Post-translational Conjugation Research Articles

Targeting Ubc9 for cancer therapy

Ubiquitin-conjugating enzyme (Ubc9) was originally thought to be a conjugating enzyme for ubiquitylation, but was later shown to be responsible for the most recently identified type of post-translational modification, (i.e., SUMO [small ubiquitin-related modifier]) conjugation or sumoylation. Like ubiquitylation, sumoylation modulates protein function through post-translational covalent attachment to lysine residues within targeted proteins. However, although ubiquitylation can lead to protein degradation through the 26S proteasome, sumoylation does not cause protein degradation; instead, it has been implicated in other cellular processes, such as regulating the activity of transcription factors, mediating nuclear translocation of proteins or the formation of subnuclear structures. Interestingly, some proteins can be modified at the same lysine residue by both SUMO and ubiquitin, but with distinct functional consequences. Given that many proteins involved in cell-cycle regulation, proliferation, apoptosis and DNA repair are targets for sumoylation, alterations of sumoylation could ultimately have an impact on cell growth, cancer development and drug responsiveness. As Ubc9 is the sole E2-conjugating enzyme required for sumoylation, and, in particular, Ubc9 is upregulated in an increasing number of human malignancies, such as ovarian carcinoma, melanoma and lung adenocarcinoma, it is a potential target for cancer therapy.

A Small Ubiquitin-Related Polypeptide Involved in Targeting RanGAP1 to Nuclear Pore Complex Protein RanBP2

We have found that the mammalian Ran GTPase–activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran–GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.

An Essential Yeast Gene Encoding a Homolog of Ubiquitin-activating Enzyme

Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.

The Saccharomyces cerevisiae DNA repair gene RAD23 encodes a nuclear protein containing a ubiquitin-like domain required for biological function.

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.

The Saccharomyces cerevisiae DNA Repair Gene RAD23 Encodes a Nuclear Protein Containing a Ubiquitin-Like Domain Required for Biological Function

In eukaryotes, the posttranslational conjugation of ubiquitin to various cellular proteins marks them for degradation. Interestingly, several proteins have been reported to contain ubiquitin-like (ub-like) domains that are in fact specified by the DNA coding sequences of the proteins. The biological role of the ub-like domain in these proteins is not known; however, it has been proposed that this domain functions as a degradation signal rendering the proteins unstable. Here, we report that the product of the Saccharomyces cerevisiae RAD23 gene, which is involved in excision repair of UV-damaged DNA, bears a ub-like domain at its amino terminus. This finding has presented an opportunity to define the functional significance of this domain. We show that deletion of the ub-like domain impairs the DNA repair function of RAD23 and that this domain can be functionally substituted by the authentic ubiquitin sequence. Surprisingly, RAD23 is highly stable, and the studies reported herein indicate that its ub-like domain does not mediate protein degradation. Thus, in RAD23 at least, the ub-like domain affects protein function in a nonproteolytic manner.

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.

Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae.

Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.

Regulation of the post-translational conjugation of amino acids to rat brain proteins

Post-translational conjugation of arginine (but not other amino acids) to proteins has been reported to occur in a high speed supernatant fraction of rat brain homogenates from which molecules of less than 5000 mol. wt have been removed. In the present study we report that removal of molecules of less than 1000 mol. wt by dialysis, does not result in incorporation of arginine into protein in amounts significantly different than in the undialysed supernatant. The addition of molecules with molecular weights greater than 1000 and less than 5000 to the active fraction, inhibits the incorporation of arginine into proteins in a concentration dependent manner suggesting that the post-translational incorporation of arginine into brain is regulated by a molecule(s) of greater than 1000 and less than 5000 mol. wt. Incorporation of lysine into proteins did not occur following removal of molecules of less than 5000 mol. wt, but did occur in the void volume fraction of a Sephacryl S-200 column (molecular weight cut-off 125,000), suggesting that the incorporation of lysine into proteins is regulated by molecules retained by the S-200 column but greater than 5000 mol. wt. When experiments were repeated using the void volume of a Sephacryl S-300 column (molecular weight exclusion, ~ 200 k), leucine and proline were incorporated in amounts similar to arginine and lysine, and serine, alanine, valine, phenylalanine and histidine were incorporated at lower but measurable levels. The data are interpreted as indicating the presence of regulatory molecules in vivo and in the high speed supernatant of rat brain, which can be removed by molecular weight exclusion chromatography and control the post-translational incorporation of individual amino acids into proteins. Comparison of the proteins modified by arginine and lysine using two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates that there exists a group of proteins modified by arginine, another group modified by lysine, and a third group which is modified by both arginine and lysine.

Purification and characterization of arginyl-tRNA-protein transferase from rabbit reticulocytes. Its involvement in post-translational modification and degradation of acidic NH2 termini substrates of the ubiquitin pathway.

Conjugation of ubiquitin to certain proteins can trigger their degradation. A major question concerns the structural features of a protein which make it susceptible to ubiquitin ligation. Recent studies have shown that the selection of proteins for degradation occurs most probably on a binding site of the ubiquitin-protein ligase (E3). It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin-ligating enzyme. Proteins with basic or bulky hydrophobic residues in the NH2-terminal position are recognized by the ligase, marked by ubiquitin, and degraded. This is not true, however, for proteins with an acidic residue in this position. We have previously shown that a tRNA-dependent post-translational conjugation of arginine to acidic NH2 termini of proteins is essential for their degradation via the ubiquitin pathway, and we speculated that this modification is required for their recognition by the ligase. In the present study we have partially purified from rabbit reticulocytes the modifying enzyme, arginyl-tRNA-protein transferase, and characterized it. We have separated the enzyme from other known components of the ubiquitin system and shown that it is specifically required for degradation of proteins with either an aspartate or glutamate residue in their NH2-terminal position. We have shown that the action of the transferase is required for conjugation of ubiquitin to the substrate and most probably for its recognition by the ligase. The enzyme in its native form has a molecular mass of about 360 kDa. It appears to be a complex between several molecules of arginyl-tRNA synthetase and arginyl-tRNA-protein transferase.

Role of arginine-tRNA in protein degradation by the ubiquitin pathway.

Degradation of intracellular proteins through the ubiquitin and ATP-dependent proteolysis pathway involves several steps. Initially, ubiquitin is covalently linked to the proteolytic substrate in an ATP-requiring reaction. Proteins marked by ubiquitin may then be selectively lysed in a reaction that also requires ATP (for reviews see refs 1-3). A major question concerns the structural features of a protein that make it a specific substrate for ubiquitin-mediated degradation. It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin ligation system, and that the half-life in vivo of a protein with an exposed amino terminus depends on its amino terminal residue. We have previously demonstrated that transfer RNA (tRNA) is essential for conjugation of ubiquitin and for the subsequent degradation of proteins with acidic amino termini (aspartate or glutamate). We now show that tRNA is required for post-translational conjugation of arginine to acidic amino termini of proteins, a modification that is essential for their degradation by the ubiquitin pathway.