Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Short-lived proteins are targeted for turnover by sequence elements known as degradation signals. Because of the large size and heterogeneity of these signals, the structural features important for their function are not well defined. In this study, we have isolated three classes of degradation signals by screening short artificial sequences for the ability to destabilize a reporter protein. Class I and class II signals were derived by inserting random nonapeptide sequences after the second residue of beta-galactosidase. Class III signals contained five-residue homopolymers at the same position. Class I beta-galactosidase turnover was inhibited in mutants lacking either the ubiquitin-conjugating enzyme Ubc2 or the ubiquitin protein ligase Ubr1. Class I random inserts functioned to promote N-terminal proteolytic processing and define a novel pathway for exposure of residues that are destabilizing according to the N-end rule. Efficient degradation of proteins containing class II signals required at least three Ubc enzymes: Ubc6, Ubc7, and either one of the related enzymes Ubc4 and Ubc5. Analysis of 56 amino acid substitutions in the class II signal suggested that it is recognized in the form of an amphipathic alpha helix. Class III signals consisted of short tracts of hydrophobic residues such as Leu and Ile. Degradation of class III proteins involved the Ubc4 and Ubc5 enzymes but not Ubc2, Ubc6, or Ubc7. Clusters of hydrophobic residues appear to be critical for the recognition of both class II and class III signals.
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PMID:Synthetic signals for ubiquitin-dependent proteolysis. 762 4

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme that is required for DNA repair, damage-induced mutagenesis, and sporulation. In addition, RAD6 mediates the multiubiquitination and degradation of amino-end rule protein substrates. The structure and function of RAD6 have been remarkably conserved during eukaryotic evolution. Here, we examine the role of the extremely conserved amino terminus, which has remained almost invariant among RAD6 homologs from yeast to human. We show that RAD6 is concentrated in the nucleus and that the amino-terminal deletion mutation, rad6 delta 1-9, does not alter the location of the protein. The amino-terminal domain, however, is essential for the multiubiquitination and degradation of amino-end rule substrates. In the rad6 delta 1-9 mutant, beta-galactosidase proteins bearing destabilizing amino-terminal residues become long lived, and purified rad6 delta 1-9 protein is ineffective in ubiquitin-protein ligase (E3)-dependent protein degradation in the proteolytic system derived from rabbit reticulocytes. The amino terminus is required for physical interaction of RAD6 with the yeast UBR1-encoded E3 enzyme, as the rad6 delta 1-9 protein is defective in this respect. The rad6 delta 1-9 mutant is defective in sporulation, shows reduced efficiency of DNA repair, but is proficient in UV mutagenesis. E3-dependent protein degradation by RAD6 could be essential for sporulation and could affect the efficiency of DNA repair.
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PMID:The extremely conserved amino terminus of RAD6 ubiquitin-conjugating enzyme is essential for amino-end rule-dependent protein degradation. 843 96

Retinoid X receptor alpha (RXR alpha) is a member of the steroid hormone receptor superfamily. Using yeast two-hybrid screening, beta-galactosidase assays, and pull-down assays, we show that RNF8, a RING finger protein recently isolated as a protein binding to a ubiquitin-conjugating enzyme, binds to RXR alpha through the N-terminal regions of both proteins. In COS7 cells, overexpressed RNF8 colocalized and interacted with RXR alpha in the nucleus, as shown by fluorescence resonance energy transfer. A point mutation of RNF8, Cys-403 to Ser (C403S), which disrupts the RING finger structure, or deletion of the N-terminal region (Delta N) of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXR alpha. Although transient overexpression of RNF8 had little effect on RXR alpha ubiquitination, RNF8 dose-dependently enhanced RXR alpha-mediated transactivation of the RXR-responsive element (RXRE)-bearing gene promoter without the addition of its ligand, 9-cis-retinoic acid (RA), and up-regulated the expression of the genes downstream of RXRE as well as an RA-response element. This transactivation-enhancing activity was not seen with either the C403S point mutant or the Delta N deletion mutant of RNF8. These results suggest a novel function of RNF8 as a regulator of RXR alpha-mediated transcriptional activity through interaction between their respective N-terminal regions.
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PMID:The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity. 1498 Oct 89

The nuclear matrix protein Msx2-interacting nuclear target protein (MINT) is a transcription factor that regulates the expression of key transcriptional effectors in diverse signaling pathways. To further understand the function and mechanism of the MINT-mediated transcription regulation, the yeast two-hybrid system was employed to screen proteins that interact with the C-terminal fragment of MINT. From a cDNA library of human lymph nodes, a cDNA encoding the ubiquitin-conjugating enzyme UbcH8 was identified. Using different truncated versions of MINT, we show that the C-terminal Spen paralog and ortholog C-terminal domain (SPOC) domain, which has been demonstrated to mediate interactions between MINT and a panel of other molecules, might be responsible for interaction between MINT and UbcH8 in yeast, as confirmed by the beta-galactosidase assay. The interaction between MINT and UbcH8 in mammalian cells was further proved by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, and co-immunoprecipitation assay. Using a reporter system, we found that MINT-mediated transcription suppression was sensitive to MG132, an inhibitor of the proteosome system. These results suggest a novel mechanism of MINT-mediated transcription regulation, and might be helpful for understanding functions of MINT.
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PMID:The Spen homolog Msx2-interacting nuclear target protein interacts with the E2 ubiquitin-conjugating enzyme UbcH8. 1658 36