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Ubiquitin and its roles: Cellular degradation of proteins is carried out by two main pathways: the lysosomal and the proteasomal. The latter pathway is more specific due to the use of ubiquitin as a marker. Ubiquitin is a small, highly conserved protein that can be attached to other proteins via an isopeptide bond between its C-terminus and a lysine residue on the protein. Several other ubiquitins are then added on by Gly76-Lys48 isopeptide linkage between individual monomers. Thus marked, the protein is recognized by a multienzyme complex, known as the 26S proteasome, and degraded. The ubiquitin chain is released and disassembled. Many kinds of proteins are degraded by this pathway including damaged proteins, cyclins, p53 tumor suppressor, and CFTR chloride channel. This pathway functions in the stress response and the production of antigens for the class I MHCs (1).
Ubiquitinylation is not limited to degraded proteins. Ubiquitin has been found on certain cell membrane receptors such as the platelet-derived growth factor receptor and the T-cell antigen receptor (1). Two types of histones, H2A and H2B, can also be ubiquitinylated at different points in the cell cycle. Ubiquitinylation of these proteins has not been shown to result in their degradation. The attachment of ubiquitin is reversible. In the case of histones, ubiquitin is present during interphase but is removed in mitosis. Mutants defective in ubiquitin conjugating enzymes fail to condense their chromosomes and arrest in S-phase. These and other observations led to the hypothesis that ubiquitin may be involved in maintaining chromosome structure (2). The genes encoding ubiquitin are unusual in that they code for a fusion protein of either several ubiquitin monomers or for a ubiquitin and a ribosomal zinc finger protein. The fusion protein has to be cleaved to produce functional monomers (1). Ubiquitin C-terminal Hydrolases (UCHs): The disassembly of polyubiquitin chains, cleavage of ubiquitin gene products and removal of ubiquitin from proteins requires the presence of enzymes that can cleave peptide bonds at the C-terminus of ubiquitin. Two families of such enzymes have been identified. All of these enzymes are thiol proteases that use the cysteine nucleophile to cleave peptide bonds. The enzymes thus far characterized have been shown to have substrate specificity for the type of peptide or amino acid attached to the C-terminus of ubiquitin and the type of linkage by which it is attached (1). The substrate specificity and tissue specific expression of these enzymes indicate that they play an important role in the regulation of the ubiquitin system. Two human isozymes of the UCH family 1 have been identified, cloned, expressed and characterized in our laboratory (3, 4). These enzymes are small globular proteins that possess conserved active site cysteine, histidine, and aspartate (4). They have substrate specificity for short peptides or extended chains at the C-terminus of ubiquitin and have been shown to disassemble ubiquitin gene products (5).
In this poster, we describe the identification, sequencing of, and construction of an expression vector for a gene encoding a new member of UCH family 1. We also suggest that this enzyme may function in removal ubiquitin from histone H2A. The expression of this protein was undertaken to study its substrate specificity and structure and to produce antibodies for the study of its functions in vivo.
Identification and Sequencing of the UCH LX cDNA: Two cDNA clones containing the 5' and 3' ends of the human UCH LX gene were identified by BLAST homology search of the GeneBank EST database. These clones were obtained from IMAGE consortium and digested with two pairs of restriction enzymes to confirm their identity. Sequencing was carried out by the dideoxynucleotide chain termination method using 35S, a dATP, as an isotope marker. A total of 9 cDNA specific primers and 2 vector specific primers were used. Inosine triphosphate and 7-deaza dGTP nucleotide analogues were used to resolve G/C compressions. The sequence was analyzed by the GeneMark program to identify the start codon. A brain cDNA library was screened using a sequence specific and a vector specific primer for a 5' extension of the cDNA. Construction of an expression vector: Once the sequence was thought to be complete, the gene was put together in pSP64 vector, and an NdeI site was introduced at the start codon by PCR for future cloning into the pRSETB expression vector. UCH LX was cut out of pSP64 with NdeI and BamHI and cloned into complementary sites in pRSETB vector. Top10 strain of E.coli was transformed with the pRSETB_UCH LX plasmid, and clones were screened for correct insertion by NdeI/BamHI digest. The correct clones were transformed into the expression strain DE3. The expression vector pRSETB contains T7 promoter, ribosomal binding site, and T7 terminator sequences. The expression strain possesses a genomic copy of phage T7 RNA polymerase under the control of a lac operator. Upon induction with IPTG, T7 polymerase is produced and can transcribe the gene of interest at a high level, overexpressing the protein. Purification of uH2A, a potential substrate: The ubiquitinylated histone H2A was partially purified from rabbit liver using the procedure of Hunter and Cary (6). In brief, the liver tissue was homogenized and centrifuged. The resulting pellet was washed with several buffers to remove non-histone protein, histone H1 and DNA. The core histone pellet was dissolved in 6M deionized urea and applied to Sephadex G-100 gel filtration column. Fractions were screened for uH2A by SDS-PAGE, and the protein was detected by size. The fractions of interest were pooled, dialyzed, and concentrated in an Amicon ultrafiltration cell in preparation for the next purification step.
The UCH LX cDNA was found to be 3.4 kb long, containing a 1125 bp putative coding sequence, a 39 bp 5' and a 2.2 kb 3' untranslated region. The 3' untranslated region has not been sequenced in its entirety. No further 5' extension of the cDNA was identified by a PCR screen of a brain cDNA library. The cDNA encodes a putative 388 amino acid protein of molecular weight around 43 KD. Homologies between UCH LX and the human UCH isozymes L1 and L3 are greatest around the catalytic core of the enzymes. All sequences possess cysteine, histidine, and aspartate residues that are believed to be necessary for the catalytic activity of UCH thiol proteases (4). The sequence of UCH LX possesses strong homology to a putative protein sequence in C. elegans. A nearly identical partial cDNA sequence from a mouse has also been identified. A complete cDNA sequence of UCH LX has been constructed and cloned into pRSETB expression vector. Expression of the protein in E. coli DE3 expression host is underway. The size of the UCH LX and its acidic pI are similar to a previously reported bovine enzyme, Isopeptidase (7). Isopeptidase is 38,000k in size as determined by gel-filtration chromatography and has a pI of 5-5.5. This enzyme has been shown to remove ubiquitin from histone H2A and is implicated in the regulation of its ubiquitinylation state during the cell cycle. The enzyme has never been purified to homogeneity, and its sequence has not been determined. To investigate the ability of recombinant UCH LX to cleave uH2A, the substrate has been partially purified from rabbit liver.
The ability of UCH LX to hydrolyze ubiquitin ethyl ester (a model substrate for UCHs) will be examined. If the enzyme is found to be active, its ability to hydrolyze uH2A and other substrates will be studied.
Purification of the protein by ion exchange and ubiquitin affinity chromatography will also be attempted. If UCH LX is shown to deubiquitinate histone H2A, its regulation during the cell cycle can be studied to learn more about the dynamics of chromosome condensation. Due to the larger size of this protein, its activity and substrate specificity can be significantly different from that of other UCH family 1 enzymes. The characterization of UCH LX will increase our knowledge of this enzyme family and its role in the regulation of the ubiquitin system.
- Wilkinson, K. D., Annual Reviews in Nutrition, 15, 161-89 (1995)
- Bradbury, E. M., BioEssays 14, 9-16 (1992)
- Mayer, A. N. and K. D. Wilkinson, Biochemistry, 28, 166 (1989)
- Larsen, C. N., J. S. Price, K. D. Wilkinson, Biochemistry, 35, 6735-6744 (1996)
- Larsen, C. N., B. A. Krantz, K. D. Wilkinson, (in press)
- Hunter A. J. and P. D. Cary, Analytical Biochemistry, 150, 394-402 (1985)
- Matsui S. I., A. A. Sandberg, S. Negoro, B. K. Seon, G. Goldstein, Proceedings of the National Academy of Sciences, USA, 79, 1535-1539 (1982)
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