The Stability of Ribosome Biogenesis Factor WBSCR22 Is Regulated by Interaction with TRMT112 via Ubiquitin-Proteasome Pathway

ABSTRACT Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.

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Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

Frontiers in Pharmacology ◽

10.3389/fphar.2021.763950 ◽

2021 ◽

Vol 12 ◽

Author(s):

Edanur Sen ◽

Krishna P. Kota ◽

Rekha G. Panchal ◽

Sina Bavari ◽

Erkan Kiris

Keyword(s):

Small Molecules ◽

Ubiquitin Proteasome Pathway ◽

Lead Compounds ◽

Botulinum Neurotoxins ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

The Stability ◽

Dose Dependent ◽

In Cells

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

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The Molecular Basis of Ubiquitin-Conjugating Enzymes (E2s) as a Potential Target for Cancer Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms22073440 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3440

Author(s):

Xiaodi Du ◽

Hongyu Song ◽

Nengxing Shen ◽

Ruiqi Hua ◽

Guangyou Yang

Keyword(s):

Therapeutic Target ◽

Molecular Basis ◽

Biological Processes ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Conjugating Enzymes ◽

The Stability ◽

Ubiquitin Conjugating Enzymes ◽

Effective Drugs

Ubiquitin-conjugating enzymes (E2s) are one of the three enzymes required by the ubiquitin-proteasome pathway to connect activated ubiquitin to target proteins via ubiquitin ligases. E2s determine the connection type of the ubiquitin chains, and different types of ubiquitin chains regulate the stability and activity of substrate proteins. Thus, E2s participate in the regulation of a variety of biological processes. In recent years, the importance of E2s in human health and diseases has been particularly emphasized. Studies have shown that E2s are dysregulated in variety of cancers, thus it might be a potential therapeutic target. However, the molecular basis of E2s as a therapeutic target has not been described systematically. We reviewed this issue from the perspective of the special position and role of E2s in the ubiquitin-proteasome pathway, the structure of E2s and biological processes they are involved in. In addition, the inhibitors and microRNAs targeting E2s are also summarized. This article not only provides a direction for the development of effective drugs but also lays a foundation for further study on this enzyme in the future.

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Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin–proteasome pathway

Biochemical Society Transactions ◽

10.1042/bst0330953 ◽

2005 ◽

Vol 33 (5) ◽

pp. 953-956 ◽

Cited By ~ 30

Author(s):

Q. He ◽

Y. Liu

Keyword(s):

Circadian Clock ◽

Major Function ◽

Ubiquitin Proteasome Pathway ◽

Mutant Strains ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

The Stability ◽

Clock Protein

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin–proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCFFWD−1 complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCFFWD−1 and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

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