scholarly journals Nedd8-Activating Enzyme Is a Druggable Host Dependency Factor of Human and Mouse Cytomegalovirus

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1610
Author(s):  
Yulia Alejandra Flores-Martínez ◽  
Vu Thuy Khanh Le-Trilling ◽  
Mirko Trilling
Keyword(s):  
Antiviral Activity ◽  
Human Cytomegalovirus ◽  
Cell Clone ◽  
Resistant Cell ◽  
Ubiquitinated Proteins ◽  
Potent Antiviral Activity ◽  
Ubiquitin Proteasome ◽  
Infected Cells ◽  
Proteasome Pathway ◽  
The Stability

Human cytomegalovirus causes diseases in individuals with insufficient immunity. Cytomegaloviruses exploit the ubiquitin proteasome pathway to manipulate the proteome of infected cells. The proteasome degrades ubiquitinated proteins. The family of cullin RING ubiquitin ligases (CRL) regulates the stability of numerous important proteins. If the cullin within the CRL is modified with Nedd8 (“neddylated”), the CRL is enzymatically active, while CRLs lacking Nedd8 modifications are inactive. The Nedd8-activating enzyme (NAE) is indispensable for neddylation. By binding to NAE and inhibiting neddylation, the drug MLN4924 (pevonedistat) causes CRL inactivation and stabilization of CRL target proteins. We showed that MLN4924 elicits potent antiviral activity against cytomegaloviruses, suggesting that NAE might be a druggable host dependency factor (HDF). However, MLN4924 is a nucleoside analog related to AMP, and the antiviral activity of MLN4924 may have been influenced by off-target effects in addition to NAE inhibition. To test if NAE is indeed an HDF, we assessed the novel NAE inhibitor TAS4464 and observed potent antiviral activity against mouse and human cytomegalovirus. Additionally, we raised an MLN4924-resistant cell clone and showed that MLN4924 as well as TAS4464 lose their antiviral activity in these cells. Our results indicate that NAE, the neddylation process, and CRLs are druggable HDFs of cytomegaloviruses.

scholarly journals Serogroup-Related Escape of Yersinia enterocolitica YopE from Degradation by the Ubiquitin-Proteasome Pathway

2007 ◽  
Vol 75 (9) ◽  
pp. 4423-4431 ◽  
Author(s):  
Moritz Hentschke ◽  
Konrad Trülzsch ◽  
Jürgen Heesemann ◽  
Martin Aepfelbacher ◽  
Klaus Ruckdeschel
Keyword(s):  
Host Cell ◽  
Yersinia Enterocolitica ◽  
Virulence Proteins ◽  
Ubiquitin Proteasome Pathway ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Protein Secretion System ◽  
Proteasome Pathway ◽  
Type Iii Protein Secretion ◽  
The Stability

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.

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

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.

scholarly journals Strigolactone Analogs Are Promising Antiviral Agents for the Treatment of Human Cytomegalovirus Infection

2020 ◽  
Vol 8 (5) ◽  
pp. 703
Author(s):  
Matteo Biolatti ◽  
Marco Blangetti ◽  
Giulia D’Arrigo ◽  
Francesca Spyrakis ◽  
Paola Cappello ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Human Cytomegalovirus ◽  
Antiviral Agents ◽  
Nucleoside Analogs ◽  
Developed Countries ◽  
Promising Alternative ◽  
Late Protein ◽  
Potential Applications ◽  
Infected Cells

The human cytomegalovirus (HCMV) is a widespread pathogen and is associated with severe diseases in immunocompromised individuals. Moreover, HCMV infection is the most frequent cause of congenital malformation in developed countries. Although nucleoside analogs have been successfully employed against HCMV, their use is hampered by the occurrence of serious side effects. There is thus an urgent clinical need for less toxic, but highly effective, antiviral drugs. Strigolactones (SLs) are a novel class of plant hormones with a multifaceted activity. While their role in plant-related fields has been extensively explored, their effects on human cells and their potential applications in medicine are far from being fully exploited. In particular, their antiviral activity has never been investigated. In the present study, a panel of SL analogs has been assessed for antiviral activity against HCMV. We demonstrate that TH-EGO and EDOT-EGO significantly inhibit HCMV replication in vitro, impairing late protein expression. Moreover, we show that the SL-dependent induction of apoptosis in HCMV-infected cells is a contributing mechanism to SL antiviral properties. Overall, our results indicate that SLs may be a promising alternative to nucleoside analogs for the treatment of HCMV infections.

scholarly journals Destabilization of YopE by the Ubiquitin-Proteasome Pathway Fine-Tunes Yop Delivery into Host Cells and Facilitates Systemic Spread ofYersinia enterocoliticain Host Lymphoid Tissue

2010 ◽  
Vol 79 (3) ◽  
pp. 1166-1175 ◽  
Author(s):  
Kristin Gaus ◽  
Moritz Hentschke ◽  
Nicole Czymmeck ◽  
Lena Novikova ◽  
Konrad Trülzsch ◽  
...  
Keyword(s):  
Lymphoid Tissue ◽  
Defense Responses ◽  
Host Cells ◽  
Virulence Plasmid ◽  
Ubiquitin Proteasome Pathway ◽  
Systemic Spread ◽  
Ubiquitin Proteasome ◽  
Infected Cells ◽  
Proteasome Pathway

ABSTRACTPathogenicYersiniaspecies inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of theY. enterocoliticaserogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in theY. enterocoliticaserogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, theYersiniamutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured,Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard,Y. enterocoliticaO8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.

scholarly journals Human Fas-Associated Factor 1, Interacting with Ubiquitinated Proteins and Valosin-Containing Protein, Is Involved in the Ubiquitin-Proteasome Pathway

2005 ◽  
Vol 25 (6) ◽  
pp. 2511-2524 ◽  
Author(s):  
Eun Joo Song ◽  
Seung-Hee Yim ◽  
Eunhee Kim ◽  
Nam-Soon Kim ◽  
Kong-Joo Lee
Keyword(s):  
Scaffolding Protein ◽  
Cellular Functions ◽  
Associated Factor ◽  
Binding Partner ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitinated Proteins ◽  
Uba Domain ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Novel Protein

ABSTRACT Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.

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

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.

scholarly journals Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation

2008 ◽  
Vol 89 (10) ◽  
pp. 2416-2426 ◽  
Author(s):  
Vu Thuy Khanh Le ◽  
Mirko Trilling ◽  
Manuel Wilborn ◽  
Hartmut Hengel ◽  
Albert Zimmermann
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Cytomegalovirus ◽  
Proteasome Inhibition ◽  
Early Gene ◽  
Similar Degree ◽  
Signal Transducer ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Infected Cells ◽  
Hcmv Replication

We have investigated the role of signal transducer and activator of transcription (STAT) 2 during human cytomegalovirus (HCMV) replication and found that protein levels of STAT2 are downregulated. STAT2 downregulation was observed in HCMV clinical isolates and laboratory strains with the exception of strain Towne. The HCMV-induced loss of STAT2 protein occurred despite an increased accumulation of STAT2 mRNA; it required HCMV early gene expression. The decrease in STAT2 was sensitive to proteasome inhibition, suggesting degradation of STAT2 via the ubiquitin proteasome pathway. Notably, pUL27, the HCMV homologue of the mouse CMV pM27 protein, which mediates the selective proteolysis of STAT2, did not induce STAT2 downregulation. Moreover, preceding STAT2 degradation, alpha/beta interferon (IFN)-receptor-mediated tyrosine phosphorylation of STAT2 was inhibited in HCMV-infected cells. This effect was paralleled by impaired tyrosine activation of STAT1 and STAT3. Accordingly, IFNs affected the replication efficiency of STAT2 degrading and non-degrading HCMV strains to a similar degree. In summary, HCMV abrogates IFN receptor signalling at multiple checkpoints by independent mechanisms including UL27-independent degradation of STAT2 and a preceding blockade of STAT2 phosphorylation.

scholarly journals Pyrrolidine Dithiocarbamate Reduces Coxsackievirus B3 Replication through Inhibition of the Ubiquitin-Proteasome Pathway

2005 ◽  
Vol 79 (13) ◽  
pp. 8014-8023 ◽  
Author(s):  
Xiaoning Si ◽  
Bruce M. McManus ◽  
Jingchun Zhang ◽  
Ji Yuan ◽  
Caroline Cheung ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Degradation ◽  
Viral Myocarditis ◽  
Coxsackievirus B3 ◽  
Ros Generation ◽  
Pyrrolidine Dithiocarbamate ◽  
Ubiquitin Proteasome Pathway ◽  
Cvb3 Infection ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

ABSTRACT Coxsackievirus B3 (CVB3) is one of the most common pathogens for viral myocarditis. The lack of effective therapeutics for CVB3-caused viral diseases underscores the importance of searching for antiviral compounds. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant and is recently reported to inhibit ubiquitin-proteasome-mediated proteolysis. Previous studies have shown that PDTC inhibits replication of rhinovirus, influenza virus, and poliovirus. In the present study, we report that PDTC is a potent inhibitor of CVB3. Coxsackievirus-infected HeLa cells treated with PDTC showed a significant reduction of CVB3 viral RNA synthesis, viral protein VP1 expression, and viral progeny release. Similar to previous observation that divalent ions mediate the function of PDTC, we further report that serum-containing copper and zinc are required for its antiviral activity. CVB3 infection resulted in massive generation of reactive oxygen species (ROS). Although PDTC alleviated ROS generation, the antiviral activity was unlikely dependent on its antioxidant effect because the potent antioxidant, N-acetyl-l-cysteine, failed to inhibit CVB3 replication. Consistent with previous reports that PDTC inhibits ubiquitin-proteasome-mediated protein degradation, we found that PDTC treatment led to the accumulation of several short-lived proteins in infected cells. We further provide evidence that the inhibitory effect of PDTC on protein degradation was not due to inhibition of proteasome activity but likely modulation of ubiquitination. Together with our previous findings that proteasome inhibition reduces CVB3 replication (H. Luo, J. Zhang, C. Cheung, A. Suarez, B. M. McManus, and D. Yang, Am. J. Pathol. 163:381-385, 2003), results in this study suggest a strong antiviral effect of PDTC on coxsackievirus, likely through inhibition of the ubiquitin-proteasome pathway.

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