Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have utilized double-stranded RNA interference (RNAi) to examine the effects of reduced expression of individual subunits of the 26 S proteasome in Drosophila S2 cells. RNAi significantly decreased mRNA and protein levels of targeted subunits of both the core 20 S proteasome and the PA700 regulatory complex. Cells deficient in any of several 26 S proteasome subunits (e.g. d beta 5, dRpt1, dRpt2, dRpt5, dRpn2, and dRpn12) displayed decreased proteasome activity (as judged by hydrolysis of succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin), increased apoptosis, decreased cell proliferation without a specific block of the cell cycle, and accumulation of ubiquitinated cellular proteins. RNAi of many individual 26 S proteasome subunits promoted increased expression of many non-targeted subunits. This effect was not mimicked by chemical proteasome inhibitors such as lactacystin. Reduced expression of most targeted subunits disrupted the assembly of the 26 S proteasome. RNAi of six of eight targeted PA700 subunits disrupted that structure and caused accumulation of increased levels of uncapped 20 S proteasome. Notable exceptions included RNAi of dRpn10, a polyubiquitin binding subunit, and dUCH37, a ubiquitin isopeptidase. dRpn10-deficient cells showed a significant increase in succinyl-Leu-Leu-Val-Tyr-aminomethylcoumarin hydrolyzing activity of the 26 S proteasomes but accumulated polyubiquitinated proteins. d beta 5-Deficient cells had a phenotype similar to that of most PA700-deficient cells but also accumulated low molecular mass complexes containing subunits of the 20 S proteasome, probably representing unassembled precursors of the 20 S proteasomes. Cells deficient in several of the 26 S proteasome subunits were more resistant to otherwise toxic concentrations of various proteasome inhibitors. Our data suggest that those cells adapted to grow in conditions of impaired ubiquitin and proteasome-dependent protein degradation.
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PMID:Analysis of Drosophila 26 S proteasome using RNA interference. 1173 92

Time-resolved NMR spectroscopy is used to studychanges in protein conformation based on the elapsed time after a change in the solvent composition of a protein solution. The use of a micromixer and a continuous-flow method is described where the contents of two capillary flows are mixed rapidly, and then the NMR spectra of the combined flow are recorded at precise time points. The distance after mixing the two fluids and flow rates define the solvent-protein interaction time; this method allows the measurement of NMR spectra at precise mixing time points independent of spectral acquisition time. Integration of a micromixer and a microcoil NMR probe enables low-microliter volumes to be used without losing significant sensitivity in the NMR measurement. Ubiquitin, the model compound, changes its conformation from native to A-state at low pH and in 40% or higher methanol/water solvents. Proton NMR resonances of the His-68 and the Tyr-59 of ubiquitin are used to probe the conformational changes. Mixing ubiquitin and methanol solutions under low pH at microliter per minute flow rates yields both native and A-states. As the flow rate decreases, yielding longer reaction times, the population of the A-state increases. The micromixer-NMR system can probe reaction kinetics on a time scale of seconds.
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PMID:Micromixer-based time-resolved NMR: applications to ubiquitin protein conformation. 1262 91

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.
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PMID:Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. 1271 48

Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.
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PMID:Endocytosis of receptor tyrosine kinases is driven by monoubiquitylation, not polyubiquitylation. 1271 35

Downregulation of protein tyrosine kinases is a major function of the multidomain protein c-Cbl. This effect of c-Cbl is critical for both negative regulation of normal physiological stimuli and suppression of cellular transformation. In spite of the apparent importance of these effects of c-Cbl, their own regulation is poorly understood. To search for possible novel regulators of c-Cbl, we purified a number of c-Cbl-associated proteins by affinity chromatography and identified them by mass spectrometry. Among them, we identified the UBA- and SH3-containing protein T-cell Ubiquitin LigAnd (TULA), which can also bind to ubiquitin. Functional studies in a model system based on co-expression of TULA, c-Cbl, and EGF receptor in 293T cells demonstrate that TULA is capable of inhibiting c-Cbl-mediated downregulation of EGF receptor. Furthermore, modulation of TULA concentration in Jurkat T-lymphoblastoid cells demonstrates that TULA upregulates the activity of both Zap kinase and NF-AT transcription factor. Therefore, our study indicates that TULA counters the inhibitory effect of c-Cbl on protein tyrosine kinases and, thus, may be involved in the regulation of biological effects of c-Cbl. Finally, our results suggest that TULA-mediated inhibition of the effects of c-Cbl on protein tyrosine kinases is caused by TULA-induced ubiquitylation and degradation of c-Cbl.
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PMID:TULA: an SH3- and UBA-containing protein that binds to c-Cbl and ubiquitin. 1510 35

Cbl proteins are ubiquitin protein ligases, which ubiquitinate activated tyrosine kinases and target them for degradation. Both c-Cbl and Cbl-b have an ubiquitin associated (UBA) domain at their C-terminal end. We observed that high molecular weight ubiquitinated proteins constitutively coimmunoprecipitated with transfected and endogenous Cbl-b, but not c-Cbl. The binding site for these ubiquitinated proteins was mapped to the UBA domain of Cbl-b (UBAb). GST-fusion proteins containing the UBAb interacted with ubiquitinated proteins and polyubiquitin chains in vitro, whereas those containing the UBA domain of c-Cbl (UBAc) did not. The UBAb had a much greater affinity for polyubiquitin chains than for monoubiquitin. Analysis of the UBAb and UBAc demonstrate that the affinity for ubiquitin is determined by multiple amino-acid differences between the two domains. Overexpression of the UBAb, but not overexpression of the UBAc, inhibited a variety of ubiquitin-mediated processes such as degradation of ubiquitinated proteins (i.e. EGFR, Mdm-2, and Siah-1). This in vivo result is consistent with the differences in ubiquitin binding observed in vitro between the UBAb and UBAc. This difference in ubiquitin-binding may reflect distinct regulatory functions of c-Cbl and Cbl-b.
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PMID:Cbl-b interacts with ubiquitinated proteins; differential functions of the UBA domains of c-Cbl and Cbl-b. 1527 20

Regulation of tyrosine kinase-mediated cellular activation through antigen receptors is of great biological and practical significance. The evolutionarily conserved Cbl family ubiquitin ligases have emerged as key negative regulators of activated tyrosine kinase-coupled receptors, and their impaired function switches a normal immune response into autoimmunity. Cbl proteins facilitate the ubiquitinylation of activated tyrosine kinases and other signaling proteins and of the signaling chains of receptors themselves; monoubiquitin tag promotes sorting of activated receptors and associated proteins into internal vesicles of the multivesicular body, facilitating their lysosomal degradation, whereas polyubiquitin tag promotes proteasomal degradation. Notably, increased expression of Cbl proteins and other ubiquitin ligases is a component of anergic signaling program in T cells. Thus, controlled destruction of the signaling apparatus has emerged as a key to fine-tuning antigen receptor signaling. Further studies of this pathway are likely to elucidate the pathogenesis of autoimmune diseases and offer new therapeutic targets.
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PMID:The Cbl family and other ubiquitin ligases: destructive forces in control of antigen receptor signaling. 1534 16

Ubiquitin plays a fundamental role not only in proteasome-mediated protein degradation but also in the targeting of membrane proteins for degradation inside the lysosome. Ubiquitination provides a key signal for endosomal sorting of membrane proteins into the MVB (multi-vesicular body), which delivers its cargo to the proteolytic interior of the lysosome. Attachment of single ubiquitin molecules, rather than ubiquitin chains, to one or multiple lysines of the cytoplasmic domains of many growth factor receptors, ion channels and other membrane transporters is sufficient to target these proteins to a complex sorting apparatus on the endosome. This machinery selects ubiquitinated proteins for lysosomal sorting through consecutive interactions with a variety of ubiquitin-binding domains. The major ubiquitin ligase (E3) responsible for ubiquitination in this pathway in yeast is the HECT [homologous to E6-AP (E6-associated protein) C-terminus]-ligase, Rsp5, whereas in mammalian cells the RING (really interesting new gene)-ligase Cbl has been implicated in the down-regulation of several RTKs (receptor tyrosine kinases). Ubiquitinated receptors can be rescued from degradation by the activity of DUBs (deubiquitinating enzymes), which may provide a proofreading mechanism that enhances the fidelity of this sorting and degradation process. DUBs also allow for recycling of the ubiquitin moieties from proteins prior to their final commitment to the MVB and lysosome interior.
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PMID:Ubiquitin and endocytic protein sorting. 1625 Aug 99

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Primary destabilizing N-terminal residues (Nd(p)) are recognized directly by the targeting machinery. The recognition of secondary destabilizing N-terminal residues (Nd(s)) is preceded by conjugation of an Nd(p) residue to Nd(s) of a polypeptide substrate. In eukaryotes, ATE1-encoded arginyl-transferases (R(D,E,C*)-transferases) conjugate Arg (R), an Nd(p) residue, to Nd(s) residues Asp (D), Glu (E), or oxidized Cys residue (C*). Ubiquitin ligases recognize the N-terminal Arg of a substrate and target the (ubiquitylated) substrate to the proteasome. In prokaryotes such as Escherichia coli, Nd(p) residues Leu (L) or Phe (F) are conjugated, by the aat-encoded Leu/Phe-transferase (L/F(K,R)-transferase), to N-terminal Arg or Lys, which are Nd(s) in prokaryotes but Nd(p) in eukaryotes. In prokaryotes, substrates bearing the Nd(p) residues Leu, Phe, Trp, or Tyr are degraded by the proteasome-like ClpAP protease. Despite enzymological similarities between eukaryotic R(D,E,C*)-transferases and prokaryotic L/F(K,R)-transferases, there is no significant sequelogy (sequence similarity) between them. We identified an aminoacyl-transferase, termed Bpt, in the human pathogen Vibrio vulnificus. Although it is a sequelog of eukaryotic R(D,E,C*)-transferases, this prokaryotic transferase exhibits a "hybrid" specificity, conjugating Nd(p) Leu to Nd(s) Asp or Glu. Another aminoacyl-transferase, termed ATEL1, of the eukaryotic pathogen Plasmodium falciparum, is a sequelog of prokaryotic L/F(K,R)-transferases (Aat), but has the specificity of eukaryotic R(D,E,C*)-transferases (ATE1). Phylogenetic analysis suggests that the substrate specificity of R-transferases arose by two distinct routes during the evolution of eukaryotes.
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PMID:Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen. 1649 67

UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48- and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.
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PMID:The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation. 1652 Mar 78


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