scholarly journals Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Aixin Song ◽  
Zachary Hazlett ◽  
Dulith Abeykoon ◽  
Jeremy Dortch ◽  
Andrew Dillon ◽  
...  
Keyword(s):  
Active Site ◽  
Human Cells ◽  
26S Proteasome ◽  
Deubiquitinating Enzyme ◽  
Ubiquitin Chain ◽  
Proteasome Degradation ◽  
Chain Architecture ◽  
Cultured Human Cells ◽  
Branched Chain ◽  
Proteolytic Stress

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

scholarly journals Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

2021 ◽  
Author(s):  
Aixin Song ◽  
Zachary Hazlett ◽  
Dulith Abeykoon ◽  
Jeremy Dortch ◽  
Andrew Dillon ◽  
...  
Keyword(s):  
Active Site ◽  
Human Cells ◽  
26S Proteasome ◽  
Deubiquitinating Enzyme ◽  
Ubiquitin Chain ◽  
Proteasome Degradation ◽  
Chain Architecture ◽  
Cultured Human Cells ◽  
Branched Chain ◽  
Proteolytic Stress

AbstractUCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

scholarly journals Deubiquitinating enzyme USP3 controls CHK1 chromatin association and activation

2018 ◽  
Vol 115 (21) ◽  
pp. 5546-5551 ◽  
Author(s):  
Yu-Che Cheng ◽  
Sheau-Yann Shieh
Keyword(s):  
Nuclear Localization ◽  
Active Site ◽  
Kinase Activity ◽  
Genotoxic Stress ◽  
Inhibitory Effect ◽  
Nuclear Localization Sequence ◽  
Deubiquitinating Enzyme ◽  
Ubiquitin Chain ◽  
Critical Residue ◽  
Localization Sequence

Checkpoint kinase 1 (CHK1), a Ser/Thr protein kinase, is modified by the K63-linked ubiquitin chain in response to genotoxic stress, which promotes its nuclear localization, chromatin association, and activation. Interestingly, this bulky modification is linked to a critical residue, K132, at the kinase active site. It is unclear how this modification affects the kinase activity and how it is removed to enable the release of CHK1 from chromatin. Herein, we show that the K63-linked ubiquitin chain at CHK1’s K132 residue has an inhibitory effect on the kinase activity. Furthermore, we demonstrate that this modification can be removed by ubiquitin-specific protease 3 (USP3), a deubiquitinating enzyme that targets K63-linked ubiquitin chains. Wild-type USP3, but not the catalytically defective or nuclear localization sequence-deficient mutants, reduced CHK1 K63-linked ubiquitination. Conversely, USP3 knockdown elevated K63-linked ubiquitination of the kinase, leading to prolonged CHK1 chromatin association and phosphorylation. Paradoxically, by removing the bulky ubiquitin chain at the active site, USP3 also increased the accessibility of CHK1 to its substrates. Thus, our findings on the dual roles of USP3 (namely, one to release CHK1 from the chromatin and the other to open up the active site) provide further insights into the regulation of CHK1 following DNA damage.

Alpha-Tocopherol Protects Cultured Human Cells from the Acute Lethal Cytotoxicity of Dioxin

2002 ◽  
Vol 72 (3) ◽  
pp. 147-153 ◽  
Author(s):  
Kei-Ichi Hirai ◽  
Jie-Hong Pan ◽  
Ying-Bo Shui ◽  
Eriko Simamura ◽  
Hiroki Shimada ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Damage ◽  
Smooth Endoplasmic Reticulum ◽  
Dehydroascorbic Acid ◽  
Human Cells ◽  
Alpha Tocopherol ◽  
Cervical Cancer Cells ◽  
Cultured Human Cells ◽  
Nuclear Damage ◽  
Conjunctival Epithelial Cells

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30–40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0–31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78–97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinate in cultured human cells and the role of glutathione

2008 ◽  
Vol 228 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Takafumi Ochi ◽  
Kayoko Kita ◽  
Toshihide Suzuki ◽  
Alice Rumpler ◽  
Walter Goessler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Human Cells ◽  
Cultured Human Cells

scholarly journals Multiple Fates of L1 Retrotransposition Intermediates in Cultured Human Cells

2005 ◽  
Vol 25 (17) ◽  
pp. 7780-7795 ◽  
Author(s):  
Nicolas Gilbert ◽  
Sheila Lutz ◽  
Tammy A. Morrish ◽  
John V. Moran
Keyword(s):  
Genetic Instability ◽  
Noncoding Rnas ◽  
Human Cells ◽  
Genomic Rearrangements ◽  
U6 Snrna ◽  
Human Dna ◽  
Cultured Human Cells ◽  
Repair Pathways ◽  
Genomic Locations ◽  
Host Parasite

ABSTRACT LINE-1 (L1) retrotransposons comprise ∼17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ∼1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.

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