Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ubiquitin-proteasome pathway (UPP) controls a wide range of signal transduction cascades by targeting key regulatory proteins for 26S proteasome-mediated degradation. Several observations suggest that protein deubiquitination may modulate this process; however, few experiments have been performed to test this idea. An excellent model system for studying the regulatory role of the UPP is signal transduction via the nuclear factor-kappa B (NF-kappa B) family of transcription factors. The principal inhibitor of NF-kappa B, I kappa B alpha, is polyubiquitinated and degraded in response to diverse stimuli. In this study, we sought to determine whether I kappa B alpha deubiquitination also occurs. We established an in vitro deubiquitination assay using polyubiquitinated I kappa B alpha as the substrate. Our data provide evidence of an I kappa B alpha-directed deubiquitinating activity present in lysates of several cell lines. This activity was inhibited by ubiquitin aldehyde, a specific inhibitor of deubiquitinating enzymes, as well as by alkylating reagents or heat, but was unaffected by the inhibition of several other classes of proteases. Cell lysates and the deubiquitinating enzyme, UCH-L3, hydrolyzed ubiquitin 7-amido-4-methylcoumarin, a model substrate for assaying deubiquitinating activities. However, UCH-L3 had no detectable activity toward ubiquitinated I kappa B alpha, thus suggesting a degree of enzymatic specificity in the deubiquitination of I kappa B alpha. This assay will be useful for the study of I kappa B alpha deubiquitination. Moreover, this assay can be adapted to monitor the deubiquitination of other proteins modified by ubiquitin conjugation.
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PMID:A novel in vitro assay for deubiquitination of I kappa B alpha. 1191 73

The ubiquitin pathway functions in the process of protein turnover in eukaryotic cells. This pathway comprises the enzymes that ubiquitinate/deubiquitinate target proteins and the proteasome that degrades ubiquitin-conjugated proteins. Ubiquitin C-terminal hydrolases (UCHs) are thought to be essential for maintaining ubiquitination activity by releasing ubiquitin (Ub) from its substrates. Mammalian UCH-L1 and UCH-L3 are small proteins that share considerable homology at the amino acid level. Both of these UCHs are highly expressed in the testis/ ovary and neuronal cells. Our previous work demonstrated that UCH-L1-deficient gracile axonal dystrophy (gad) mice exhibit progressively decreasing spermatogonial stem cell proliferation, suggesting that UCH isozymes in the testis function during spermatogenesis. To analyze the expression patterns of UCH isozymes during spermatogenesis, we isolated nearly homogeneous populations of spermatogonia, spermatocytes, spermatids, and Sertoli cells from mouse testes. Western blot analysis detected UCH-L1 in spermatogonia and Sertoli cells, whereas UCH-L3 was detected in spermatocytes and spermatids. Moreover, reverse transcription-polymerase chain reaction analysis of UCH isozymes showed that UCH-L1 and UCH-L4 mRNAs are expressed in spermatogonia, whereas UCH-L3 and UCH-L5 mRNAs are expressed mainly in spermatocytes and spermatids. These results suggest that UCH-L1 and UCH-L3 have distinct functions during spermatogenesis, namely, that UCH-L1 may act during mitotic proliferation of spermatogonial stem cells whereas UCH-L3 may function in the meiotic differentiation of spermatocytes into spermatids.
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PMID:Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice. 1508 87

To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.
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PMID:Delta12-Prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition. 1519 90

The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis.
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PMID:Two closely related ubiquitin C-terminal hydrolase isozymes function as reciprocal modulators of germ cell apoptosis in cryptorchid testis. 1546

UCH-L3 belongs to the ubiquitin C-terminal hydrolase family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. A murine Uchl3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. To elucidate the function of UCH-L3, we investigated histopathological changes and expression of apoptosis- and oxidative stress-related proteins during retinal degeneration. In the normal retina, UCH-L3 was enriched in the photoreceptor inner segment that contains abundant mitochondria. Although the retina of Uchl3-deficient mice showed no significant morphological abnormalities during retinal development, prominent retinal degeneration became manifested after 3 weeks of age associated with photoreceptor cell apoptosis. Ultrastructurally, a decreased area of mitochondrial cristae and vacuolar changes were observed in the degenerated inner segment. Increased immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment indicated mitochondrial oxidative stress. Expression of cytochrome c, caspase-1, and cleaved caspase-3 did not differ between wild-type and mutant mice; however, immunoreactivity for endonuclease G was found in the photoreceptor nuclei in the mutant retina. Hence, loss of UCH-L3 leads to mitochondrial oxidative stress-related photoreceptor cell apoptosis in a caspase-independent manner. Thus, Uchl3-deficient mice represent a model for adult-onset retinal degeneration associated with mitochondrial impairment.
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PMID:Photoreceptor cell apoptosis in the retinal degeneration of Uchl3-deficient mice. 1681 67

Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes.
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PMID:Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy. 1707 95

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. The two ubiquitin C-terminal hydrolase (UCH) enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. These two UCH isozymes have 52% amino acid identity and share significant structural similarity. A new function of these two closely related UCH enzymes during spermatogenesis which is associated with germ cell apoptosis has been analyzed. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. During spermatogenesis, apoptosis controls germ cell numbers and eliminates defective germ cells to facilitate testicular homeostasis. In this paper, I review the distinct function of the two UCH isozymes in the testis of gad and Uchl3 knockout mice, which are strongly but reciprocally expressed during spermatogenesis. In addition, the importance of UCHL1-dependent apoptosis for normal spermatogenesis and sperm quality control is discussed.
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PMID:The new function of two ubiquitin C-terminal hydrolase isozymes as reciprocal modulators of germ cell apoptosis. 1746 Mar 51

UCH-L3 (ubiquitin C-terminal hydrolase-L3) is a de-ubiquitinating enzyme that is a component of the ubiquitin-proteasome system and known to be involved in programmed cell death. A previous study of high-throughput drug screening identified an isatin derivative as a UCH-L3 inhibitor. In this study, we attempted to identify a novel inhibitor with a different structural basis. We performed in silico structure-based drug design (SBDD) using human UCH-L3 crystal structure data (PDB code; 1XD3) and the virtual compound library (ChemBridge CNS-Set), which includes 32,799 chemicals. By a two-step virtual screening method using DOCK software (first screening) and GOLD software (second screening), we identified 10 compounds with GOLD scores of over 60. To address whether these compounds exhibit an inhibitory effect on the de-ubiquitinating activity of UCH-L3, we performed an enzymatic assay using ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) as the substrate. As a result, we identified three compounds with similar basic dihydro-pyrrole skeletons as UCH-L3 inhibitors. These novel compounds may be useful for the research of UCH-L3 function, and in drug development for UCH-L3-associated diseases.
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PMID:Identification of novel chemical inhibitors for ubiquitin C-terminal hydrolase-L3 by virtual screening. 1776 21

Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein-folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C-terminal hydrolase UCH-L3, which is an integral part of the ubiquitin-proteasome system. The structure of UCH-L3 contains five distinct crossings of its polypeptide chain, and it adopts a 5(2)-knotted topology, making it a fascinating target for folding studies. Here, we provide the first in depth characterization of the stability and folding of UCH-L3. We show that the protein can unfold and refold reversibly in vitro without the assistance of molecular chaperones, demonstrating that all the information necessary for the protein to find its knotted native structure is encoded in the amino acid sequence, just as with any other globular protein, and that the protein does not enter into any deep kinetic traps. Under equilibrium conditions, the unfolding of UCH-L3 appears to be two-state, however, multiphasic folding and unfolding kinetics are observed and the data are consistent with a folding pathway in which two hyperfluorescent intermediates are formed. In addition, a very slow phase in the folding kinetics is shown to be limited by proline-isomerization events. Overall, the data suggest that a knotted topology, even in its most complex form, does not necessarily limit folding in vitro, however, it does seem to require a complex folding mechanism which includes the formation of several distinct intermediate species.
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PMID:Untangling the folding mechanism of the 5(2)-knotted protein UCH-L3. 1947 99

Ubiquitin C-terminal hydrolases (UCHs) are one of five sub-families of de-ubiquitinating enzymes (DUBs) that hydrolyze the C-terminal peptide bond of ubiquitin. UCH37 (also called UCH-L5) is the only UCH family protease that interacts with the 19S proteasome regulatory complex and disassembles Lys48-linked poly-ubiquitin from the distal end of the chain. The structures of three UCHs, UCH-L1, UCH-L3, and YUH1, have been determined by X-ray crystallography. However, little is known about their physiological substrates. These enzymes do not hydrolyze large adducts of ubiquitin such as proteins. To identify and characterize the hydrolytic specificities of their substrates, the crystal structure of the UCH37 catalytic domain (UCH-domain) was determined and compared with that of the other UCHs. The overall folding patterns are similar in these UCHs. However, helix-3 is collapsed in UCH37 and the pattern of electrostatic potential on the surface of the putative substrate-binding site (P'-site) is different. Helix-3 comprises an edge of the P'-site. As a result, the P'-site is wider than that in other UCHs. These differences indicate that UCH37 can interact with larger adducts such as ubiquitin.
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PMID:Crystal structure of the de-ubiquitinating enzyme UCH37 (human UCH-L5) catalytic domain. 1983 45


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