scholarly journals E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Sarah Müncheberg ◽  
Ron T. Hay ◽  
Wing H. Ip ◽  
Tina Meyer ◽  
Christina Weiß ◽  
...  
Keyword(s):  
Gene Expression ◽  
Infected Cell ◽  
Viral Replication ◽  
Ubiquitin Ligase ◽  
Posttranslational Modification ◽  
E3 Ubiquitin Ligase ◽  
Human Adenovirus ◽  
Productive Infection ◽  
Dependent Manner ◽  
Ubiquitin Ligase Complex

ABSTRACTHuman adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCEDaxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

scholarly journals A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

2009 ◽  
Vol 83 (23) ◽  
pp. 12172-12184 ◽  
Author(s):  
Frédéric Dallaire ◽  
Paola Blanchette ◽  
Philip E. Branton
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Human Adenovirus ◽  
Productive Infection ◽  
Viral Mrnas ◽  
Lung Carcinoma Cells ◽  
Proteomic Approach ◽  
Ubiquitin Ligase Complex ◽  
Cullin 5 ◽  
Human Lung Carcinoma

ABSTRACT It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.

scholarly journals Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

2009 ◽  
Vol 83 (11) ◽  
pp. 5329-5338 ◽  
Author(s):  
Frédéric Dallaire ◽  
Paola Blanchette ◽  
Peter Groitl ◽  
Thomas Dobner ◽  
Philip E. Branton
Keyword(s):  
Ubiquitin Ligase ◽  
Small Cell Lung Carcinoma ◽  
E3 Ubiquitin Ligase ◽  
Human Adenovirus ◽  
Cellular Protein ◽  
Viral Mrnas ◽  
Cell Lung Carcinoma ◽  
Proteomic Screen ◽  
Ubiquitin Ligase Complex ◽  
Cellular Mrnas

ABSTRACT The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.

scholarly journals Regulation of photoprotection gene expression in Chlamydomonas by a putative E3 ubiquitin ligase complex and a homolog of CONSTANS

2019 ◽  
Vol 116 (35) ◽  
pp. 17556-17562 ◽  
Author(s):  
Stéphane T. Gabilly ◽  
Christopher R. Baker ◽  
Setsuko Wakao ◽  
Thien Crisanto ◽  
Katharine Guan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
High Light ◽  
Forward Genetics ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Low Light ◽  
Green Algal ◽  
Ubiquitin Ligase Complex

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii. The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes.

scholarly journals The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Frédéric Dallaire ◽  
Sabrina Schreiner ◽  
G. Eric Blair ◽  
Thomas Dobner ◽  
Philip E. Branton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Viral Gene Expression ◽  
Adenovirus Vector ◽  
Human Adenovirus ◽  
Viral Gene ◽  
E2f Transcription Factors

ABSTRACT During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication.

scholarly journals The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

mSphere ◽  
2015 ◽  
Vol 1 (1) ◽  
Author(s):  
Frédéric Dallaire ◽  
Sabrina Schreiner ◽  
G. Eric Blair ◽  
Thomas Dobner ◽  
Philip E. Branton ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Viral Gene Expression ◽  
Human Adenovirus ◽  
Viral Gene ◽  
Ubiquitin Ligase Complex ◽  
Cellular Dna ◽  
Adenovirus Replication ◽  
Do So

ABSTRACT Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication by enhancing the action of E1A products. Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication by enhancing the action of E1A products.

scholarly journals HIV/Simian Immunodeficiency Virus (SIV) Accessory Virulence Factor Vpx Loads the Host Cell Restriction Factor SAMHD1 onto the E3 Ubiquitin Ligase Complex CRL4DCAF1

2012 ◽  
Vol 287 (15) ◽  
pp. 12550-12558 ◽  
Author(s):  
Jinwoo Ahn ◽  
Caili Hao ◽  
Junpeng Yan ◽  
Maria DeLucia ◽  
Jennifer Mehrens ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virulence Factor ◽  
Ubiquitin Ligase ◽  
Catalytic Domain ◽  
E3 Ubiquitin Ligase ◽  
Dependent Manner ◽  
Heterologous Proteins ◽  
C Terminus ◽  
Ubiquitin Ligase Complex ◽  
Species Specific

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4DCAF1 E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4DCAF1-Vpx E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4DCAF1 and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

scholarly journals Aromatase is a novel neosubstrate of cereblon responsible for immunomodulatory drug–induced thrombocytopenia

Blood ◽  
2020 ◽  
Vol 135 (24) ◽  
pp. 2146-2158
Author(s):  
Taro Tochigi ◽  
Toshihiro Miyamoto ◽  
Kiwamu Hatakeyama ◽  
Teppei Sakoda ◽  
Daisuke Ishihara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Dependent Manner ◽  
Immunomodulatory Drugs ◽  
Drug Induced ◽  
Chromosome 5Q ◽  
Ubiquitin Ligase Complex ◽  
Immunomodulatory Drug ◽  
5Q Deletion

Abstract Immunomodulatory drugs (IMiDs) are key agents for the treatment of multiple myeloma and myelodysplastic syndrome with chromosome 5q deletion. IMiDs exert their pleiotropic effects through the recruitment of neosubstrates to cereblon, a substrate receptor of the E3 ubiquitin ligase complex; therefore, identification of cell-specific neosubstrates is important to understand the effects of IMiDs. In clinical practice, IMiDs induce thrombocytopenia, which frequently results in the discontinuation of IMiD treatment. In the current study, we sought to identify the molecular mechanism underlying thrombocytopenia induced by IMiD treatment. We found that IMiDs strongly impaired proplatelet formation, a critical step in functional platelet production, through the inhibition of autocrine estradiol signaling in human megakaryocytes. Furthermore, we identified aromatase, an indispensable enzyme for estradiol biosynthesis, as a novel neosubstrate of cereblon. IMiDs promoted the recruitment of aromatase to cereblon, resulting in the degradation of aromatase in a proteasome-dependent manner. Finally, aromatase was significantly degraded in the bone marrow of patients with multiple myeloma who developed thrombocytopenia with IMiD treatment. These data suggest that aromatase is a neosubstrate of cereblon that is responsible for IMiD-induced thrombocytopenia.

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