HUMAN PROTEINS RESPONSIBLE FOR NEDD8 ACTIVATION AND CONJUGATION

Abstract: 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 NEDD8. The invention provides compositions and methods for detecting and/or modulating the activation and/or conjugation of NEDD8, as well as compositions and methods for discovering molecules which are useful in detecting and/or modulating the activation and/or conjugation of NEDD8. The present invention arises from the purification and characterization of novel NEDD8 activating and conjugating enzymes.

Description: HUMAN PROTEINS RESPONSIBLE FOR NEDD8 ACTIVATION AND CONJUGATION BACKGROUND OF THE INVENTION Field of the invention 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. Summary of the related art Covalent modification of proteins through their conjugation with other proteins is an important biological mechanism for regulating protein metabolism and biological activity. Hershko and Ciechanover, Annu. Rev. Biochem. 6V. 761-807 (1992) discloses conjugation of ubiquitin, one of the most conserved eukaryotic proteins, to other proteins through an enzymatic mechanism, as well as its role in protein degradation. Rock et al. Cell 78: 761-771 (1994) discloses that ubiquitination of protein antigens is required for processing of such antigens. Murray, Cell 81: 149- 152 (1995), teaches that ubiquitination of cyclin is involved in cell cycle regulation. Scheffner et al., Cell 75: 495-505 (1993) discloses that ubiquitination of p53 is involved in degradation of this tumor suppressor. The enzymatic pathway for ubiquitination has been reasonably well defined. Jentsch, Annu. Rev. Genet. 26: 179-207 (1992) discloses that ubiquitination requires initial activation of a conserved C-terminal glycine residue by the ubiquitin activating enzyme, El, through formation of ubiquitin adenylate in an ATP- dependent process which liberates PPi, followed by thiol ester formation at a thiol site in El with release of AMP. Ubiquitin is then transferred to a thiol site in ubiquitin conjugating enzyme, E2, through formation of a thiol ester bond. 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. 272: 14001-14004 (1997), discloses that sentrin, a ubiquitin-like protein, appears to be processed similarly to ubiquitin, but has a smaller target protein repertoire than ubiquitin. Okura el al, J. Immunol. T l: 4277-4281 (1996) teaches that sentrin protects cells against anti-FAS and tumor necrosis factor-mediated cell death. Loeb and Haas, J. Biol. Chem. 267: 7806-7813 (1992), discloses that ubiquitin cross-reactive protein (UCRP), which contains two ubiquitin domains, is conjugated to a large number of intracellular proteins- Kumar et al., Biochem. Biophys. Res. Commun. 185: 1155- 1161 (1992), discloses another ubiquitin-like protein, called NEDDS, for Neural precursor cell-Expressed Developmentally Down regulated. Kamitani et al, J. Biol. Chem. 272: 28557-2S562 (1997), teaches that NEDDS is predominantly expressed in the nucleus and is conjugated to target proteins through a mechanism analogous to ubiquitination. These proteins, which covalently modify other cellular proteins, are important components of biological regulatory processes. The nuclear expression pattern and developmental regulation of NEDDS make it a particularly compelling candidate as an important regulatory molecule. There is a need, therefore to understand the role of NEDDS in biological regulation. Unfortunately, the lack of understanding about the specific proteins involved in NEDD8 conjugation has resulted in a lack of effective tools to probe the role of NEDD8- There is, therefore, a need for better tools to utilize in elucidating the role of NEDDS in biological regulation. Ideally, such tools would allow modulation of the activation and/or conjugation of NEDDS. BRIEF SUMMARY OF THE INVENTION 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 In a first aspect, the invention provides purified atιng protein beta suburut (NAEl-beta) The primary airuno acid sequence of a preferred embodiment of such NAEl-beta protein is shown in Figure 1 In a second aspect, the invention provides NAEl-beta expression elements Such 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 The purified protein and its structural information provided herein enables the preparation of NAEl-beta-binding molecules (NAElBBMs) Thus, in a third aspect, 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 Thus, in a fourth aspect, 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 Thus, in a fifth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta nucleic acid in a biological sample In preferred embodiments, 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 In a sixth aspect, the invention provides methods for identifying modulating ligands of NAEl-beta Some NAElBBMs are capable of acting as antagonists or agonists of NAEl-beta Thus, 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-actιvatιon/conjugatιon 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 In a seventh aspect, 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 In certain embodiments, 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 In an eighth aspect, 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 In a ninth aspect, 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 In a tenth aspect, the invention provides methods for identifying NAE1- alpha binding molecules (NAElABMs) 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. 221 11339-11346 (1996)) 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 Thus, in an eleventh aspect, 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 In preferred embodiments, the method according to this aspect of the invention is used to detect the presence or absence, and/or quantity of NAE1 heterodimer or NAE1 heterodimer/NEDDS complex in a biological sample 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 Thus, in a twelfth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha nucleic acid in such a biological sample In preferred embodiments, 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 In an thirteenth aspect, the invention provides methods for identifying modulating ligands of NAEl-alpha Some NAElABMs are capable of acting as antagonists or agonists of NAEl-alpha Thus, 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 In a fourteenth aspect 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 In a fifteenth aspect, the invention provides modulating ligands of NAEl- alpha Certain preferred 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 Preferably, such inhibition or enhancement is specific, as described above In certain embodiments, 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 In a sixteenth aspect, 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 In a seventeenth aspect, the invention provides allelic variants ot NAE-1 alpha This aspect of the invention further includes NAEl-alpha allelic \ aπant 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 In a eighteenth aspect, the invention provides methods for modulating auxin response in plants The present inventors have discovered that 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 In a nineteenth aspect, 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 proteιn-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 In an twentieth aspect, the invention provides t o new purified NEDDS- conjugating enzymes and allelic v ariants thereof 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 In a twenty-first aspect, 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 purified protein and its structural information provided herein enables the preparation of NCEl and NCE2 binding molecules, respectively NCElBMs and NCE2BMs Thus, in a twenty-second aspect, 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/NΕDDS complex in a biological sample Thus, in a twenty-third aspect, 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 Thus, in a twenty-fourth aspect, the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2 nucleic acid in a biological sample In preferred embodiments, 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 In a twenty-fifth aspect, 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 Thus, the method according to this aspect of the invention comprises providing NCElBMs or NCE2BMs to an assay system for NCEl or NCE2 participation in the NEDDS- activation/con j ugation 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 inv ention includes modulating ligands ot NCEl or NCE2 identified by this method according to the invention In a twenty-sixth aspect, the invention prov ides 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 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 In certain embodiments, such 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 In a twenty-seventh aspect, 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 In a twenty-eighth aspect, 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 BRIEF DESCRIPTION OF THE DRAWINGS 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 NEDDS and NCE2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 patents and publications cited herein reflect the knowledge in the art and are hereby incorporated by reference in entirety Any inconsistency between these patents and publications and the present disclosure shall be resolv ed in favor of the present disclosure In a first aspect, the invention provides purified NEDDS-activating protein beta subunit (NAEl-beta) The primary amino acid sequence of a preferred embodiment of such NAEl-beta protein is shown in Figure 1 However, 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 at least 5-fold and most preferably at least 10-fold Alternatively, an allelic v ariant can retain such biological activity and comprise a peptide sequence hav ing at least 70% amino acid identity to the peptide sequence corresponding to residues 46-118 in Figure 1, or at least 45% amino acid identity to the peptide sequence corresponding to residues 119-166, at least 55% amino acid identity to the peptide sequence corresponding to residues 175-239, or at least 35% amino acid identity to the peptide sequence corresponding to residues 276-375 Preferably such biologically active portion comprises at least the PXCT motif, wherein X can be any amino acid, preferably a hydrophobic amino acid, more preferably methiorune, leucine, or isoleucine, and most preferably methiorune More preferably, such biologically active portion comprises amino acid sequence of residues 214-217, more preferably comprises at least about 25 additional amino acids of NAEl-beta, ev en more preferably at least about 50 additional amino acids of NAEl-beta, still more preferably at least about 75 additional amino acids of NAEl-beta, et even more preferably at least about 100 additional amino acids of NAEl-beta, most preferably at least about 150 additional ammo acids from NAEl-beta Such allelic variants have the biological activity of NAEl-beta, as discussed above, which is the cataly tic monomer of the NAE1 heterodimer In alternative preferred embodiments, 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 g , international publication W095/ 18974) "Purified", as used herein means having less than about 25% by weight, and preferably less than about 10% by weight contamination with other proteins Such purified proteins may be obtained from natural sources, from recombinant expression, or by chemical synthesis "Protein", as used herein and hereinbelow is intended to encompass any polypeptide having at least 10 ammo acid residues In a second aspect, 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 For purposes of the invention, 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)) For view ing aligned sequences, the program GeneDoc Version 2 2 was used A sequence is "specifically homologous" to another sequence if it is sufficiently homologous to specifically hvbπdize 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 A sequence "specifically hybridizes" to another sequence if it hybridizes to form Watson-Crick or Hoogsteen base pairs either in the body, or under conditions which approximate physiological conditions w ith respect to ionic strength, e , 140 mM NaCl, 5 mM MgCl 2 Preferably, such specific hybridization is maintained under stringent conditions, e g , 0 2X SSC at 6S°C A ' recombinant expression element" is a nucleic acid sequence which encodes NAEl-beta, or a portion encoding at least 15 contiguous ammo acids thereof, or a dominant negative 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 a chromosome, provided however that it cannot be the native chromosomal locus tor NAEl-beta Preferred recombinant expression elements are vectors, which may include an origin of replication and are thus rep catable in one or more cell ty e Certain preferred recombinant expression elements are expression vectors and further comprise at least a promoter and passive terminator, thereby allo ing transcription of the recombinant expression element in a bacterial, fungal, plant insect or mammalian cell Preferred recombinant expression elements hav at least 75% nucleic acid sequence identity with the nucleic acid sequence set forth in SEQ ID NO 1, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99%, and encode a protein or peptide having either NAEl-beta biological activity, as described above, or activity as a dominant negative mutant thereof, as further described below "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)) Preferably, 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 as described in the following examples by at least 50%, preferably by at least 75%, more preferably by at least 90% and most preferably by at least 99% Preferably, such inhibitory portion comprises an ammo acid sequence spanning residue 216, more preferably comprises at least about 25 additional amino acids of NAEl-beta, or at least about 50 additional amino acids of NAEl-beta, or at least about 75 additional amino acids of NAEl-beta, or at least about 100 additional amino acids of NAEl-beta, or even at least about 150 additional amino acids of NAEl-beta For purposes of this aspect of the invention, the term spanning residue 216' means comprising amino acid residues in both the N-terminal and C-terminal directions from residue 216, as that residue is shown in Figure 1 Preferably , residue 216 itself may be substituted by one or more amino acids, more preferably from about 1 to about 50 ammo acids, or residue 216 may be absent Preferably the amino acids in the N-terminal and C-termmal directions from residue 216 are each independently within 20 amino acids of residue 216, as shown in Figure 1, more preferably within 10, even more preferably w ithin 5 and most preferably are immediately adjacent residue 216 as shown in Figure 1 The purified protein and its structural information prov ided herein enables the preparation of NAEl-beta-binding molecules (NAElBBMs) Thus, in a third aspect, 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 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 and amino acid sequence disclosed herein prov ided by the invention and determining w hether such rationally designed molecules bind specifically to NAEl-beta Molecules that bind specifically to NAEl-beta are molecules that bind to NAEl-beta with greater affinity than to other unrelated proteins Preferably, 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 NAElBBMs identified by the methods according to the invention As used herein, a"NAEl-beta-bιndιng molecule", or "NAE1BBM", 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"mιxed population of molecules' refers to a plurality of molecules wherein more than one type of molecule is present In certain preferred embodiments, an NAE1BBM according to the inv ention is a peptide or a peptidomimetic For purposes of the invention, 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 In certain preferred embodiments, such an NAE1BBM comprises a complementarity determining region of an antibody which binds under physiological conditions to a peptide-containing epitope of NAEl-beta, or a peptidomimetic of such a complementarity determining region For purposes of the invention, a complementarity determining region of an antibody" is that portion of an antibody which binds under physiological conditions to an epitope, including any framework regions necessary for such binding, and which is preferably comprised of a subset of amino acid residues encoded by the human heav y chain V, D and J regions, the human light chain V and J regions, and/or combinations thereof Examples of such preferred embodiments include an antibody, or an antibody deriv ative, which may more preferably be a monoclonal antibody a human antibody, a humanized antibody, a single-chain antibody, a chimeπc antibody, or an antigen-binding antibody fragment Those skilled in the art are enabled to make any such antibody deriv atives using standard art-recognized techniques For example, Jones et al , Nature 3_21 522-525 (19S6) discloses replacing the CDRs of a human antibody w ith those from a mouse antibody Marx, Science 229 455- 456 (19S5) discusses chimeric antibodies hav ing mouse variable regions and human constant regions Rodw ell, Nature 342 99-100 (1989) discusses low er molecular weight recognition elements derived from antibody CDR information Clackson, Br J Rheumatol 3052 36-39 (1991) discusses genetically engineered monoclonal antibodies, including Fv fragment derivatives, single chain antibodies, fusion proteins chimeric antibodies and humanized rodent antibodies Reichman et al , Nature 332 323-327 (19SS) discloses a human antibody on hich rat hyperv anable regions have been grafted Verhoeyen et al , Science 239 1534-1536 (19SS) teaches grafting of a mouse antigen binding site onto a human antibody In addition, those skilled in the art are enabled to design and produce pcptidomimetics hav ing binding characteristics similar or superior to such complementarity determining region (see e g , Horwell el al , Bioorg Med Chem 4 1573 (1996), Liskamp et al , Reel Trav Chim Pays- Bas I 113 (1994) Gante et al , Angew Chem Int Ed Engl 33 1699 (1994), Seebach et al , Helv Chim Acta Z2 913 (1996)) Accordingly, all such antibody derivatives and peptidomimetics thereof are contemplated to be within the scope of the present invention Compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers Additional preferred NAElBBMs according to the invention 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 Thus, in a fourth aspect, 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 Such 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 Such 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 In certain preferred embodiments, the method according to this aspect of the invention takes the form of a conventional ELISA or RIA In another preferred embodiment, the method employs either direct or indirect immunofluorescence Additional preferred embodiments utilize in vivo imaging of cells expressing NAEl-beta using conventional imaging agents directly or indirectly bound to an NAEIBBM according to the invention 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 Thus, in a fifth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAEl-beta nucleic acid in a biological sample In preferred embodiments, 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 In a sixth aspect, 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 Thus, 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 carried out using an in vitro activity system, as later described herein Preferably, such interference or enhancement results in a reduction of NEDDS activation/conjugation of at least 50%, more preferably at least 90%, and most preferably, at least 99%, or an increase of NEDDS activation/conjugation of at least 50%, preferably at least 2-fold, more preferably at least 5-fold In a sev enth aspect, 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 Other preferred modulating ligands are NAElBBMs which act as agonists, enhancing the ability of NAEl-beta to participate in the NEDDS-activation/conjugation pathway Preferably, such inhibition or enhancement is specific, . c , 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 Preferably, the 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 In certain embodiments, such 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 In an eighth aspect, 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 The term "biological system", as used herein, 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 In a ninth aspect, 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 For purposes of the invention, the term ohgonucleotide includes polymers of two or more deoxvπbonucleotide, or any modified nucleoside, including 2'-halo-nucleosιdes, 2 -O-substituted πbonucleosides, deazanucleosides or any combination thereof Preferably, 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 In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotπester, 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 and adamantane For purposes of the invention the term "2'-0-substιtuted' means substitution of the 2 position of the pentose moiety with a halogen (preferably Cl, Br, or F), or an O-lower al y l group containing 1-6 saturated or unsaturated carbon atoms, or with an O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, ary or allyl group may be unsubstituted or may be substituted, e g , with halo, hydroxy, tπfluoromethyl, cy ano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups, or such 2 substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a 2'-H group Certain embodiments of such ohgonucleotides are useful in hybridization assays Other embodiments are useful as antisense ohgonucleotides for use in animal model or human therapeutic settings In a tenth aspect, the invention provides methods for identify ing NAEl- alpha binding molecules (NAElABMs) 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. 271: 11339-11346 (1996)) 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. The terms "bind specifically", "population of molecules" and "mixed population of molecules" are as described previously. Structural aspects of NAElABMs are as discussed above for NAElBBMs, except that NAElABMs bind under physiological conditions to NAEl-alpha. Preferably, 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. This aspect of the invention includes NAElABMs identified by the methods according to the invention As used herein, a"NAEl-alpha-binding molecule", or "NAE1ABM", is a molecule or macromolecule which binds under physiological conditions to NAEl- alpha. The terms "binds under physiological conditions", "population of molecules", and "mixed population of molecules" are as used previously. In certain preferred embodiments, an NAE1ABM according to the invention is a peptide or a peptidomimetic. For purposes of the invention, the term peptide" is as used previously In certain preferred embodiments, 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 For purposes of the invention, 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 Thus, in an eleventh aspect, 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 Such detection is preferably through the direct or indirect binding of a tag or label on the NAEIABM The term "direct or indirect binding" is as used previously Preferred 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 In certain preferred embodiments, the method according to this aspect of the invention takes the form of a conventional ELISA or RIA In another preferred embodiment, the method employ s 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 Thus, in a twelfth aspect, the invention provides methods for determining the presence or absence and/or quantity of NAEl-alpha nucleic acid in a biological sample In preferred embodiments, 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. In a thirteenth aspect, the invention provides methods for identifying modulating ligands of NAEl-alpha Some NAElABMs are capable of acting as antagonists or agonists of NAEl-alpha Thus, 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, such interference or enhancement results in a reduction of NEDDS activation/conjugation of at least 50%, more preferably at least 90% and most preferably, at least 99% or an increase of NEDDS activation/conjugation of at least 50%, preferably at least 2-fold, more preferably at least 5-fold In a fourteenth aspect 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 The term ' purified" is as used previously In a fifteenth aspect, 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 Preferably, 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 Preferably, 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 concentration required to produce an unrelated biological effect In certain embodiments, such NAElABMs preferably interact with NAEl-alpha 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 In a sixteenth aspect, 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-alpha or a recombinant expression unit which expresses NAEl-alpha or an antagonist thereof to a biological system in which NEDDS is conjugated to a NEDDS conjugating enzyme or a target protein The term "biological system", as used herein, 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 In a seventeenth aspect, the invention provides allelic variants of NAE-1 alpha An ' allelic variant' , as used herein, 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 Each of these terms is as used previously In a eighteenth aspect, 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 In a nineteenth aspect, the invention provides methods for modulating the biological function of APP and/or beta peptide accumulation in a biological system. The present inventors have discovered that 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. In a twentieth aspect, 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 However, 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. of that of NCEl or NCE2, preferably at least 25% as fast, more preferably at least 507> as fast, and most preferably at least 757- as fast Preferably, 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 Such allelic variants have the biological activity of NCEl or NCE2, as discussed above In alternative preferred embodiments, 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 e g , international publication W095/1S974) The terms "purified" and "protein" are as used prev iously In a twenty-first aspect, 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 a chromosome, provided however that it cannot be the native chromosomal locus tor NCEl or NCE2 Preferred recombinant expression elements are vectors, which may include an origin of replication and are thus rephcatable in one or more cell type Certain preferred recombinant expression elements are expression vectors, and further comprise at least a promoter and passive terminator, thereby allowing transcription of the recombinant expression element in a bacterial, fungal, plant, insect or mammalian cell Preferred recombinant expression elements have at least 757. 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. ammo acid sequence identity to about 99% identity to the amino acid sequence set forth in SEQ ID NO 3 or SEQ ID NO 5, or to a portion or protein conjugate thereot which inhibits the biological activity of NCEl or NCE2 to form a thioester linkage with NEDDS under conditions as described in the following examples by at least 50%, preferably by at least 75%, more preferably by at least 90% and most preferably by at least 997o Preferably, such 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 purified protein and its structural information provided herein enables the preparation of NCEl and NCE2 binding molecules, respectively NCElBMs and NCE2BMs Thus, in a twenty second aspect, 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 Preferably, 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 In certain preferred embodiments, an NCEIBM or NCE2BM according to the invention is a peptide or a peptidomimetic For purposes of the invention, the term ' peptide" is as used previously In certain preferred embodiments, 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 For purposes of the invention, the term "complementarity determining region of an antibody" is as used previously. Accordingly, all such antibody derivatives and peptidomimetics thereof are contemplated to be within the scope of the present invention 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 Thus, in a twenty-third aspect, 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 The term ' direct or indirect binding" is as used previously Preferred 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 In certain preferred embodiments, the method according to this aspect of the invention takes the form of a conventional ELISA or RIA In another preferred embodiment, 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 Thus, in a twenty-fourth aspect, the invention provides methods for determining the presence or absence and/or quantity of NCEl or NCE2 nucleic acid in a biological sample In preferred embodiments, 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 In a twenty-fifth aspect, 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 Thus, 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- activation/coiηugation pathway" can conveniently be carried out using an in vitro activity system, as later described herein Preferably, such interference or enhancement results in a reduction of NEDDS activation/conjugation of at least 507>, more preferably at least 907>, and most preferably, at least 997.., or an increase of NEDDS activation/conjugation of at least 507., preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 10-fold In a twenty-sixth aspect, 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 Preferably, 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 Preferably, the concentration ot 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 concentration required to produce an unrelated biological effect In certain embodiments, such NCElBMs or NCE2BMs preferably interact with, respectively, 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 a target protein In a twenty-seventh aspect, 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 The term "biological system", as used herein, 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 In a twenty-eighth aspect, the invention provides ohgonucleotides that are specifically complementary to a portion of a nucleotide sequence show n in Figure 2 or Figure 5 For purposes of the invention, 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 In a twenty -ninth aspect the invention provides a purified complex ot NCEl and NEDDS, or of NCE2 and NEDDS The terms ' complex" and "purified ' are as used previously The following examples are intended to further illustrate certain particularly preferred embodiments of the invention and are not intended to limit the scope of the invention Searches of the human EST database utilized the program BLAST (Altschul et al , Nucleic Acids Res 25. 3389-3402 (1997)) Searches for transmembrane helices used the program Antheprot V 3 0 Gilbert Deleague, Institute de Biologie et Chemie des Proteines 69 367 Lyon cdex 07, France Example 1 Preparation of Human NEDDS Nucleotide sequence coding the N-terminal 76 residues of human NeddS was obtained from a human leukocyte cDNA Library (Life Technologies Tech-Lιne 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. In the second round, 5'-ggg aat tec ata tgc taa tta aag tga aga cgc and 5'-ccc aag ctt tea tec tec tct cag age caa cac were used as the forward and reverse primer, respectively The second PCR product was digested w ith Ndel and Hindlll and ligated to the large fragment of a similarly digested PT7-7 vector The construct was transformed into the E co strain BL21(DE3)/pLy sS (Novagen) NeddS expression was induced by the addition of 0 5 mM IPTG The SlOO fraction of bacterial extracts w as applied to a Q-Sepharose column in 50 M HEPES, pH 7 5 and the flow-through which contained NeddS was collected, concentrated by ultrafiltration and fractionated by size exclusion chromatography on Superdex G75. 3S Example 2 Identification of NEDDS-Activating Enzvme To identify the human NeddS-activating enzy me, w e first tested for the presence of this enzvme activity by monitoring the incorporation ot NeddS in the form of a thioester linkage into proteins derived from Hela cells On the basis of the chromatographic behavior of recombinant human NeddS, we generated from Hela cell extracts two protein fractions (FI and FII) which are expected to be devoid of endogenous NeddS as follow s To remove NeddS, 400 mg of protein from Hela cell SlOO fraction was applied to a 70 ml Q-Sepharose column, equilibrated with 50 mM HEPES, pH 8 0 with 1 mM DTT Proteins in the flow-through fraction were precipitated in 907. ammonium sulfate, dialyzed and fractionated bv size exclusion chromatography on Superdex G75 Fractions which eluted earlier than NeddS were pooled and concentrated bv ultrafiltration to 15mg/ml and is designated here as FI Proteins retained by the Q-Sepharose were eluted by inclusion of 0 b M NaCl in the equilibration buffer The collected proteins were precipitated with 907. ammonium sulfate and dialyzed against 25 mM Hepes, pH7 5, and 1 mM DTT and concentrated to 15 mg/ml of protein This fraction is designated here as 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! T-Nedd8 with FII, but not with FI, produced a radiolabeled band on SDS-gel which migrated at 59 kDa Formation of this radiolabeled species required the presence of ATP, and this species could not be detected when DTT was included in the SDS-gel sample buffer Thus, FII contains an activity which attaches NeddS to a protein via a DTT-sensitive linkage Incubation of 12 T-NeddS with FI and FII together resulted in the formation of two additional radiolabeled bands on SDS-gel, migrating at 30 and 97 kDa Only the 30 kDa species exhibited DTT sensitivity 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 Example 3 Purification of NEDDS-Activ ating Enzvme To purify the protein in FII which forms the DTT-sensitive linkage to NeddS, we immobilized NeddS to CH-Sepharose 4B gels and used DTT to elute proteins that were initially retained by the gel matrix, as follows 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 Tπs-HCl buffer, pH 7 5), buffer A with 0 5M NaCl, and buffer A A buffer containing 50 mM Tπs-HCl, pH 9 0 and 10 mM DTT was used to elute bound proteins Analysis of the eluted proteins by SDS-PAGE and silver-staining revealed the presence of two major proteins that migrated at 60 and 49 kDa A third major protein, migrating at 43 kDa, eluted as a broad peak When the eluted proteins were analyzed by gel filtration chromatography, the 43 kDa protein eluted as a large aggregate at the void volume while p60 and p49 were found to co-elute with a retention time similar to that of the 110 kDa ubiquitin-activating enzyme, suggesting that these two proteins form a heterodimer To determine w hich one of these two proteins forms the DTT-sensitive linkage w ith NeddS, proteins purified from the NeddS-affinity chromatography step were tested The result is consistent with p49 being the NeddS acceptor This protein is quantitatively absent only w hen ATP or AMPPNP was included in the reaction and only if the electrophoresis w as carried out in the absence of DTT The fact that no new discrete protein band was detected under conditions in which p49 was absent is likely due to the presence of p60 which precludes the detection of proteins that would migrate vs ith similar mobility In a separate experiment, the use of 12C T-NeddS in the reaction led to the detection ot a DTT-sensitiv e 59 kDa band, confirming the presence of a NeddS-containing thioester The ability of AMPPNP to substitute for ATP suggests that NEDDS activation, similar to ubiquitin and SUMO-1, involves the intermediate formation of an enzyme-bound NeddS-adeny late prior to thioester linkage Example 4 Sequence Determination of NEDDS-Activating Enzyme To obtain the identity of p49, this protein was excised from an SDS-gel, digested with trypsin and peptides were eluted and purified by HPLC as follow s The peak fractions from NeddS-affinity chromatography step were concentrated and separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and bands corresponding to p49 and p60 were excised. 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 (TFA), essentially as described in (J Pohl et al, FEBS Lett 272, 200, 1990 ) The masses of the peptides were determined by matrix-assisted laser desorption lonization mass spectrometry (MALDI-TOF) using a Bruker Instruments model ProFlex MALDI-TOF instrument operated in the reflectron mode, 2,5-dιhydroxybenzoιc acid was used as the sample matrix The sequences of the peptides were determined by automated Edman degradation on a PE-Applted Biosystems model Procise-HT sequencer system operated in the pulsed- quid mode using manufacturer's supplied sequencing cycles Two tryptic peptide sequences were determined (shown as underlined in Figure 1), and these sequences were used to search the protein as w ell as the expressed sequence tag (EST) data bases Although these sequences did not match known proteins in the data bases, two groups of EST clones could be identified whose translated amino acid sequence yielded perfect matches to either one of the two tryptic peptides Further homology search with these EST sequences identified additional EST clones with overlapping sequences Analy sis of these EST clones enabled us to obtain a contiguous open reading frame (ORF) that encodes a 442-resιdue protein which contains the two tryptic peptide sequences The nucleotide sequence of this ORF was confirmed by direct nucleotide sequencing of two EST clones (AA40S62 and R57021) Analysis of this protein sequence revealed three regions of homology with human Ubal. 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 These similarities are expected if the activation of NeddS utilizes a mechanism similar to that of ubiquitin and Smt3 Since p49 forms a heterodimer with p60 and functions as a protein component of NeddS activation, we designate it as Nae. eta and p60 as Nae. al ha Searches of the data banks with this protein sequence identified an open reading frame in S pυmbe, and one in C elegan which code for similar size proteins In addition, a S cerevisiae 299-resιdue protein, despite its smaller size, also shows extensive homology with this human protein These are likely homologues of Nae t , in different species since identical and highly conserved residues among these four proteins are interspersed throughout most of the protein whereas their homology to Ubal and Uba2 is limited to defined regions only Example 5 Identification of Nae-alpha The similarity between NeddS- and Smt3-actιvatιng enzvme in their subunit structure suggested that p60 or Nae.- lphι - would also contain a sequence stretch that shares homology to the N-terminal portion of Ubal. Using procedures similar to those with p49, three tryptic peptide sequences were obtained for p60 These sequences FTVVATQLPEXTXL, EHFQSYDLDHME, and QTPSFWILA yielded perfect matches to residues 123-138, 194-205 and 300-308 in the 534-resιdue APP-BP1 In addition mass spectrometry of 15 of the try ptic peptides revealed matches w ithin 1 Da of the expected mass of tryptic peptides of APP-BP1 These matches covered 377. of the APP-BP1 sequence Thus, w e concluded that Nae alpln is indeed APP-BP1 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) The second PCR used the primers CGGGAATTCCATATGATCAAGCTGTTCTCGCTG (forward) and CGCCCAAGCTTCTATTTCAGGCAGCGCTCAAAG (reversed) The PCR product was digested with Ndel and Hindlll and gated with similarly digested plasmid pT7-7 The resulting clone, pT7-7-UbcH12, was sequenced to determine the nucleotide sequence [SEQ ID NO 3] and deduced amino acid sequence [SEQ ID NO 4] shown in Figure 1 Figure 2 shows the alignment of NCEl w ith yeast Ubcl2 NCEl shows 417. identity and 637. homology with yeast Ubcl2 Example 7 Expression and purification of NCEl BL21 (DE3) bacterial cells (Novagen, MadisonΛVI, 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 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 For 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) and ImM DTT NCEl protein was retained by the gel and eluted using a linear NaCl gradient in the gel equilibration buffer Fractions containing NCEl protein were determined by assaying for NEDDS thioester formation NCEl was found to elute at 0 OS M NaCl Activ e fractions ere 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 NEDDS-thioester formation NCEl eluted at a volume expected for a 19kDa protein, suggesting that it exists as a monomer SDS-PAGC analysis with Coomassie stain indicated that the preparation was predominantly (>90%) NCEl protein Purified NCEl protein migrated on an S7> TRICINE gel at a molecular weight of 21 kDa (data not shown) Extending the N-terminus of NCEl with the amino acid sequence MHHHHHH resulted in an NCEl v riant protein that retained activ ity in NEDDS-thioester formation The six histidinc residues provide a nickel binding site and allowed this v ariant to be purified w ith Ni-NTA or other metal affinity chromatography procedures 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. SDS-Tricine PAGE, followed by silv r staining The results are shown in Figure 4 Lanes 1-4 are reaction mixtures 1-4 Lanes 5-8 are reaction mixtures 1-4 which were incubated with 10 mM DTT and heated to 90°C for two minutes prior to electrophoresis These results show that NCEl migrates at a slower rate in the presence of NEDDS and Nae, and that this is reversible by DTT Ubiquitin activating enzy me, El, cannot substitute for NAE in prov iding this result These data support the view that NCEl is a NEDDS conjugating enzv me which forms a thioester with NEDDS in the presence of activating enzyme NAE Reaction No Proteins 1 NAE + NEDDS 2 NCEl + NEDDS 3 NCEl + NEDDS + NAE 4 NCEl + NEDDS + ubiquitin activating enzyme, El Example 9 Identification and cloning of NCE2 The human EST database was searched using as query sequence 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 The ov erlapping sequences yielded an open reading frame w hich encodes a protein of 185 ammo acids (predicted molecular mass = 21076 Da) Based upon homology to known human Ubc proteins, this gene is a member of the human Ubc gene family The contiguous nucleotide sequence of NCE2 was obtained using nested PCR on a human leukocyte cDNA library The first PCR used the primers AGCCCAGGGTAAAGGCAGCA (forward) and CATGTTAGAGACAAACTGTA (reversed) The second PCR used the primers GGGAATTCCATATGCTAACGCTAGCAAGTAA (forward) and CCATCGATTCATCTGGCATAACGTTTGA (reversed) The PCR product was then cloned into the Ndel/Hindlll sites of pT7-7 to generate the plasmid pT7-7-HSUBC17 The sequence ot the NCE2 gene and its deduced amino acid sequence are shown in Figure 4 No close homolog exists in the yeast genome The protein has 467, identity and 64"/. homology with a C elegant gene (Genebank Accession # CE 275850) of unknown function (see figure 5) Example 10 Expression and purification of NCE2 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, ιsopropy'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 For anion exchange chromatography, 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. 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. SDS-PAGE analysis with Coomassie stain indicated that the preparation was predominantly (>907>) NCE2 protein. Purified NCE2 protein migrated on an 87. TRICINE gel at a molecular weight of 21 kDa (data not shown). Example 11 Thioester formation between NCE2 and NEDDS The ability of NCE2 to form a thioester bond with NEDDS was assessed as follows. NCE2 protein, either purified or from bacterial lysate, was incubated with ι 5 I-NEDD8 (10 ή cpm/μg) in a buffer containing 25 mM HEPES (pH 7.0), 10 mM MgCl 2 , 1 mM ATP and 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. In the reaction containing NCE2 (lane 3), the autoradiograph showed two radiolabeled bands with apparent molecular masses of 7 and 29 kDa, which are the expected molecular masses of NEDDS and NEDD8-NCE2, respectively. Only the 7kDa band was detected when the sample was incubated in 10 mM DTT prior to electrophoresis, consistent with the 29 kDa band being a NEDDS-NCE2 thioester. Analogous reactions containing NCEl in place of NCE2 (lanes 2 and 4) are shown for comparison. These results demonstrate that NCE2 is capable of forming a thioester bond with NEDDS, but not with ubiquitin, in a NAE-dependent reaction These data support the view that NCE2 is a NEDDS conjugating enzyme Example 12 Preparation of dominant negative mutants 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 seπne 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 4S

Claims: What is claimed is 1 Purified NEDDS-activating protein beta subunit 2 The purified NEDDS-activating protein beta subunit according to claim 1 having the amino acid sequence shown in Figure 1 3 An NAEl-beta expression element 4 The NAEl-beta expression element selected from isolated or recombinant nucleic acid sequences encoding NAEl-beta or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such nucleic acid sequences, and recombinant expression units which express NAEl-beta, antisense transcripts, or dominant negative mutants thereof 5 A method for identifying NAElBBMs comprising contacting purified NAEl- beta according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NAEl-beta 6 An NAEIBBM identified by the method according to claim 5 7 A method for determining the presence or absence and/or quantity of NAEl- beta, NAEl heterodimer, or NAEl heterodimer/NEDDS complex in a biological sample, the method comprising 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 8 A method for determining the presence or absence and/or quantity of NAEl- beta nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAEl-beta nucleic acid 9 A method for identifying modulating ligands of NAEl-beta comprising 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 10 A modulating ligand of NAEl-beta 11 The modulating ligand of NAEl-beta identified by the method according to claim 9 12 The modulating ligand of NAEl-beta according to claim 10, which interacts 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 enzv me 13 An antagonist of NAEl-beta w hich interferes w ith the expression of the NAEl-beta gene 14 A method for identifying NAElABMs comprising 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 15 An NAEIABM identified by the methods according to claim 14 16 A method for determining the presence or absence and/or quantity of NAEl- alpha, NAEl heterodimer, or NAEl heterodimer/NEDDS complex in a biological sample comprising providing a detectable NAEIABM to a biological sample, allowing the detectable NAEIABM to bind to NAEl-alpha, 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 NAEIABM and NAEl-alpha, NAEl-heterodimer, or NAEl heterodimer/NEDDS complex 17 A method for detecting the presence or absence of NAEl-alpha nucleic acid in a biological sample in which NEDDS conjugation is suspected, the method comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NAEl-alpha nucleic acid 18 A method for identifying modulating ligands of NAEl-alpha comprising 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 19 A modulating ligand of NAEl-alpha 20 A modulating ligand hich w as identified by the method of claim IS 21 The modulating ligand according to claim 19, which interacts with NAEl- alpha to inhibit the formation ot NAEl heterodimer, the formation ot NEDDS adenvlate, the formation of a thiol ester bond between NEDDS and NAEl, and/or transfer of NEDDS to NEDDS-conjugating enzyme 22 An antagonist of NAEl-alpha which interferes with the expression of the NAEl-alpha gene 23 A method for modulating the activation and/or conjugation of NEDDS comprising providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit hich expresses NAEl-beta or NAEl-alpha or an antagonist thereof to a biological sy stem in which NEDDS is conjugated to another protein 24 A method for modulating auxin response in plants comprising providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAEl-alpha or an antagonist thereof to a plant that is undergoing auxin treatment 25 A method for modulating APP function and/or beta peptide accumulation in a biological system comprising providing a modulating ligand of NAEl-beta or NAEl-alpha or a recombinant expression unit which expresses NAEl-beta or NAE1- alpha or an antagonist thereof to a biological system 26 A purified complex of NAEl-beta and NAEl-alpha, or a purified complex of portions thereof 27- A purified complex of NAEl-beta, NAEl-alpha and NEDDS, or a purified complex of portions thereof. 28. An allelic variant of NAEl-alpha. 29. An NAEl-alpha allelic variant expression element selected from 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 which express NAEl-beta or antisense transcripts or dominant negative mutants thereof. 30. Purified NEDDS-conjugating enzyme 1 31. The purified NEDDS-conjugating enzyme 1 according to claim 30 having the amino acid sequence shown in Figure 1. 32. An NCEl expression element. 33. The NCEl expression element according to claim 32 selected from isolated or recombinant nucleic acid sequences encoding NCEl or dominant negative mutants thereof, or expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, vectors comprising any such NCEl expression elements. 34. A method for identifying NCElBMs comprising contacting purified NCEl and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NCEl. 35. An NCEIBM identified by the method according to claim 34. 36 A method for determining the presence or absence and/or quantity of NCEl or NCE1/NEDDS complex in a biological sample, the method comprising providing a detectable NCEIBM to a biological sample, allowing the detectable NCEIBM to bind to NCEl, or NCEl /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 and NCEl or NCEl /NEDDS complex 37 A method for determining the presence or absence and/or quantity of NCEl nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NCEl nucleic acid 3S A method for identifying modulating ligands of NCEl comprising providing NCElBMs to an assay system for NCEl participation in the NEDDS- activation/conjugation pathway, and determining w hether such NCElBMs interfere with or enhance the ability of NCEl to participate in the NEDD8- activation/conjugation pathway 39 A modulating ligand of NCEl 40 The modulating ligand of NCEl identified by the method according to claim 3S 41 The modulating ligand of NCEl according to claim 39, which interacts with NCEl to inhibit or enhance the formation of a thiol ester bond betw een NEDDS and NCEl, and/or transfer of NEDDS to NEDDS target protein 42 An antagonist of NCEl which interferes with the expression of the NCEl gene 43 Purified NEDDS-conjugating enzyme 2 44 The purified NEDDS-conjugating enzyme 2 according to claim 43 having the amino acid sequence shown in Figure 4 45 An NCE2 expression element 46 The NCE2 expression element according to claim 45 selected from isolated or recombinant nucleic acid sequences encoding NCE2 or dominant negative mutants thereof, or expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such NCE2 expression units 47 A method for identifying NCE2BMs comprising contacting purified NCE2 and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to NCE2 4S An NCE2BM identified by the method according to claim 47 49 A method for determining the presence or absence and/or quantity of NCE2 or NCE2/NEDDS complex a biological sample, the method comprising providing a detectable NCE2BM to a biological sample, allowing the detectable NCE2BM to bind to NCE2, 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 NCE2BM and NCE2 or NCE2/NEDDS complex 50 A method for determining the presence or absence and/or quantity ot NCE2 nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence which is specifically complementary to NCE2 nucleic acid 51 A method for identifying modulating ligands of NCE2 comprising providing NCE2BMs to an assay system for NCE2 participation in the NEDDS- activation/conjugation pathway, and determining whether such NCE2BMs interfere with or enhance the ability of NCE2 to participate in the NEDDS- activation/ conjugation pathway 52 A modulating ligand of NCE2 53 The modulating ligand of NCE2 identified by the method according to claim 51 54 The modulating ligand of NCE2 according to claim 24, which interacts with NCE2 to inhibit or enhance the formation of a thiol ester bond betw een NEDDS and NCE2, and/or transfer of NEDDS to NEDDS target protein 55 An antagonist of NCEl which interferes with the expression of the NCEl gene 56 A purified complex of NCEl and NEDDS, or a purified complex of portions thereof 57 A dominant negative mutant of NCEl or NCE2 58 An ohgonucleotide that is specifically complementary to a portion of NCEl or NCE2

Inventors: CHAU VINCENT, CHAU, VINCENT
Applicants: PROSCRIPT INC, PROSCRIPT, INC.
Application year: 1998
Main classification: C12N15
IPC codes: C07K14, C12N9, C12N15, C12P21, C12Q1, G01N33