  • dextrose %
  • 3-amino-l,2,4-triazole (10 mM, Sigma) and stored at 30°C in the dark.
  • Glutathione-S-transferase (GST) fusion proteins of Hrs-2 (GST-Hrs-2) were prepared using previously described methods (Pevsner et al., 1994). Briefly, the partial human Hrs coding sequence (SEQ ID NO: 5), isolated as described above, was cloned into the pGEX-derived (Smith and Johnson, 1988) vector, pGEX-KG (Guan and Dixon, 1991). The resultant vectors were used to transform XL-1 Blue E. coli cells (Stratagene, La Jolla, CA).
  • Bacterial clones containing the protein sequences were selected and grown at 37°C, with vigorous agitation, for approximately 4 hours in 1-liter of liquid culture (Luria Broth (LB); Howard Hughes Media Supply Facility, Stanford University, Stanford, CA). 1 ml of 100 mM isopropyl-l-thio-/3-D-galactoside (IPTG) was added to induce protein expression, and the culture was incubated for approximately another three hours.
  • LB Lia Broth
  • IPTG isopropyl-l-thio-/3-D-galactoside
  • the cells were pelleted and resuspended in 10 ml ice-cold Buffer A, lysed with a French Press (SLM Aminco, Rochester, NY) until translucent, centrifuged briefly to pellet cellular debris, and the supernatant transferred to a fresh tube.
  • the fusion protein was purified by affinity chromatography as follows. Five ml of a 50%
  • the fusion protein was typically eluted from the beads using the thrombin cleavage protocol
  • Phenylmethylsulfonyl fluoride (0.6 mM final concentration) was then added to the protein elution, and the sample was concentrated to 0.5 ml using a "CENTRIPREP” concentrator (Amicon Inc., Beverly, MA).
  • the Hrs-2 protein was then size fractionated by gel filtration on a "SUPEROSE 12" sizing column (Pharmacia, Piscataway, NJ) using a buffer consisting of 50 mM Tris, 500 mM NaCl, and 0.05 % "TWEEN 20" .
  • Hrs-2 His-tagged Hrs-2 was purified using methods described previously (Sollner, et al., 1993b). Briefly, the coding region of the Hrs-2 sequence was cloned into the £r ⁇ RI site of pRSET-B (Invitrogen, Carlsbad, CA). The resulting plasmid and pREP4 (Qiagen, Chatsworth, CA) were transformed into E. coli (HB101, Stratagene, La Jolla, CA).
  • His 6 -Hrs-2-expressing cells were collected, washed in Buffer A, disrupted in a French Press, and the suspension was clarified by centrifugation at 100,000 Xg for 1 hour. The supernatant was passed over a Ni-NTA-agarose column (Qiagen; 5 ml bed volume) and the His 6 -Hrs-2 was eluted with a two step imidazole wash -50 mM followed by 500 mM imidazole. Protein concentrations for the above-isolated proteins were estimated by Coomassie blue staining of protein bands after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) using bovine serum albumin (BSA) as a standard.
  • SDS- PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • Anti-Hrs-2 antisera were prepared in rabbits with gel-purified recombinant Hrs-2 protein, prepared as described above, using standard methods (Harlow, et al., 1988). Briefly, -250 ⁇ g of protein isolated from a polyacrylamide gel was suspended in — 1 ml PBS and injected subcutaneously (sc) once every three weeks for 12 weeks. The initial injection contained complete Freund's adjuvant, while subsequent injections contained incomplete Freund's adjuvant. Serum was isolated after 12 weeks and used as described below.
  • NTPase reactions contained Hrs-2 (10 ⁇ l of Superose 12 column fractions pooled from monomer and oligomer peaks), unlabeled NTPs at various concentrations, 1.5-2.0 ⁇ Ci of the corresponding [ ⁇ - 32 P] NTPs, 1 mM MgCl 2 , 0.1 mM EDTA, 0.2% (w/v) BSA, and 50 mM Tris-HCl, pH 7.4. Reactions were conducted for 20 min at 25 °C and were terminated by addition of an ice-cold slurry containing 5% (w/v) activated charcoal and 50 mM NaH 2 P0 4 , pH 2.0.
  • Radioactivity in supernatants of reaction mixtures was determined by scintillation counting. Blank values were determined using column elution buffer in the reaction mixture. Steady state NTPase activity of Hrs-2 is expressed as molar turnover number (moles of NTP hydrolyzed per mol Hrs-2 per minute). Data shown are the mea ⁇ +S.D. of one representative experiment performed in triplicate. Very similar results were obtained in two-three independent experiments with different preparations of Hrs-2.
  • Hrs-2 were incubated with immobilized GST-SNAP-25 (0.3 ⁇ M, 4°C for 60 min) in the presence and absence of Zn 2+ (1 mM) or to immobilized GST (X), or a single concentration of Hrs-2 (2 ⁇ M) was incubated with immobilized GST-SNAP-25 in the presence of various free calcium concentrations. After washing, the beads were boiled in SDS-containing sample buffer and proteins were separated by SDS- PAGE. After transfer to nitrocellulose, blots were probed with Hrs-2 antibodies and 125 I-labeled secondary antisera. Hrs-2 was visualized/quantitated using a phosphorimager.
  • Rat brain post-nuclear supernatant was prepared as follows. Frozen rat brains were homogenized in 20 ml of Buffer B with a Teflon-glass homogenizer. The homogenate was centrifuged at 100,000 x g for 1 hr. The pellet was dissolved in 1 ml 20 mM Tris, pH 8.0 and 150 mM NaCl and solubilized with 1 % Triton X-100 at a final protein concentration of — 2 mg/ml.
  • solubilized sample was incubated at 4 °C for 30 min before centrifuging at 20,000 x g for 20 min. Following centrifugation, the solubilized brain membranes were pre-cleared by incubation with protein A beads (1 ml solubilized membranes per 200 ⁇ l protein A beads) for 60 min at 4 °C.
  • Immunoprecipitation was performed by incubating the detergent solubilized rat brain post nuclear supernatant with the antibody-conjugated beads for 16 hours at 4°C. The beads were then washed three times with 500 ⁇ l 20 mM Tris, pH 8.0, 150 mM NaCl containing 0.7% Triton X-100 and resuspended in 30 ⁇ l of sample buffer. The sample was separated by SDS-PAGE (10%), transferred to nitrocellulose, and probed with antibodies. Proteins were visualized using chemiluminescence (Amersham ECL).
  • Hrs-2 novel proteins described herein were therefore termed "Hrs-2".
  • Hrs-2 sequences suggest that the previously- published sequence of Hrs is either incorrect or is a splice variant of Hrs-2 in the carboxy terminus.
  • both a rat and a human clone were identified.
  • the coding sequence of the rat clone encodes a 925 amino acid peptide with a predicted molecular mass of 106,764 kDa.
  • SEQ ID NO:3 The nucleotide sequence encoding rat Hrs-2 is presented herein as SEQ ID NO:3, with the amino acid sequence represented as SEQ ID NO:4.
  • the human clone initially recovered in the two hybrid screen contained all of the rat clone except the amino terminal 149 amino acids.
  • the nucleotide sequence of the human clone is presented as SEQ ID NO:5; the amino acid sequence as SEQ ID NO:6.
  • Figure 1 is a schematic illustration of the domain structure of Hrs-2.
  • the predicted ORF from the rat cDNA contains domains with potential functional significance.
  • a zinc finger (Zn finger) composed of 8 cysteine residues is present in the amino terminal half of the protein. This region surrounding the 8 cysteine residues that form two zinc coordination domains (Zn 2+ finger) is well conserved among a variety of proteins suggested to be involved in protein trafficking to the yeast vacuole (e.g. Fabl, VPS 27, Vac lp) or to the endosome in eukaryotic cells (e.g. pl62).
  • a putative nucleotide binding site consisting of GXXXXGK (SEQ ID NO:7), RDET (SEQ ID NO:8), DXXG (SEQ ID NO:9), and TQKXD (SEQ ID NO: 10) is also present (Bourne, et al. , 1991).
  • the first three domains of the putative nucleotide binding site are identical to the consensus (G1,G2,G3) while the fourth domain (G4) is a variant found in some GTP-binding proteins (Bourne, et al., 1991).
  • the G4 domain is thought to confer selectivity for the nucleotide although some proteins specific for GTP completely lack the G4 domain (Bourne, et al.
  • Hrs-2 Characterization and Expression Pattern of Hrs-2
  • the expression of Hrs-2 was evaluated by immunoblot analysis of various rat tissues (indicated) using a polyclonal antibody raised against Hrs-2 bacterial fusion protein. Immunoblot analysis revealed a single band of approximately 115 kDa, with the greatest abundance in brain.
  • a Western blot experiment was also conducted with rat brain post nuclear supernatant (PNS) using SNAP-25 antisera. Taken together, the results indicate that brain postnuclear supernatant contains both Hrs-2 and SNAP-25.
  • Hrs-2 antiserum was also used to perform immunoprecipitation studies from rat brain PNS. These experiments showed that both Hrs-2 and SNAP-25 are present in the rat brain PNS sample. Anti-Hrs-2 antisera ( ⁇ Hrs-2), but not rabbit IgG, resulted in precipitation of both Hrs-2 and SNAP-25. These results provide evidence for the interaction of Hrs-2 and SNAP-25 in vivo.
  • (X 1000) for Hrs-2 were: 0.125 ⁇ M, 238; 0.25 ⁇ M, 767; 0.5 ⁇ M, 1652; l ⁇ M, 5724; 2 ⁇ M, 7728; 4 ⁇ M,
  • Pixel values (X 1000) for Hrs-2 + Zn 2+ were: 14, 20, 187, 413, 847, 1422, 1772.
  • the stoichiometry of the interaction between Hrs-2 and SNAP-25 was calculated to be in the range of 0.1-0.55: 1 (Hrs-2: SNAP-25).
  • the stoichiometry was determined by quantifying the amount of Hrs-2 bound to SNAP-25 when one of the proteins was immobilized on glutathione agarose beads and the other was soluble. The range was due to slightly different results obtained depending on whether Hrs-2 or SNAP25 was immobilized.
  • the zinc finger or other domains of Hrs-2 may bind calcium.
  • FIG. 3A shows that recombinant Hrs-2 eluted from the gel filtration column in two peaks.
  • the top of the figure shows the profile of fractions of eluate probed with the anti-Hrs-2 antibody, the bottom part shows the protein elution profile (A 280 ) from the gel filtration column.
  • the position of one peak was consistent with monomeric protein while the other peak migrated at a position consistent with a multimer.
  • Figs. 3B and 3C show the NTPase activity of monomeric Hrs-2 (right column peak in Fig. 3A), while Fig. 3C shows the NTPase activity of oligomeric Hrs-2 (left column peak in A).
  • the symbols in both figures are as follows: [ ⁇ ], ATPase; [ A] , GTPase; [ ⁇ ], UTPase.
  • the results show that both peaks possess ATPase, GTPase, and UTPase activities.
  • the nucleotidase activity of the oligomeric form of Hrs-2 demonstrates that ATP is the preferred substrate.
  • Hrs-2 for ATP are similar to what has been previously reported for NSF, another oligomeric ATPase that physically interacts with other components of the secretory apparatus (Whiteheart, et al. , 1994; Tagaya, et al. , 1993; Morgan, et al. , 1994). Additional experiments were performed to assess the effects non-hydrolyzable nucleotide analogs on the ATPase activity of monomeric Hrs-2. ATPase activity was assessed in the presence of l ⁇ M ATP with various concentrations of ATP7S and GTP ⁇ S. The results are shown in Figs. 3D
  • ATP7S ATP7S
  • GTP7S 3E
  • Hrs-2 with similar potency and in a monophasic manner, indicating the presence of a single nucleotide binding site. This is consistent with the proposed single nucleotide binding site in the deduced amino acid sequence.
  • the ATPase activity of the oligomeric form was also inhibited by ATP7S and GTP7S, as well as EDTA, suggesting that Hrs-2 requires Mg 2+ for this enzyme activity.
  • Figs. 4A-4D show hippocampal neurons immunolabeled with anti-Hrs-2 antibodies and stained with secondary antibodies conjugated to DTAF.
  • Fig. 4B shows hippocampal neurons immunolabeled with anti-synaptophysin antibodies and stained with secondary antibodies conjugated to Texas Red.
  • Fig. 4C shows hippocampal neurons immunolabeled with anti- Hrs-2 antibodies and stained with secondary antibodies conjugated to DTAF.
  • Hrs-2 protein is most abundantly expressed in brain ( Figure IB) where apparently colocalizes with SNAP-25 and synaptic vesicles in axons and nerve terminals, consistent with its role in transmitter secretion.
  • CAG GCC AGG CCG GGC CAC CAC CAA CCC TGC TAC TCC TCG TAC CAG CCT 2022 Gin Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser Ser Tyr Gin Pro 645 650 655 660
  • Lys Pro Leu lie Cys Leu Thr Ser His Phe Leu Pro Leu Leu Pro Cys 790 795 800
  • Asp Leu lie Arg Gin Gly Asp Thr Gin Ala Lys Tyr Ala Val Asn Ser 35 40 45 lie Lys Lys Lys Val Asn Asp Lys Asn Pro His Val Ala Leu Tyr Ala 50 55 60
  • GCC ATG TTT GCT GCT GAA AGA GCC CCT GAC TGG GTG GAC GCT GAG GAA 48 Ala Met Phe Ala Ala Glu Arg Ala Pro Asp Trp Val Asp Ala Glu Glu 1 5 10 15
  • CAG GCC CAA CCA GCT TTC CTG GCA CCT TTA GCC
  • CAG CAG GTA GTC AGA 1344 Gin Ala Gin Pro Ala Phe Leu Ala Pro Leu Ala Gin Gin Val Val Arg 435 440 445
  • GCC AGG CCG GGC CAC CAC CAA CCC TGC TAC TCC TCG TAC CAG CCT ACT 1536 Ala Arg Pro Gly His His Gin Pro Cys Tyr Ser Ser Tyr Gin Pro Thr 500 505 510
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • ORIGINAL SOURCE :
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • CAA GGC GAA CAA CTG GAA CGC ATT GAG GAA GGG ATG GAC CAA ATC AAT 367 Gin Gly Glu Gin Leu Glu Arg He Glu Glu Gly Met Asp Gin He Asn 55 60 65
  • GATACCATGT GTCTTTTGTT TTCTCCGGCT CTCTTTCTTT GCCAAAGGTT GTACATAGTG 1041
  • AAAGCAACAA CTACGCATGC TCAGCATTGG GACACTGTCA AGATTAAGTC ATACCAGCAA 1521 AACCTGCAGC TGTGTCACCT TCTTCTGTCA ACATACAGAC TGATCATAAT GATCCCTTCT 1581

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Abstract

L'invention concerne des polynucléotides et des polypeptides Hrs-2, ainsi que des méthodes d'identification d'un composé capable de moduler la sécrétion, régulée par le calcium, des vésicules de sécrétion, telle que la sécrétion de vésicules synaptiques comportant des neurotransmetteurs.
PCT/US1998/003789 1997-02-26 1998-02-26 Methodes et compositions de modulation de la secretion vesiculaire WO1998038210A2 (fr)

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US6132977A (en) * 1998-03-13 2000-10-17 University Of New Mexico Measurement of a CNS protein in cerebrospinal or amniotic fluid
WO2006058781A3 (fr) * 2004-12-03 2007-03-22 Proteosys Ag Utilisation de finasteride, dutasteride et de composes associes dans la prevention ou le traitement de troubles neurologiquement associes
EP1644739B1 (fr) * 2003-07-04 2008-04-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede de mise en evidence d un evenement moleculaire dans une cellule grace a des proteines marqueurs fluorescentes

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WO1996015149A2 (fr) * 1994-11-10 1996-05-23 University Of Washington Procedes et compositions de criblage d'agents bloquant les canaux calciques presynaptiques
WO1998021333A2 (fr) * 1996-11-15 1998-05-22 Cedars-Sinai Medical Center Acide nucleique codant des proteines de fixation de la schwannomine et produits associes

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WO1996015149A2 (fr) * 1994-11-10 1996-05-23 University Of Washington Procedes et compositions de criblage d'agents bloquant les canaux calciques presynaptiques
WO1998021333A2 (fr) * 1996-11-15 1998-05-22 Cedars-Sinai Medical Center Acide nucleique codant des proteines de fixation de la schwannomine et produits associes

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DATABASE EMBL SEQUENCES EMBL, Heidelberg, FRG Accession No. O14964, 8 January 1998 LU L. ET AL,: "Human Hrs" XP002073825 *
DEBELLO W.M. ET AL.: "SNAP-mediated protein-protein interactions essential for neurotransmitter release" NATURE, vol. 373, 16 February 1995, pages 626-630, XP002073821 *
KOMADA M. & KITAMURA N.: "Growth Factor-induced tyrosine phosphorylation of Hrs, a novel 115-kDa protein with a structurally conserved putative zinc finger domain" MOL. CELL BIOL., vol. 15, November 1995, pages 6213-6221, XP002073820 cited in the application *
SCHIAVO G. ET AL.: "Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses" PROC. NATL. ACAD- SCI. USA, vol. 94, 4 February 1997, pages 997-1001, XP002073822 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6132977A (en) * 1998-03-13 2000-10-17 University Of New Mexico Measurement of a CNS protein in cerebrospinal or amniotic fluid
EP1644739B1 (fr) * 2003-07-04 2008-04-23 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procede de mise en evidence d un evenement moleculaire dans une cellule grace a des proteines marqueurs fluorescentes
WO2006058781A3 (fr) * 2004-12-03 2007-03-22 Proteosys Ag Utilisation de finasteride, dutasteride et de composes associes dans la prevention ou le traitement de troubles neurologiquement associes
US7998970B2 (en) 2004-12-03 2011-08-16 Proteosys Ag Use of finasteride, dutasteride and related compounds for the prevention or treatment of neurologically-associated disorders

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