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J. Biol. Chem., Vol. 279, Issue 18, 18935-18943, April 30, 2004
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From the Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712
Received for publication, November 7, 2003 , and in revised form, February 6, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-amino group of the substrate. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-amino groups of lysine side chains of the substrate, or in some cases to the N-terminal 
-amino group. Additional ubiquitin molecules can be conjugated sequentially at specific lysine residues of ubiquitin to form multi-ubiquitinated proteins, which are the principal substrate of the 26 S proteasome.
Three groups of proteins cooperate in catalyzing ubiquitination: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating proteins (Ubc1 proteins), and the E3 ubiquitin ligases (1). The E1 enzyme uses ATP to form a ubiquitin-adenylate intermediate at the terminal carboxyl group of ubiquitin. A ubiquitin-thioester is then formed at the active site cysteine of the E1 enzyme, with release of AMP. The E1 enzyme transfers ubiquitin to the active site cysteine of the highly conserved family of E2 proteins, maintaining a ubiquitin-thioester linkage. The E3 ubiquitin-ligases interact with both the E2 proteins as well as with substrate proteins. Whereas several classes of E3 enzymes are now recognized (SCF, APC, CBC, RING, CHIP, and HECT E3s), they fall into two groups with respect to overall mechanism. The first group, which includes all classes except for the HECT E3s, are thought to function essentially as docking proteins, orienting the E2 and substrate so that -amino groups of substrate lysines can attack the E2-ubiquitin thioester, forming a ubiquitin-isopeptide bond. HECT E3s participate more directly in catalysis and are defined by a conserved C-terminal domain of 
350 amino acids (the HECT domain, Ref. 2). Like the E1 and E2 proteins, HECT E3s also have an active site cysteine that forms a ubiquitin-thioester intermediate, accepting ubiquitin from an activated E2 enzyme (3). Substrates are then ubiquitinated by transfer from the E3 to the substrate, rather than from the E2 to the substrate. The HECT domain can be re-charged with ubiquitin while the substrate remains bound, and therefore multiple ubiquitins can be ligated to the substrate.
Human E6AP was the first HECT E3 to be identified and characterized (4, 5). The human papillomavirus (HPV) E6 protein of the cancer-associated HPV types (e.g. types HPV16 and 18) binds to and redirects the activity of E6AP toward a set of cellular proteins that are not normally targeted by E6AP (5). The most notable of these substrates is the p53 tumor suppressor, although several cellular proteins have been reported to be ubiquitinated by the E6/E6AP complex (reviewed in Ref. 6.) Whereas few of the normal physiologic (E6-independent) functions or substrates of E6AP have been identified (7-9), it is now clear that mutations in the gene or alteration in expression of human E6AP (gene designation UBE3A) is the cause of Angelman syndrome (AS), a severe neurological disorder (10, 11). In a remarkable case of tissue specific imprinting, only the maternal allele of E6AP/UBE3A is expressed in subregions of the brain, including the hippocampal and Purkinje neurons (12, 13). AS patients lack a functional maternal allele of E6AP, and therefore lack E6AP protein in these regions of the brain. The AS phenotype is therefore hypothesized to reflect the lack of E6AP-mediated ubiquitination of one or more proteins in the brain. The identification of these substrates and an understanding of the biochemistry of E6AP and HECT E3s, in general, are critical to understanding the molecular basis of this devastating disease.
HECT E3s are found in all eukaryotic organisms, and the human genome encodes 50 HECT E3s (2). HECT E3 proteins range in size from 90 to over 500 kDa, with the 350 amino acid HECT domain being the only defining element. The HECT domain is always located at the C terminus, with the active site cysteine located 32-34 amino acids from the end of the protein. The Saccharomyces cerevisiae genome encodes five HECT E3s. The best characterized is Rsp5p, which contains three WW domains in the central portion of the protein. WW domains are protein-protein interaction motifs with affinity for polyproline containing ligands (14), and in some cases the WW domains mediate direct substrate binding (15, 16). In other cases, as in the targeting of Spt23p by Rsp5p, enzyme-substrate recognition may be mediated by additional factors because an obvious WW domain recognition element does not exist in Spt23p. Yet the WW domains are required for Spt23p targeting (17).
The isolated 350 amino acid HECT domain of E6AP and Rsp5p can form a thioester-linked ubiquitin adduct when supplied with E1, an appropriate E2 protein, ATP, and ubiquitin, indicating that all information necessary to interact with the upstream components of the ubiquitin system resides within the HECT domain (18). At least three separable biochemical activities of the HECT domain can be defined: 1) binding to a specific E2, 2) transfer of ubiquitin from the E2 active site cysteine to the HECT active site cysteine (transthiolation), and 3) transfer of ubiquitin from the HECT active site to lysine side chains on the substrate (catalysis of isopeptide bond formation). The x-ray crystal structure of the E6AP HECT domain bound to UbcH7 directly demonstrated the basis of the E2-HECT domain interaction (19). The overall structure of this HECT-E2 complex is "U" shaped, where the HECT domain represents the base and one arm of the U, and the E2 the second arm. A common conserved feature of the E2s that function with HECT E3s is a phenylalanine (Phe63 of UbcH7) on the tip of loop L1 of the E2. The Phe63 side chain of UbcH7 fits into a deep hydrophobic groove of the HECT domain.
The E6AP HECT-UbcH7 x-ray crystal structure did not reveal an obvious mechanism for transfer of ubiquitin from either the E2 to the HECT domain, or from the HECT domain to the substrate. A striking feature of this complex was that the active site cysteines of the E2 and HECT domain were separated by 41 Å (19). Clearly, a dramatic structural change would be required to bring the thiol groups into proximity during transfer of ubiquitin from the E2 to E3. The most likely source of flexibility is the unstructured loop that connects the HECT domain N lobe (the base of the U) with the C lobe (one arm of the U) that contains the active site cysteine. Indeed, this is strongly supported by a more recent x-ray crystal structure of a second HECT domain, that of the WWP1 protein (20). In this structure, the N and C lobes of WWP1 are individually very similar to those of E6AP; however, the relative orientation of the N and C lobes is different from that seen in the E6AP structure. Modeling an E2 into the WWP1 structure based on the E6AP-UbcH7 structure, the active site cysteines would be substantially closer than in the E6AP structure (16 compared with 41 Å), but clearly additional conformational changes would still be necessary to bring them into proximity for the transthiolation reaction. The orientation of the N and C lobes seen in the WWP1 structure is more likely to be representative of the relative position of these lobes during transthiolation than the orientation seen in the E6AP structure. It is possible that the orientation of the N and C lobes seen in the E6AP structure may represent the structure at the last phase of the reaction, during the transfer of ubiquitin from the E3 to the substrate.
Whereas understanding the basis of ubiquitin transfer from the E2 to the HECT domain remains unresolved, the final phase of the ubiquitination reaction, the transfer of ubiquitin from the HECT active site to the substrate protein, can nevertheless be studied independently. We reported previously that E6AP deleted of the C-terminal six amino acids was unable to catalyze substrate ubiquitination, even though the protein was competent for formation of the ubiquitin-thioester intermediate (2). These amino acids of E6AP are partially disordered in the E6AP structure and follow a 13-amino acid -helix (19). Perhaps related to the disordered nature of the C terminus, crystals of the WWP1 HECT domain could not be obtained until the last five amino acids were deleted (20). The last six amino acids of E6AP (residues 847-852; KGFGML) contain only two residues that are conserved among most HECT E3s, Gly848, and Phe849 (see Fig. 1A). For purposes of comparison between different HECT E3s we will often refer to these residues by their position relative to the C terminus (e.g. Phe849, the fourth from last amino acid of E6AP, will be -4F). Whereas the -4F residue is conserved among almost all HECT E3s, in some cases it is found at either the -2 or -5 position. The only exceptions to this are a small number of HECT E3s that have an extended and divergent C-terminal sequence (e.g. human Herc1, Herc2; see Fig. 1A). This subgroup of HECT E3s is not a subject of the current study, primarily because functional assays for these proteins have not been established. Four of five S. cerevisiae HECT E3s (Rsp5p, Ufd4p, Hul5p, and Tom1p) possess the conserved -4F, while Hul4p has a tyrosine at the -4 position that, as suggested by results presented below, is likely to functionally substitute. We show here that -4F is the only determinant within the last six amino acids that is specifically required for the final step in catalysis of substrate ubiquitination by both human E6AP and yeast Rsp5p, that its precise positioning is critical to function, and that C-terminal extensions to the HECT domain result in a similar biochemical defect as alterations of the -4F. We present a model for the possible function of the -4F in promoting substrate ubiquitination.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Protein Expression and Purification—GST-Rsp5p fusion proteins were expressed in E. coli by standard methods and affinity-purified on glutathione-Sepharose (Amersham Biosciences). Rsp5p proteins were cleaved from GST using PreScission protease (Amersham Biosciences) under the manufacturer's recommended conditions. GST-E6AP proteins were expressed in High5 insect cells (Invitrogen) using recombinant baculoviruses. Cell lysates were made 36-h post-infection in Nonidet P-40 lysis buffer (100 mM Tris, pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT), affinity-purified on glutathione-Sepharose, and cleaved from GST with PreScission protease. GST-ubiquitin was purified from E. coli lysate on glutathione-Sepharose and eluted with PBS containing 25 mM reduced glutathione. Human E1 ubiquitin-activating enzyme was expressed in insect cells infected with recombinant E1-expressing baculovirus. The protein was partially purified by DEAE batch chromatography, eluting with buffer containing 400 mM NaCl. Arabidopsis thaliana Ubc8 protein was prepared as described previously (5). HPV-33E6 protein was expressed as a GST fusion protein in the baculovirus/insect cells system, purified on glutathione-Sepharose, and cleaved from GST with PreScission protease. In vitro translation reactions were carried out in coupled transcription-translation reactions using either rabbit reticulocyte lysate or wheat germ systems (Promega) and 35S-labeled methionine.
In Vitro Ubiquitin-Thioester and Ubiquitination Assays—For thioester assays, E6AP or Rsp5p proteins (1 µg) were incubated in 50-µl reactions containing 25 mM Tris (pH 7.5), 50 mM NaCl, 0.1 µM DTT, 4 mM MgCl2, 4 mM ATP. Reactions contained E1 and A. thaliana Ubc8 enzymes (250 ng each), with or without 2 µg of GST-ubiquitin. The reactions were incubated at room temperature for 10 min, then divided into two portions, and stopped with SDS-PAGE loading buffer, with or without 100 mM DTT. Samples were electrophoresed on 8% SDS-PAGE gels, transferred to polyvinylidene difluoride membrane, and immunoblotted with either anti-RSP5 mouse antibody or anti-E6AP rabbit antibody. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG and visualized with the SuperSignal chemiluminescence detection system (Pierce). Ubiquitination assays are carried out under the same conditions as the ubiquitin-thioester assays but for 30 min, and in presence of unmodified bovine ubiquitin (Sigma). 5 µl of in vitro translated WBP2 (rabbit reticulocyte lysate) or p53 (wheat germ extract) was used as source of substrates. 500 ng of purified bacterially expressed and purified FLAG-WBP2 was used for Rsp5p ubiquitination assays, where indicated. The reaction products were analyzed by SDS-PAGE followed by autoradiography or immunoblotting using an anti-FLAG antibody (F3165 Sigma).
In Vivo Complementation Assays—As described previously (18), the rsp5-1 strain, FW1808 (MAT, his4-912
R5, lys2-128, ura3-52, rsp5-1), was used to assay for the ability of plasmid-expressed RSP5 variants to complement the growth arrest of this strain at 36 °C. All of the variants shown in Fig. 2 were subcloned into the pYES2 vector (Invitrogen) under control of the GAL1 promoter, and all contained an N-terminal hemagglutinin epitope. pYES2 plasmids were transformed into FW1808 or the closely related RSP5 strain FY56 (MAT, his4-912, lys2-128, ura3-52), with selection on uracil drop-out plates. Growth at 36 °C was scored on uracil drop-out plates with 2% galactose as the carbon source. Protein expression for all variants was assayed by preparing total cell extracts from cultures grown at 30 °C in 2% galactose containing uracil drop-out liquid medium, as described previously (18). Extracts were analyzed by SDS-PAGE and immunoblotting using anti-HA and anti-Rsp5p antibodies.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The 2.7 Å structure of the human E6AP HECT domain (amino acids 493-852) in complex with the UbcH7 E2 enzyme was reported previously (19). Ubiquitin was not present in this complex, so its position when bound to either the E2 or HECT E3 was unknown. A model of the S. cerevisiae Ubc1p E2 enzyme in complex with ubiquitin has been reported more recently (22). The structures of UbcH7 and Ubc1p are very similar and both belong to the subgroup of E2 enzymes known to function with HECT E3s. By aligning the E2 structures from the HECT-E2 and E2-ubiquitin structures it is possible to predict the position of ubiquitin within the HECT-UbcH7 structure when bound to UbcH7 (22). In this predicted ternary complex, ubiquitin lies to the side of the U-shaped structure formed by the HECT-E2 complex and does not cross the line-of-sight between the active site cysteines of the E2 and E3 (Fig. 1B). Fig. 1B also highlights the position of the -4F within the predicted HECT-E2-ubiquitin structure.
Analysis of C-terminal Mutations in E6AP and Rsp5p—To explore the function of the HECT C terminus in ubiquitination, a set of mutations was created in the context of both full-length human E6AP and yeast Rsp5p (Fig. 2). The set of mutants included single amino acid alterations within the last five amino acids, small deletions from the C terminus, or extensions to the C terminus. E6AP and Rsp5p proteins were expressed as GST fusion proteins, either in a baculovirus insect cell system for E6AP, or in bacteria for Rsp5p. These expression systems were determined to be optimal for obtaining biochemically active protein for the respective wild-type proteins. After purification of the proteins on glutathione-Sepharose the E3 proteins were cleaved from GST by site-specific proteolysis. Fig. 3 shows a Coomassie Blue-stained SDS-PAGE gel of purified E6AP and Rsp5p, both of the wild-type and the active site cysteine to alanine (C-A) mutated protein. All of the E6AP and Rsp5p proteins included in our analyses expressed similar amounts of protein relative to the wild-type control and no differences were observed in solubility of the altered proteins.
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Most Angelman syndrome mutations that result in single amino acid changes within the E6AP open reading frames are found within the region encoding the HECT domain, and all mutations tested so far disrupt to some degree the catalytic activity of E6AP in vitro (Ref. 19).2 One Angelman syndrome mutation has been identified that affects the stop codon of E6AP, resulting in a terminally extended protein (24). To determine whether E6AP with a C-terminal extension would behave similarly to variants where the -4F is altered, the stop codon of E6AP was deleted within the E6AP cDNA sequence, resulting in a protein that is extended by eight amino acids (NKTKQNKT; E6AP-Ext8, see Fig. 2). As shown in Fig. 4, this protein, like the -4F to A mutant, could form a ubiquitin-thioester intermediate but could not catalyze p53 ubiquitination. To determine whether this was because of the actual sequence of the extension or if another extension would have a similar effect, the FLAG epitope (DTKDDDDK) was placed at the C terminus of E6AP. Again, substrate ubiquitination was blocked by this mutation without affecting thioester formation. These results suggest that C-terminal extensions interfere with the function of the -4F in ubiquitination.
A similar set of C-terminal mutants was made in Rsp5p and assayed for thioester formation and substrate ubiquitination. In addition to confirming the generality of our results with a second HECT E3, the advantage of performing these experiments with Rsp5p was that a genetic system exists for easily assessing in vivo function. As reported previously, it is difficult to detect DTT-sensitive ubiquitin adducts in vitro with wild-type Rsp5p because Rsp5p very efficiently ubiquitinates itself at internal lysine residues (2). This is shown in Fig. 5, where purified Rsp5p proteins were incubated with E1, E2, ATP, and GST-ubiquitin and the products were analyzed by SDS-PAGE in the absence or presence of DTT, followed by immunoblotting with anti-Rsp5p antibody. A ladder of high molecular ubiquitinated Rsp5p products was seen with wild-type Rsp5p, and these conjugates remained after incubation with high concentrations of DTT, as expected for isopeptide-linked multi-ubiquitin conjugates. In contrast, the active site mutant (C777A) did not form any conjugates, either in the absence or presence of DTT. Whereas there is no evidence that the self-ubiquitination reaction is biologically meaningful, this in vitro assay simultaneously reflects the ability of the protein to form a thioester intermediate and to catalyze isopeptide-linked ubiquitination. Based on the E6AP results, we predicted that alterations that disrupted the -4F would not affect thioester formation but would block stable Rsp5p multi-ubiquitination. Therefore, we would expect to see only a DTT-sensitive mono-ubiquitinated conjugate in this assay, and this was indeed the case (Fig. 5), although a small amount of self-ubiquitination was seen for the F806A and F806P proteins. Whereas tyrosine could not substitute for phenylalanine in E6AP, the analogous F806Y mutation in Rsp5p resulted in a protein that could catalyze self-ubiquitination. This suggests that at least in some HECT E3s a tyrosine can functionally substitute for phenylalanine, consistent with the fact that a small number of HECT E3s do contain a tyrosine at the -4 position (Fig. 1A). Rsp5p deleted of the last three amino acids of Rsp5p (806stop) inactivated Rsp5p self-ubiquitination activity in vitro, in contrast to the equivalent mutation in E6AP, which did not affect p53 ubiquitination. The 806Astop mutation, which combines the -4F to alanine mutation with truncation of the last three amino acids, as well as further truncations (805stop and 803stop), were also negative for self-ubiquitination. Rsp5p with a FLAG epitope at the C terminus retained self-ubiquitination activity, although the level of activity was consistently decreased relative to wild type.
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All of the genes encoding the Rsp5p variants shown in Fig. 2 were introduced into both the temperature-sensitive rsp5-1 mutant strain (FW1808) and a closely related RSP5 strain (FY56) on a multicopy plasmid, with expression being driven by the GAL1 promoter. Protein expression at 30 °C was analyzed by immunoblotting of total cell extract, and all plasmid-expressed proteins were expressed at similar levels, 5-fold higher than the endogenous Rsp5p protein (not shown). The rsp5-1 strain arrests within 1 h of shift to 36 °C, and there were no differences in expression levels of the exogenous Rsp5p proteins up to 6 h after shift to high temperature. The ability of the various mutant alleles to complement the temperature-sensitive growth phenotype was scored at 36 °C on galactose-containing agar plates. The only C-terminal mutant allele tested from the set shown in Fig. 2 that, like the wild-type control, complemented the rsp5-1 mutation was the -4F to tyrosine (F806Y) mutant. The Rsp5-FLAG allele, which had decreased self-ubiquitination activity and decreased activity against WBP2, did not complement. The 806A/805F, 806A/807F, and 806A/809F mutants, all of which had substantially reduced ubiquitination activity against WBP2 in vitro, also did not complement. The biochemical activities and in vivo complementation results are tabulated in Fig. 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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What, specifically, might the -4F do to promote transfer of ubiquitin from the HECT active site cysteine to the substrate? The final step in the ubiquitination reaction is the nucleophilic attack on the ubiquitin-thioester carbonyl carbon by the -amino nitrogen of the lysine residue that is to be ubiquitinated. Given the separation of the -4F from the active site cysteine in the crystal structure (18 Å; see Fig. 1B), and the nature of the phenylalanine side chain, it would seem unlikely that the -4F participates directly in catalysis. Assuming that the overall structure of the C lobe of the HECT domain is similar in the absence and presence of bound ubiquitin, we favor a model in which the -4F may contact ubiquitin whereas it is linked to the active site cysteine and play a role in orienting and positioning the tethered ubiquitin molecule. The active site cysteine of E6AP lies in a four-residue loop at the base of the junction formed by the N and C lobes of the HECT domain. When bound to the cysteine thiol, the orientation of the bulk of the ubiquitin molecule may be critical for allowing access of the incoming substrate lysine to the thioester bond. We propose that the -4F may contact ubiquitin and position it in an optimal orientation to allow for approach of the incoming lysine side chain. Thus, alteration of the -4F may prevent substrate ubiquitination by mis-orienting ubiquitin and thereby blocking access of the substrate lysine to the active site.
Whereas the above model is speculative, it predicts that specific amino acid substitutions on the surface of ubiquitin that mediate interaction with the -4F would result in a similar ubiquitination defect. Similarly, specific amino acid substitutions on the surface of ubiquitin might suppress the effect of alteration of the -4F. Given the nature of the phenylalanine side chain, hydrophobic patches on the surface of ubiquitin might be good candidate sites for -4F interaction. We have preliminarily tested alterations at hydrophobic surface residues of ubiquitin described by Beal et al. (26) at residues Leu8, Ile44, and Val70; purified proteins kindly provided by C. Pick-art). The altered ubiquitin molecules could be conjugated to substrates by E6AP and Rsp5p only slightly less efficiently than wild-type ubiquitin, however the defects were proportional to their ability to form the ubiquitin thioester intermediate (data not shown). Thus, these particular hydrophobic surface alterations do not mimic the affect of altering the -4F. Additional surface residues of ubiquitin are currently being investigated.
It has been suggested that the terminal C group of the HECT domain might participate in catalysis by acting as a general base to facilitate deprotonation of the incoming lysine residue side chain of the substrate (20). Thus, the terminal carboxylate might be a second determinant necessary specifically for substrate ubiquitination, and it is conceivable that this might also be linked to or cooperate in some way with -4F function. Rigorous testing of a potential role of the terminal carboxylate in ubiquitination will require chemical modification of the carboxyl group in an otherwise normal HECT E3. With respect to this hypothesis, however, it is perhaps interesting that eight amino acid C-terminal extensions to E6AP resulted in proteins with identical biochemical characteristics to the -4F to A mutants of these proteins: they could form the ubiquitin-thioester intermediate but could not transfer ubiquitin to the substrate. Therefore, if there is cooperation between the -4F and the terminal carboxylate, the separation between the phenylalanine side chain and the carboxylate might be expected to be critical. Alternatively, if the -4F contacts ubiquitin, the affect of the C-terminal extensions might be explained by a simple steric inhibition of the extended sequence on the -4F-ubiquitin interaction.
A single Angelman syndrome patient was identified in which the maternal E6AP allele contained a 15-bp deletion beginning with the last nucleotide before the stop codon and extending into the 3'-untranslated region (3142del15) (24). The investigators who identified this mutation suggested that the predicted protein would terminate in a string of lysines encoded by the poly(A) tail of mRNA (4); however, rigorous characterization of E6AP mRNAs (27) indicates the protein predicted from the 3142del15 alteration would be extended by 39 amino acids. Whereas this 39 amino acid-extended protein has not been analyzed biochemically, we predict, based on the results presented here, that if it is able to form the ubiquitin-thioester intermediate it would be unable to transfer ubiquitin to substrates. Indeed, Rsp5p with a large C-terminal extension (an 190 amino acid "TAP" tag; Ref. 28) is still able to form a ubiquitin-thioester in vitro, but cannot transfer that ubiquitin to itself in a self-ubiquitination assay (not shown). Together, this suggests that the protein encoded by the 3142del15 Angelman syndrome-associated mutation is likely to be specifically defective in catalysis of ubiquitin-isopeptide conjugates.
The HECT domain -4F is the first determinant to be identified that is required for the last step in transfer of ubiquitin to substrate, but is not required for upstream steps in the enzymatic cycle. Whereas other such determinants may yet be identified, the ability to separate the transthiolation step from the ubiquitination step in vitro will permit more detailed biochemical investigations of each of both of these steps. In addition, in cases where there may be an advantage to therapeutically inactivating HECT E3s, such as in HPV E6-expressing cervical cancers and their associated metastases, the identification of the essential role of the -4F highlights a specific and exposed target for potential drug interaction.
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FOOTNOTES |
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Present address: Laureate Pharma, L.P., 201 College Road East, Princeton, NJ 08540.
To whom correspondence should be addressed. Tel.: 512-232-7700; Fax: 512-232-3432; E-mail: huibreg{at}icmb.utexas.edu.
1 The abbreviations used are: Ubc, ubiquitin-conjugating proteins; HPV, human papillomavirus; GST, glutathione S-transferase; DTT, dithiothreitol; AS, Angelman syndrome.
2 C. Salvat and J. M. Huibregtse, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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