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WO1999032624A1 - Proteines humaines responsables de l'activation et de la conjugaison de nedd8 - Google Patents

Proteines humaines responsables de l'activation et de la conjugaison de nedd8 Download PDF

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Publication number
WO1999032624A1
WO1999032624A1 PCT/US1998/027141 US9827141W WO9932624A1 WO 1999032624 A1 WO1999032624 A1 WO 1999032624A1 US 9827141 W US9827141 W US 9827141W WO 9932624 A1 WO9932624 A1 WO 9932624A1
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WO
WIPO (PCT)
Prior art keywords
nael
nedds
beta
ncel
nce2
Prior art date
Application number
PCT/US1998/027141
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English (en)
Inventor
Vincent Chau
Original Assignee
Millennium Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals, Inc. filed Critical Millennium Pharmaceuticals, Inc.
Priority to EP98964164A priority Critical patent/EP1037988A1/fr
Priority to IL13687398A priority patent/IL136873A0/xx
Priority to JP2000525543A priority patent/JP2001526890A/ja
Priority to AU19350/99A priority patent/AU758077B2/en
Priority to CA002315240A priority patent/CA2315240A1/fr
Publication of WO1999032624A1 publication Critical patent/WO1999032624A1/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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention relates to covalent modification of proteins through their conjugation with other proteins. More particularly, the invention relates to the modulation of such conjugation involving the protein NEDDS.
  • Ubiquitin is then transferred to an epsilon amino group of a lysine residue in the target protein through an amide linkage, usually with the involvement ofubiquitin- protein isopeptide ligase, E3. Hopkin, J. Natl. Inst. Health Res. 9_: 36-42 (1997), teaches that target specificity is regulated by the particular combination of E2 and E3 protein, with more than 30 E2 proteins and 10 E3 proteins being known at present. Ubiquitin is not the only protein which is used to modify other proteins through covalent linkage, however. Kamitani et al, J. Biol. Chem.
  • NEDDS another ubiquitin-like protein, called NEDDS, for Neural precursor cell-Expressed Developmentally Down regulated.
  • the invention provides compositions and methods for detecting and/or modulating the conjugation of NEDDS and/or its transfer to a target protein, as well as compositions and methods for discovering molecules which are useful in detecting and/or modulating the conjugation of NEDDS and/or its transfer to a target protein
  • the present invention arises from the purification and characterization of novel NEDDS activating and conjugating enzymes
  • the invention provides purified at ⁇ ng protein beta suburut (NAEl-beta)
  • NAEl-beta The primary airuno acid sequence of a preferred embodiment of such NAEl-beta protein is shown in Figure 1
  • NAEl-beta expression elements include, without limitation, isolated or recombinant nucleic acid sequence:-, encoding NAEl-beta or nucleic acid sequences specificalU homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences and recombinant expression units which express NAEl-beta or, antisense transcripts or dominant negative mutants thereof
  • NAElBBMs NAEl-beta-binding molecules
  • the invention provides methods for identif ing NAElBBMs
  • One preferred method according to this aspect of the invention comprises screening for NAElBBMs by contacting purified NAEl-beta according to the lm ention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAEl-beta
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NAEl-beta based upon structural information from the purified NAEl-beta protein provided by the invention and determining w hether such rationally designed molecules bind specifically to NAEl-beta
  • This aspect of the invention includes NAElBBMs identified by the methods according to the invention
  • NAElBBMs can be used in conventional assays to detect the presence or absence, and/or quantity of NAEl-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NAE1BBM to a biological sample, allowing the detectable NAE1BBM to bind to NAEl-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAE1BBM and NAEl-beta, NAEl-heterodimer, or NAE1 heterodimer/NEDDS complex
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAEl-beta can also be used in conventional assays to detect the presence or absence of NAEl-beta nucleic acid in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta nucleic acid in a biological sample
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAEl-beta nucleic acid
  • the invention provides methods for identifying modulating ligands of NAEl-beta
  • Some NAElBBMs are capable of acting as antagonists or agonists of NAEl-beta
  • the method according to this aspect of the invention comprises providing NAElBBMs to an assay system for NAEl-beta participation in the NEDDS-activation/ conjugation pathway, and determining w hether such NAElBBMs interfere with or enhance the ability of NAEl-beta to participate in the NEDD8-act ⁇ vat ⁇ on/conjugat ⁇ on pathway
  • the NAElBBMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as tor example in a screening procedure
  • This aspect of the invention includes modulating ligands of NAEl-beta identified by this method according to the invention
  • the invention provides modulating ligands of NAE1- beta Preferred modulating ligands are NAElBBMs which act as antagonists, interfering with the ability ot NAEl-beta to participate in the NEDDS- activation/conjugation path av
  • Other preferred modulating ligands are NAElBBMs which act as agonists, enhancing the ability of NAEl-beta to participate in the NEDDS-activation/conjugation pathway
  • such NAElBBMs preferably interact w ith NAEl-beta to inhibit or enhance the formation of NAE1 heterodimer, the formation of NEDDS adenylate, the formation of a thiol ester bond between NEDDS and NAE1, and/or transfer of NEDDS to NEDD8- conjugating enzyme
  • the invention provides methods for modulating the activation and/or conjugation of NEDDS
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAEl-beta or a recombinant expression unit which expresses NAEl-beta or an antagonist thereof to a biological system in which NEDDS is conjugated to another protein
  • the invention provides ohgonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 1 Preferred embodiments include hybridization probes and antisense ohgonucleotides
  • the invention provides methods for identifying NAE1- alpha binding molecules (NAElABMs)
  • NAElABMs NAE1- alpha binding molecules
  • the present inventors have identified the alpha subunit of the NAE1 heterodimer (NAEl-alpha) Surprisingly, it has an amino acid sequence which is substantially identical to a protein previously identified as amyloid precursor protein binding protein 1 (APP-BP1, see Chow et al , J Biol Chem.
  • One preferred method according to this aspect of the invention comprises screening for NAElABMs by contacting purified NAE1- alpha and populations of molecules or mixed populations of molecules and determining the presence of molecules hich bind specifically to NAEl-alpha
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NAEl-alpha based upon structural information from the NAEl-alpha protein identified bv the present inventors and determining whether such rationally designed molecules bind specifically to NAEl- alpha
  • This aspect of the invention includes NAElABMs identified by the methods according to the invention
  • NAElABMs can be used in conventional assavs to detect the presence or absence, and/or quantity of NAEl-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NAE1ABM to a biological sample, allowing the detectable NAE1ABM to bind to NAEl-alpha, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAE1ABM and NAEl-alpha, NAE1- heterodimer, or NAE1 heterodimer/NEDDS complex
  • the method according to this aspect of the invention is used
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAEl-alpha can also be used in conventional assays to detect the presence or absence of NAEl-alpha nucleic acid in a biological sample in which NEDDS conjugation is suspected
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha nucleic acid in such a biological sample
  • such assays are nucleic acid hybridization and/or amplification assay s, such assa ⁇ s comprising providing to the biological sample a nucleic acid sequence which is specifically complementarv to NAEl-alpha nucleic
  • the invention provides methods for identifying modulating ligands of NAEl-alpha
  • Some NAElABMs are capable of acting as antagonists or agonists of NAEl-alpha
  • the method according to this aspect of the invention comprises providing NAElABMs to an assay system for NAEl-alpha participation in the NEDDS-activation/conjugation pathway, and determining whether such NAElABMs interfere with or enhance the abilitv of NAEl-alpha to participate in the NEDDS-activation/conjugation pathway
  • the NAElABMs are preferably provided as a population of molecules (most preferablv rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure
  • This aspect of the invention includes antagonists or agonists of NAEl-alpha identified by this method according to the invention
  • the invention provides a purified complex of NAEl- beta and NAEl-alpha, or of NAEl-beta, NAEl-alpha and NEDDS, or a purified complex of
  • the invention provides modulating ligands of NAEl- alpha
  • modulating ligands are NAElABMs which act as antagonists which interfere with the ability of NAEl-alpha to participate in the NEDDS-activation/conjugation pathway
  • Other preferred modulating ligands are NAElABMs which act as agonists which enhance the ability of NAEl-alpha to participate in the NEDDS-activation/conjugation pathway
  • such inhibition or enhancement is specific, as described above
  • such modulating ligands preferably interact with NAEl-alpha to inhibit or enhance the formation of NAE1 heterodimer, the formation of NEDDS adenylate, the formation of a thiol ester bond between NEDDS and NAE1, and/or transfer of NEDDS to NEDDS-conjugating enzyme
  • the invention provides methods for modulating the activation and/or conjugation of NEDDS
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ltgand NAEl-alpha or a recombinant expression unit hich expresses NAEl-alpha or an antagonist thereof to a biological system in which NEDDS is conjugated to another protein
  • the invention provides allelic variants ot NAE-1 alpha
  • This aspect of the invention further includes NAEl-alpha allelic ⁇ a ⁇ ant expression elements
  • Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NAEl-alpha, or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences, and recombinant expression units w hich express NAEl-beta or antisense transcripts or dominant negative mutants thereof
  • the invention provides methods for modulating auxin response in plants
  • N ⁇ El-alpha shares 39% identity and 61% conserved residues w ith Auxl in A Thaliai i w hich is involved in signal transduction in the auxin response in plants This suggests that antagonists of NAEl-beta and/or NAEl-alpha should down-regulate the auxin response, and that expression of NAEl-beta and/or NAEl-alpha should up-regulate the auxin response
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand ot NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAE1 or an antagonist thereof to a plant that is under auxin treatment
  • the invention provides methods for modulating the biological role of APP and/or beta peptide accumulation in a biological system
  • the present inventors hav e discovered that NAEl-alpha is substantial the same protein as amyloid precursor protein binding prote ⁇ n-1 (APP-BP1) This suggests that antagonists or agonists of NAEl-beta and/or NAEl-alpha should modulate APP function, including its role in beta peptide accumulation
  • One prererred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAE1 or an antagonist thereof to a biological system
  • the invention provides t o new purified NEDDS- conjugating enzymes and allelic v ariants thereof
  • NEDDS-conjugating enzymes NEDDS-conjugating enzymes
  • NCE2 NEDDS-conjugating enzyme
  • the invention provides NEDDS-conjugation enzyme expression elements
  • Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NCEl or NCE2 or dominant negative mutants thereof, or capable of expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such recombinant expression elements, preferably expression vectors
  • the invention provides methods for identifying NCElBMs and NCE2BMs
  • One preferred method according to this aspect of the invention comprises screening for NCElBMs or NCE2BMs by contacting purified NCEl or NCE2 according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NCEl or NCE2
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NCEl or NCE2 based upon structural information from the purified NCEl or NCE2 prov ided by the invention and determining whether such rationally designed molecules bind specifically to NCEl or NCE2.
  • This aspect of the invention includes NCElBMs and NCE2BMs identified by the methods according to the invention
  • NCElBMs and NCE2BMs can be used in conventional assays to detect the presence or absence, and/or quantity of NCEl or NCE2, or NCEl or NCE2/N ⁇ DDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2, or NCEl or NCE2/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NCEIBM or NCE2BM to a biological sample, allowing the detectable NCEIBM or NCE2BM to bind to, respectively NCEl or NCE2, or respectively NCEl or NCE2/ NEDDS complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NCEIBM or NCE2BM and NCEl or NCE2, or NCEl or NCE2/NEDDS complex.
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NCEl or NCE2 can also be used in conventional assays to detect the presence or absence of NCEl or NCE2 nucleic acid in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2 nucleic acid in a biological sample
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NCEl or NCE2 nucleic acid
  • the invention provides methods for identifying modulating ligands of NCEl or NCE2 Some NCElBMs or NCE2BMs are capable of acting as antagonists or agonists of, respectively NCEl or NCE2
  • the method according to this aspect of the invention comprises providing NCElBMs or NCE2BMs to an assay system for NC
  • the invention provides modulating ligands of NCEl or NCE2
  • Preferred modulating ligands are NCElBMs or NCE2BMs which act as antagonists, interfering with the ability of NCEl or NCE2 to participate in the
  • NCElBMs or NCE2BMs which act as agonists, enhancing the ability of, respectively NCEl or NCE2 to participate in the NEDDS-activation/conjugation pathway
  • NCElBMs or NCE2BMs preferably interact with NCEl or NCE2 to inhibit or enhance the formation of a thiol ester bond between NEDDS and NCEl or NCE2 and/or transfer of NEDDS to its target protein
  • the invention provides methods for modulating the conjugation of NEDDS or its transfer to a target protein
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NCEl or NCE2 oi a recombinant expression unit which expresses NCEl or NCE2 or an antagonist thereof to a biological system in which NEDDS is conjugated to another protein
  • the invention provides ohgonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 2 or Figure 5 Preferred embodiments include hybridization probes and antisense ohgonucleotides
  • Figure 1 shows the nucleotide [SEQ- ID. NO. 1] and predicted amino acid sequence [SEQ. ID. NO. 2] for NAEl-beta, with the two tryptic peptide sequences highlighted by underline.
  • Figure 2 shows the nucleotide [SEQ. ID. NO. 3] and predicted amino acid sequence [SEQ. ID. NO. 4] for NEDDS-conjugating enzyme 1 (NCEl), with the active Cys residue indicated.
  • Figure 3 shows the alignment of NCEl with yeast Ubcl2.
  • Figure 4 shows results of an assay for thioester bond formation between NEDD-8 and NCEl.
  • Figure 5 shoyvs the nucleotide [SEQ. ID. NO. 5] and predicted ammo acid sequence [SEQ. ID. NO. 6] for NEDDS-conjugating enzyme 2 (NCE2), with the active Cys residue indicated.
  • Figure 6 shows homology between NCE2 and a C. clegans gene of unknown function.
  • Figure 7 shows the sequence alignment of NCEl and NCE2 with known Ubc proteins
  • Figure 8 shows results of an assay for thioester bond formation between
  • the invention relates to covalent modification of proteins through their conjugation ith other proteins More particularly, the invention relates to the modulation of such conjugation involving the protein NEDDS
  • the invention provides compositions and methods for detecting and/or modulating the conjugation of NEDDS and/or its transfer to a target protein, as well as compositions and methods for discovering molecules which are useful in detecting and/or modulating the conjugation of NEDDS and/or its transfer to a target protein
  • the present invention arises from the purification and characterization of novel NEDDS activating and conjugating enzymes
  • the invention provides purified NEDDS-activating protein beta subunit (NAEl-beta)
  • NEDDS-activating protein beta subunit or "NAEl-beta”
  • the primary amino acid sequence of a preferred embodiment of such NAEl-beta protein is shown in Figure 1
  • the term "NEDDS-activating protein beta subunit”, or "NAEl-beta” is intended to include allelic variants thereof
  • An "allelic variant”, as used herein, is a protein having at least about 75% amino acid sequence, preferably at least about 85%, more preferably at least about 95%, and most preferably at least about 99% identity to the amino acid sequence set forth in SEQ ID NO 1, or to a portion or protein conjugate thereof which retains the biological activity of NAEl-beta (as part of the NAE1 heterodimer) to form a thioester linkage with NEDDS at a rate faster than that achieved bv human ubiquitin activating enzyme 1, preferably at least 2-fold faster, more preferably
  • the invention provides NAEl-beta expression elements
  • Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NAEl-beta or dominant negativ e mutants thereof, or capable of expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such recombinant expression elements, preferably expression v ectors
  • amino acid sequence identity and homology are determined using the program Clustal V Version 1 6 to do sequence alignment (Thompson et al , Nucleic Acids Res 22 4673-4680 (1994))
  • the program GeneDoc Version 2 2 was used A sequence is "specifically homologous" to another sequence if it is sufficiently homologous to specifically hvb ⁇ dize to the exact complement of the sequence
  • a sequence is ' specifically complementary” to another sequence if it is sufficiently homologous to specifically hybridize to the sequence
  • Dominant negative mutants' are proteins derived from NAEl-beta or NAEl-alpha which inhibit the biological activity of NAE1
  • Preferred dominant negative mutants include allelic variants in which the C at position 216 is substituted, preferably by S Additional preferred dominant negativ e mutants interfere with association of native NAEl-beta with native NAEl-alpha and can be derived from either NAEl-beta and NAEl-alpha
  • Such dominant negative mutants can be prepared by art recognized procedures (see e g , Townsley et al , Proc Natl Acad Sci USA 94 2362-2367 (1997))
  • such dominant negative mutant is a protein or peptide having from 50% ammo acid sequence identity to about 99% identity to the amino acid sequence set forth in SEQ ID NO 2, or to a portion or protein conjugate thereof which inhibits the biological activity of ⁇ AE1 to form a thioester linkage with NEDDS or transfer NEDDS to a NEDDS conjugating enzvme, under conditions
  • the invention provides methods for identifying NAElBBMs
  • One preferred method according to this aspect of the invention comprises screening for NAElBBMs by contacting purified NAEl-beta according to the inv ention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAEl-beta
  • binding affinity of the molecule is at least 5-fold greater than affinity for
  • a"NAEl-beta-b ⁇ nd ⁇ ng molecule is a molecule or macromolecule hich binds under ph siological conditions to NAEl- beta "Binds under physiological conditions” means forming a cov lent or non-covalent association with an affinity of at least 10 6 M -1 , most preferably at least 10 1 ' M- 1 , either in the body, or under conditions w hich approximate physiological conditions with respect to ionic strength, e g , 140 mM NaCl, 5 mM MgCl 2
  • a "population of molecules”, as used herein, refers to a plurality of identical molecules
  • A"m ⁇ xed population of molecules' refers to a plurality of molecules wherein more than one type of molecule is present
  • an NAE1BBM according to the inv ention is a peptide or a peptidomimetic
  • a peptide is a molecule comprised ot a linear array of amino acid residues connected to each other in the linear array by peptide bonds
  • Such peptides according to the invention may include from about three to about 500 amino acids, and may further include secondary, tertiary or quaternary structures, as well as intermolecular associations with other peptides or other non-peptide molecules
  • Such intermolecular associations may be through, without limitation, covalent bonding (e g , through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hy drogen bonding, lon-dipole interactions, dipole-dipole interactions, or any combination of the above
  • such an NAE1BBM comprises a complementarity determining region of an antibody which binds under physiological conditions to a peptide-
  • compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers
  • NAElBBMs include small molecules, which can be identified using screening or rational design approaches as discussed later herein
  • NAElBBMs can be used in conventional assays to detect the presence or absence, and/or quantity of NAEl-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta, NAE1 heterodimer, or NAE1 heterodimer/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NAEIBBM to a biological sample, allowing the detectable NAEIBBM to bind to NAEl-beta, NAEl heterodimer, or NAEl heterodimer/NEDDS complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAEIBBM and NAEl-beta, NAEl-heterodimer, or NAEl heterodimer/NEDDS complex
  • a detectable NAEIBBM is an NAEIBBM which can be detected in an assay
  • detection is preferably through the direct or indirect binding of a tag or label on the NAEIBBM
  • Direct or indirect binding means that the tag or label may be directly connected to the NAEIBBM by intermolecular association, or may be connected via intermediate molecules to the NAEIBBM by intermolecular association
  • intermolecular associations may be through, without limitation, cov alent bonding (e g , through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, lon-dipole interactions, dipole-dipole interactions, or any combination of the above
  • Preferred tags and labels include, without limitation, radioisotopes, heavy metals, fluorescent labels, chemoluminescent labels, enzymes and enzyme substrates
  • Preferred biological samples include blood, serum, plasma, cells, tissue portions, and cell or tissue extracts
  • the method according to this aspect of the invention takes the form of
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAEl-beta can also be used in conventional assays to detect the presence or absence of NAEl-beta nucleic acid in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta nucleic acid in a biological sample
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAEl-beta nucleic acid
  • Particularly preferred embodiments include Northern blotting, dot or slot blotting, and polymerase chain reaction
  • the invention provides methods for identify ing modulating ligands of NAEl-beta
  • Some NAElBBMs are capable of acting as antagonists or agonists of NAEl-beta
  • the method according to this aspect of the inv ention comprises providing NAElBBMs to an assay system for NAEl-beta participation in the NEDDS-activation/conjugation pathway, and determining whether such NAElBBMs interfere with or enhance the ability of NAEl-beta to participate in the NEDDS-activation/conjugation pathway
  • the NAElBBMs are preferably prov ided as a population of molecules (most pieferably rationally designed molecules) or as a mixed population of molecules, as for example in a screening procedure
  • This aspect of the invention includes antagonists or agonists of NAEl-beta identified by this method according to the invention Assessment of ability to "interfere w ith or enhance the ability to participate in the NEDDS-activation/conjugation pathw ay" can conveniently be
  • the invention provides modulating ligands of NAEl- beta Preferred modulating ligands are NAElBBMs which act as antagonists interfering with the ability of NAEl-beta to participate in the NEDDS- activation/ conjugation pathway
  • NAElBBMs which act as antagonists interfering with the ability of NAEl-beta to participate in the NEDDS- activation/ conjugation pathway
  • Other preferred modulating ligands are NAElBBMs which act as agonists, enhancing the ability of NAEl-beta to participate in the NEDDS-activation/conjugation pathway
  • such inhibition or enhancement is specific, .
  • the modulating ligand interferes with or enhances the ability of NAEl-beta to participate in the NEDDS activation/ conjugation pathw ay at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect
  • concentration of the ligand required for NEDDS activation/conjugation modulating activ ity is at least 2- fold lower, more preferably at least 5-fold lower, even more preferably at least 10- fold lower, and most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.
  • NAElBBMs preferably interact with NAEl-beta to inhibit or enhance the formation of NAEl heterodimer, the formation of NEDDS adenylate, the formation of a thiol ester bond between NEDDS and NAEl, and/or transfer of NEDDS to NEDDS- conjugating enzyme
  • the invention provides methods for modulating the conjugation of NEDDS to NAEl or its transfer to a NEDDS conjugating enzyme or a target protein
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAEl-beta or a recombinant expression unit which expresses NAEl-beta or an antagonist thereof to a biological system in which NEDDS is conjugated to a NEDDS conjugating enzyme or a target protein
  • biological system includes in vitro cell or tissue extracts, cell cultures, tissue cultures, organ cultures, living plants and animals, including mammals, including without limitation humans and mice
  • an "antagomst” is a molecule w hich inhibits the biological activity of NAEl
  • the invention provides ohgonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 1
  • Preferred embodiments include hybridization probes and antisense ohgonucleotides
  • the term ohgonucleotide includes polymers of two or more deoxv ⁇ bonucleotide, or any modified nucleoside, including 2'-halo-nucleos ⁇ des, 2 -O-substituted ⁇ bonucleosides, deazanucleosides or any combination thereof
  • such ohgonucleotides have from about 10 to about 100 nucleosides, more preferably from about 15-50, and most preferably from about 15 to 35
  • Such monomers may be coupled to each other by any of the numerous known internucleoside linkages
  • these internucleoside linkages may be phosphodiester, phosphot ⁇ ester, phosphorothioate, or phosphoramidate linkages, or combinations thereof
  • the term ohgonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines
  • the invention provides methods for identify ing NAEl- alpha binding molecules (NAElABMs)
  • NAElABMs NAEl- alpha binding molecules
  • the present inventors have identified the alpha subunit of the NAEl heterodimer (NAEl-alpha) Surprisingly , it has an amino acid sequence which is substantially identical to a protein previously identified as amyloid precursor protein binding protein 1 (APP-BP1, see Chow et al, J. Biol. Chem.
  • One preferred method according to this aspect of the invention comprises screening for NAElABMs by contacting purified NAEl- alpha and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAEl-alpha, or preferably to NAEl heterodimer
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NAEl-alpha based upon structural information from the NAEl-alpha protein identified by the present inventors and determining whether such rationally designed molecules bind specifically to NAEl-alpha
  • This aspect of the invention includes NAElABMs identified by the methods according to the invention.
  • binding affinity of the molecule for NAEl-alpha is at least 5-fold greater than affinity for unrelated proteins, more preferably at least 10-fold greater, still more preferably at least 50-fold greater, and most preferably at least 100-fold greater.
  • NAElABMs identified by the methods according to the invention
  • a"NAEl-alpha-binding molecule or "NAE1ABM”
  • binds under physiological conditions binds under physiological conditions
  • population of molecules and “mixed population of molecules” are as used previously.
  • an NAE1ABM according to the invention is a peptide or a peptidomimetic.
  • the term peptide is as used previously
  • such an NAE1ABM comprises a complementarity determining region of an antibody which binds under physiological conditions to a peptide-containing epitope of NAEl-alpha, or a peptidomimetic of such a complementarity determining region
  • the term complementarity determining region of an antibody' is as used previously Compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or butters
  • Additional preferred NAElABMs according to the invention include small molecules, w hich can be identified using screening or rational design approaches as discussed later herein
  • NAElABMs can be used in conventional assays to detect the presence or absence, and/or quantity of NAEl-alpha, NAEl heterodimer, or NAEl heterodimer/NEDDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha, NAEl heterodimer, or NAEl heterodimer/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NAEIABM to a biological sample, allowing the detectable NAEIABM to bind to NAEl-alpha, NAEl heterodimer, or NAEl heterodimer/NEDDS complex, it any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NAEIABM and NAEl-alpha, NAEl- heterodimer, or NAEl heterodimer/NEDDS complex
  • a detectable NAEIABM is an NAEIABM which can be detected in an assay
  • tags and labels include, without limitation, radioisotopes, heavy metals, fluorescent labels, chemolumtnescent labels, enzymes and enzyme substrates
  • Preferred biological samples include blood, serum, plasma, cells, tissue portions, and cell or tissue extracts
  • the method according to this aspect of the invention takes the form of a conventional ELISA or RIA
  • the method employs either direct or indirect immunofluorescence Additional preferred embodiments utilize in vivo imaging of cells expressing NAEl-alpha using conventional imaging agents directly or indirectly bound to an NAEIABM according to the invention
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NAEl-alpha can also be used in conventional assays to detect the presence or absence of NAEl-alpha nucleic acid in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha nucleic acid in a biological sample
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAEl-alpha nucleic acid
  • Particularly preferred embodiments include Northern blotting, dot or slot blotting, and polvmerase chain reaction.
  • the invention provides methods for identifying modulating ligands of NAEl-alpha
  • Some NAElABMs are capable of acting as antagonists or agonists of NAEl-alpha
  • the method according to this aspect of the invention comprises providing NAElABMs to an assay system for NAEl-alpha participation in the NEDDS-activation/conjugation pathway, and determining whether such NAElABMs interfere with or enhance the ability of NAEl-alpha to participate in the NEDDS-activation/conjugation pathway
  • the NAElABMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure
  • This aspect of the invention includes antagonists or agonists of NAEl-alpha identified by this method according to the invention
  • Assessment of ability to "interfere with or enhance the ability to participate in the NEDD8- activation/conjugation pathway" can conveniently be carried out using an in vitro activity system, as later described herein Preferably,
  • the invention provides a purified complex of NAEl- beta and NAEl-alpha, or of NAEl-beta, NAEl-alpha and NEDDS, or a purified complex of portions thereof
  • the term ' complex means in covalent or noncovalent association, preferably with an affinity greater than lO mole
  • ' purified is as used previously
  • the invention provides modulating ligands of NAEl- alpha
  • Preferred modulating ligands are NAElABMs which act as antagonists, interfering with the ability of NAEl-alpha to participate in the NEDDS- activation/conjugation pathway
  • Other preferred modulating ligands are NAElABMs which act as agonists, enhancing the ability of NAEl-alpha to participate in the NCDDS-activation/conjugation pathway
  • such inhibition or enhancement is specific, i e
  • the modulating ligand interferes with or enhances the ability of NAEl-alpha to participate in the NEDDS activation/ conjugation pathway at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect
  • the concentration of the ligand required for NEDDS activation/conjugation modulating activity is at least 2-fold lower, more preferably at least 5-fold lower, even more preferably at least 10-fold lower, and most preferably at least 20-fold lower than the
  • biological system includes in vitto cell or tissue extracts, cell cultures, tissue cultures, organ cultures, living plants and animals, including mammals, including without limitation humans and mice
  • an "antagonist' is a molecule which inhibits the biological activity of NAEl
  • the invention provides allelic variants of NAE-1 alpha
  • An ' allelic variant' is a protein having at least about 75" amino acid sequence, preferably at least about S5%, more preferably at least about 95%, and most preferably at least about 99% identity to the ammo acid sequence of NAEl-alpha, or to a portion or protein conjugate thereof which retains the biological activity of NAEl-alpha to form a heterodimer with NAEl-beta which is active in the NEDDS activation/conjugation pathway
  • This aspect of the inv ention further includes NAEl-alpha allelic variant expression elements Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding NAEl-alpha, or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences, and recombinant expression units w hich express NAEl-beta or antisense transcripts or dominant negative mutants thereof
  • the invention provides methods for modulating auxin response in plants
  • the present inventors have discovered that NAEl-alpha shares 39% identity and 61% conserv ed residues with Auxl in A Thaliana which is involved in signal transduction in the auxin response in plants This suggests that antagonists of NAEl-beta and/or NAEl-alpha should down-regulate the auxin response, and that expression ot NAEl-beta and/or NAEl-alpha should up-regulate the auxin response (see Leyser et al , Nature 3_64_ 161-164 (1993))
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAEl or an antagonist thereof to a plant that is undergoing auxin treatment
  • the invention provides methods for modulating the biological function of APP and/or beta peptide accumulation in a biological system.
  • NAEl-alpha is substantially the same protein as amyloid precursor protein binding protem-1 (APP-BP1) This suggests that antagonists or agonists of NAEl-beta and/or NAEl-alpha should modulate APP function, including its role in beta peptide accumulation
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAEl or an antagonist thereof to a biological system.
  • the invention provides two new purified NEDD8- conjugating enzymes
  • the primary amino acid sequence of a preferred embodiment of a first such NEDDS-conjugating enzyme (NCEl) is shown in Figure 2
  • the primary amino acid sequence of a preferred embodiment of a second such NEDDS- conjugating enzyme (NCE2) is shown in Figure 5
  • the terms "NEDD8- conjugating enzyme 1", “NCEl”, “NEDDS-conjugating enzyme 2", and “NCE2" are intended to include allelic variants thereof
  • An "allelic variant”, as used herein, is a protein having at least about 50 ..
  • amino acid sequence identity more preferably at least about 757.., ev en more preferably at least about 85 'X still more preferably at least about 95%, and most preferably at least about 997. identity to the amino acid sequence set forth in SEQ ID NO 4 or SEQ ID NO 6, or to a portion or protein conjugate thereof which retains the biological activ lty ot NCEl or NCE2 to form a thioester linkage with NEDDS under conditions as described in the examples below at a rate at least 107.
  • such biologically active portion comprises an amino acid sequence spanning residue 111 in Figure 2 or residue 116 in Figure 5, more preferably comprises at least about 25 additional amino acids of respectively NCEl or NCE2, even more preferably at least about 50 additional amino acids of respectively NCEl or NCE2, still more preferably at least about 75 additional amino acids of respectively NCEl or NCE2, yet even more preferably at least about 100 additional ammo acids of respectively NCEl or NCE2, most preferably at least about 150 additional amino acids from respectively NCEl or NCE2
  • allelic variants have the biological activity of NCEl or NCE2, as discussed above
  • such allelic variants are either rationally designed or naturally occurring allelic variants, i c , they are expressed in actual individual mammals, most preferably from actual individual humans or mice Rationally designed allelic variants can be produced according to standard art-recognized procedures (see
  • the invention provides NEDDS-conjugation enzyme expression elements
  • Such elements include, w ithout limitation, isolated or recombinant nucleic acid sequences encoding NCEl or NCE2 or dominant negative mutants thereof, or capable of expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such recombinant expression elements, preferably expression vectors
  • the terms "specifically homologous”, “specifically complementary " and “specifically hybridizes” are as used previously
  • a "recombinant expression element” is a nucleic acid sequence which encodes NCEl or NCE2, or a portion encoding at least 20 contiguous amino acids thereof, or a dominant negativ e mutant thereof, or is capable of expressing an antisense molecule specifically complementary thereto, or a sense molecule specifically homologous thereto wherein the recombinant expression unit may be in the form of linear DNA or RNA, covalently closed circular DNA or RNA, or as part of
  • nucleic acid sequence identity with the nucleic acid sequence set forth in SEQ ID NO 2 OR SEQ ID NO 4, more preferably at least 907., even more preferably at least 95%, and most preferably at least 99%, and encode a protein or peptide having either NCEl or NCE2 biological activity or activity as a dominant negative mutant thereof, as further described below
  • Dominant negative mutants are proteins or peptides derived from NCEl or NCE2 which inhibit the biological activity of, respectively NCEl or NCE2
  • Preferred dominant negative mutants include variants in which the C at position 111 of NCEl or position 116 of NCE2 is substituted, preferably by S Preferred dominant negative mutants interfere w ith association of NEDDS and NCEl or NCE2 and can be derived from, respectively, NCEl or NCE2
  • Other preferred dominant negative mutants interfere with conjugation of NEDDS to a target protein and can be derived from either NCEl or NCE2
  • Such dominant negative mutants can be prepared by art recognized procedures (see c g , Townsley et al , Proc Natl Acad Sci USA 94 2362-2367 (1997)) Preferably, such dominant negative mutant is a protein or peptide having from 507.
  • inhibitory portion comprises an amino acid sequence spanning residue 111 in Figure 2 or residue 116 in Figure 5, more preferably comprises at least about 25 additional amino acids of respectively NCEl or NCE2, even more preferably at least about 50 additional amino acids of respectively NCEl or NCE2, still more preferably at least about 75 additional amino acids of respectively NCEl or NCE2, yet even more preferably at least about 100 additional ammo acids of respectively NCEl or NCE2, most preferably at least about 150 additional amino acids from respectively NCEl or NCE2
  • the invention provides methods for identifying NCElBMs and NCE2BMs
  • One preferred method according to this aspect of the invention comprises screening for NCElBMs or NCE2BMs by contacting purified NCEl or NCE2 according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NCEl or NCE2
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind NCEl or NCE2 based upon structural information from the purified NCEl or NCE2 provided by the invention and determining whether such rationally designed molecules bind specifically to NCEl or NCE2.
  • Molecules that bind specifically to NCEl or NCE2 are molecules that bind to NCEl or NCE2 with greater affinity than to other unrelated proteins
  • binding affinity of the molecule is at least 5-fold greater than affinity for unrelated proteins, more preferably at least 10-fold greater, still more preferably at least 50-fold greater, and most preferably at least 100-fold greater
  • This aspect of the invention includes NCElBMs or NCE2BMs identified bv the methods according to the invention As used herein, a"NCEl or NCE2 -binding molecule", or "NCEIBM or
  • NCE2BM is a molecule or macromolecule which binds under physiological conditions to, respectively NCEl or NCE2
  • the terms binds under physiological conditions", “population of molecules” and “mixed population of molecules” are as used previously
  • an NCEIBM or NCE2BM according to the invention is a peptide or a peptidomimetic
  • the term ' peptide is as used previously
  • such an NCEIBM or NCE2BM comprises a complementarity determining region of an antibody w hich binds under physiological conditions to a peptide-contammg epitope of, respectiv ely NCEl or NCE2, or a peptidomimetic of such a complementarity determining region
  • the term "complementarity determining region of an antibody" is as used previously.
  • compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers
  • Additional preferred NCElBMs and NCE2BMs according to the invention include small molecules, which can be identified using screening or rational design approaches as discussed later herein
  • NCElBMs and NCE2BMs can be used in conventional assay s to detect the presence or absence, and/or quantity of NCEl, or NCE2, or NCEl or NCE2/NEDDS complex in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2, or NCEl or NCE2/NEDDS complex in a biological sample
  • Such methods comprise providing a detectable NCEIBM or NCE2BM to a biological sample, allowing the detectable NCEIBM or NCE2BM to bind to NCEl, or NCEl or NCE2/NEDDS complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable NCEIBM or NCE2BM and, respectively, NCEl or NCE2, or NCEl or NCE2/NEDDS complex
  • a detectable NCEIBM or NCE2BM is an NCEIBM or NCE2B which can be detected in an assay Such detection is preferably through the direct or indirect binding of a tag or label on the NCEIBM or NCE2BM
  • tags and labels include, ithout limitation, radioisotopes, heavy metals, fluorescent labels, chemoluminescent labels, enzymes and enzyme substrates
  • Preferred biological samples include blood, serum, plasma, cells, tissue portions, and cell or tissue extracts
  • the method according to this aspect of the invention takes the form of a conventional ELISA or RIA
  • the method employs either direct or indirect immunofluorescence Additional preferred embodiments utilize in vivo imaging of cells expressing NCEl or NCE2 using conventional imaging agents directly or indirectly bound to an NCEIBM or NCE2BM according to the invention
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding NCEl or NCE2 can also be used in conventional assays to detect the presence or absence of NCEl or NCE2 nucleic acid in a biological sample
  • the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2 nucleic acid in a biological sample
  • such assays are nucleic acid hybridization and/or amplification assays, such assay s comprising providing to the biological sample a nucleic acid sequence w hich is specifically complementary to NCEl or NCE2 nucleic acid
  • Particularly preferred embodiments include Northern blotting, dot or slot blotting and poly merase chain reaction
  • the invention provides methods for identify ing modulating ligands of NCEl or NCE2
  • Some NCElBMs and NCE2BMs are capable of acting as antagonists or agonists of, respectively , NCEl and NCE2
  • the method according to this aspect of the invention comprises providing NCElBMs or NCE2BMs to an assay system for, respectively, NCEl or NCE2 participation in the NEDDS-activation/conjugation pathway, and determining whether such NCElBMs or NCE2BMs interfere with or enhance the ability of NCEl or NCE2 to participate in the NEDDS-activation/conjugation pathway
  • the NCElBMs or NCE2BMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure
  • This aspect of the invention includes antagonists or agonists of NCEl or NCE2 identified by this method according to the invention Assessment of ability to ' interfere with or enhance the ability to participate in the NEDDS
  • the invention provides modulating ligands of NCEl or NCE2
  • Preferred modulating ligands are NCElBMs or NCE2BMs which act as antagomsts, interfering with the ability of, respectively, NCEl or NCE2 to participate in the NEDDS-activation/conjugation pathway
  • Other preferred modulating ligands are NCElBMs or NCE2BMs which act as agonists, enhancing the ability of, respectively NCEl or NCE2 to participate in the NEDDS-activation/conjugation pathway
  • such inhibition or enhancement is specific, i f
  • the modulating ligand interferes with or enhances the ability of NCEl or NCE2 to participate in the NEDDS activation/ conjugation pathway at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect
  • the concentration ot the ligand required for NEDDS activation/conjugation modulating activity is at least 2-fold lower, more
  • the invention provides methods for modulating the formation of a thiol ester bond between NEDDS and NCEl or i ⁇ CE2, or transfer of NEDDS to a target protein
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of NCEl or NCE2 or a recombinant expression unit which expresses NCEl or NCE2 or an antagonist thereof to a biological system in which NEDDS is conjugated to another protein
  • biological system includes in vitro cell or tissue extracts, cell cultures, tissue cultures, organ cultures, living plants and animals, including mammals, including without limitation humans and mice
  • the invention provides ohgonucleotides that are specifically complementary to a portion of a nucleotide sequence show n in Figure 2 or Figure 5
  • the term ohgonucleotide is as used previously Certain embodiments of such ohgonucleotides are useful as antisense probes Other embodiments are useful as antisense ohgonucleotides for use in animal model or human therapeutic settings
  • the invention provides a purified complex ot NCEl and NEDDS, or of NCE2 and NEDDS
  • the terms ' complex" and "purified ' are as used previously
  • Nucleotide sequence coding the N-terminal 76 residues of human NeddS was obtained from a human leukocyte cDNA Library (Life Technologies Tech-L ⁇ ne SN1 , Inc) by nested polymerase chain reaction, using 5'-ccg tgt gca gcc cca aac tgg and 5'-aca ggg taa aga ggt aaa atg as the first round forward and reverse primer, respectively.
  • FII Fraction II was generated by collecting proteins that ere retained by an aruon-exchange gel (Q-Sepharose) while FI was obtained by further fractionation of unretained proteins by gel filtration Incubation of 12!
  • FII contains an activity which attaches NeddS to a protein via a DTT-sensitive linkage
  • One interpretation of this result is the presence ot a NeddS-con j ugating enzyme in FI which serves to accept NeddS from its activating enzvme in FII to form a 30 kDa thioester
  • NeddS-aftiruty gel was prepared by coupling purified NeddS to activated CH Sepharose 4B (Pharmacia) according to manufacturer's instructions and lead to the coupling of 5 mg of NeddS/ ml of gel beads 100 mg of FII protein in a 9 ml reaction buffer containing MgATP and an ATP regenerating system was applied to 1 ml of NeddS-immobihzed gel beads at room temperature The column was washed sequentially with 5 bed volumes of buffer A (50 mM T ⁇ s-HCl buffer, pH 7 5), buffer A with 0 5M NaCl, and buffer A A buffer containing 50 mM T ⁇ s-
  • the gel slices were digested with trypsin, peptides were extracted and purified by microbore reversed-phase HPLC (PE-Apphed Biosystems model 140A/ 1000S system) on Zorbax SB-CIS silica columns (1x150 mm), using linear gradients of acetonitrile in 0 0S7, aqueous trifluoroacetic acid
  • Region I contains the putative ATP binding site found in Ubal which is also present in yeast Uba2, and region II contains the PXCT sequence motif found in Ubal in which the cysteine residue was identified by mutational analysis to form thiolester linkage with ubiquitin
  • region II contains the PXCT sequence motif found in Ubal in which the cysteine residue was identified by mutational analysis to form thiolester linkage with ubiquitin
  • Example 5 Identification of Nae-alpha
  • the similarity between NeddS- and Smt3-act ⁇ vat ⁇ ng enzvme in their subunit structure suggested that p60 or Nae.- lph ⁇ - would also contain a sequence stretch that shares homology to the N-terminal portion of Ubal.
  • Example 6 Identification and cloning of NCEl
  • the putative human homolog of yeast Ubcl2 was identified bv searching the human EST database for clones having coding sequences that are homologous to the yeast protein
  • An initial search using the yeast protein sequence identified several clones Clone AA261836, which contains a coding sequence v ery similar to a region of the yeast protein was used to search for further EST clones
  • the search led to the construction of a contiguous consensus sequence from ov erlapping clones ⁇ s hich predicts a gene to encode a protein having 1S3 amino acids, w ith a predicted molecular mass of 20S99 Da
  • the contiguous nucleotide sequence w as obtained using nested PCR on a human leukocyte cDNA library
  • the first PCR used primers having the sequence GCAGGATGATCAAGCTGTTCTCGC (forward) and CGTGGCGGGGGTGGGTATGCGCCA (reversed
  • Example 7 Expression and purification of NCEl BL21 (DE3) bacterial cells (Novagen, Madison ⁇ VI, catalog no 69450-1) w ere transformed w ith pT7-7-UbcH12 plasmid using conventional procedures
  • the transformed bacteria w ere induced to express the NCEl protein by adding, to a final concentration of 1 mM, isopropyl-b-D-thiogalactopyranoside (IPTG) to an exponentially growing culture
  • IPTG isopropyl-b-D-thiogalactopyranoside
  • the culture was allowed to grow for an additional 3 hours at 37°C NCEl protein was purified from lysed cells by sequential anion exchange and size exclusion chromatography
  • anion exchange chromatography the bacterial extract was loaded at a protein/gel ratio of 15 mg protein/ml gel onto Q- Sepharose (Pharmacia, Piscataway, NJ) equilibrated w ith 50 mM HEPES (pH 7 8)
  • Example 8 Thioester formation between NCEl and NEDDS Proteins (as indicated below ) w ere incubated in a reaction buffer containing 25 mM Hepes (pH7 0), 10 mM Mg 2 - and 1 mM ATP for 5 minutes at 30 3 C The reaction was stopped by addition of SDS sample loading buffer Each sample was divided into two aliquots, to one of which was added DTT to a final concentration of 10 mM The DTT-containing sample was heated in a 95°C bath for tw o minutes Samples were separated on 107.
  • HPNITETICLSLLREHSIDGTGWA This is the sequence of clone AA306113 and bears similarity to the active site of proteins in the UBC protein family
  • Clones were identified which had sequences overlapping the sequence of clone AA306113
  • the identified sequences of the overlapping EST clones were aligned bv the program CLUSTALW (See Thompson et al , Nucleic Acids Res 22 4673-46S0 (1994), or bv the program SeqMan (DNASTAR, Inc , Madison, WI) to yield a consensus sequence, CON1 CON 1 w s used to perform searches for additional clones ith overlapping sequences
  • BL21 (DE3) bacterial cells were transformed with pT7-7-UbcH17 plasmid using conventional procedures
  • the transformed bacteria were induced to express the NCE2 protein bv adding, to a final concentration of 1 M, ⁇ sopropy'1-b-D- thiogalactopyranoside (IPTG) to an exponentially growing culture
  • the culture w as allowed to grow for an additional 3 hours at 37°C NCE2 protein w as purified from lysed cells by sequential anion exchange and size exclusion chromatographv
  • the bacterial extract was loaded at a protein/gel ratio of 15 mg protein/ml gel onto Q-Sepharose (Pharmacia) equilibrated with 50 mM HEPES (pH 7 8) and ImM DTT NCE2 protein was retained bv the gel and eluted using a linear NaCl gradient in the gel equilibration buffer.
  • NCE2 Fractions containing NCE2 protein were determined by assaying for NEDDS thioester formation. NCE2 was found to elute at O.S M NaCl. Active fractions were pooled and concentrated by microfiltration and then subjected to size exclusion chromatography on Superdex-75 (Pharmacia) using a column buffer of 50 mM HEPES (pH 7.8), 1 mM DTT and 50 mM NaCl. Fractions were assayed for ⁇ 5 I- NEDDS-thioester formation. NCE2 eluted at a volume expected for a 21 kDa protein, suggesting that it exists as a monomer.
  • NCE2 protein either purified or from bacterial lysate
  • ⁇ 5 I-NEDD8 10 ⁇ cpm/ ⁇ g
  • 10 mM MgCl 2 10 mM MgCl 2
  • 1 mM ATP 20nM purified NAEl or ubiquitin-activating enzyme.
  • the reaction was allowed to proceed at 30°C for 5 minutes.
  • the reaction was stopped by adding SDS-sample buffer either with or without 10 mM DTT.
  • the samples were subjected to SDS-PAGE and autoradiography.
  • the active site cysteine of a cloned NCEl or NCE2 is assigned bv examining the sequence alignment with known Ubc proteins (see Figure 6 for alignment)
  • the active site cysteine is replaced by a se ⁇ ne using standard site-specific mutagenesis
  • the mutant protein is expressed in bacteria and purified
  • the ability of the mutant protein to form a stable oxygen ester with NEDDS is established as described in Examples 8 and 11 above, except that the bond formation is not labile in DTT
  • Dominant negative mutant activity is then established by introducing the mutant protein in increasing concentrations in an assay as described in Examples 8 and 11 abov e and demonstrating dose-dependent inhibition of NEDDS/NCE1 or NCE2 complex formation

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Abstract

L'invention porte sur la modification covalente des protéines par leur conjugaison à d'autres protéines. Plus spécifiquement, l'invention porte sur la modulation de la conjugaison de la protéine NEDD8. L'invention porte également sur des compositions et des procédés de détection et/ou de modulation de l'activation et/ou de la conjugaison de NEDD8, ainsi que sur des compositions et des procédés permettant de rechercher les molécules qui sont utiles dans la détection et/ou la modulation de l'activation et/ou la conjugaison de NEDD8. Cette invention résulte de la purification et de la caractérisation des nouvelles enzymes d'activation et de conjugaison des protéines NEDD8.
PCT/US1998/027141 1997-12-19 1998-12-18 Proteines humaines responsables de l'activation et de la conjugaison de nedd8 WO1999032624A1 (fr)

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EP98964164A EP1037988A1 (fr) 1997-12-19 1998-12-18 Proteines humaines responsables de l'activation et de la conjugaison de nedd8
IL13687398A IL136873A0 (en) 1997-12-19 1998-12-18 Proteins responsible for nedd8 activation
JP2000525543A JP2001526890A (ja) 1997-12-19 1998-12-18 Ｎｅｄｄ８活性化および共役化を担うヒトタンパク質
AU19350/99A AU758077B2 (en) 1997-12-19 1998-12-18 Human proteins responsible for NEDD8 activation and conjugation
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DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103407, Database accession no. AA401865 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103408, Database accession no. AA401968 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103409, Database accession no. AA261836 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103410, Database accession no. AA577116 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103411, Database accession no. AA384235 *
DATABASE EMBL NUCLEOTIDE SEQU 1 January 1900 (1900-01-01), XP002103412, Database accession no. AA671071 *
KAMITANI T ET AL: "Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein.", JOURNAL OF BIOLOGICAL CHEMISTRY, (1997 NOV 7) 272 (45) 28557-62. JOURNAL CODE: HIV. ISSN: 0021-9258., United States, XP002103404 *
KUMAR, SHARAD ET AL: "Cloning of a cDNA which encodes a novel ubiquitin-like protein", BIOCHEM. BIOPHYS. RES. COMMUN. (1993), 195(1), 393-9 CODEN: BBRCA9;ISSN: 0006-291X, XP002103405 *
OSAKA F ET AL: "A new NEDD8 -ligating system for cullin-4A.", GENES AND DEVELOPMENT, (1998 AUG 1) 12 (15) 2263-8. JOURNAL CODE: FN3. ISSN: 0890-9369., United States, XP002103402 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1840215A1 (fr) * 2004-12-28 2007-10-03 Daiichi Pharmaceutical Co., Ltd. Procede inhibant l'activite de la telomerase et inhibiteur
EP1840215A4 (fr) * 2004-12-28 2008-03-26 Daiichi Seiyaku Co Procede inhibant l'activite de la telomerase et inhibiteur

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EP1037988A1 (fr) 2000-09-27
AU1935099A (en) 1999-07-12
IL136873A0 (en) 2001-06-14
JP2001526890A (ja) 2001-12-25
AU758077B2 (en) 2003-03-13

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