scholarly journals E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes

2020 ◽  
Vol 118 (1) ◽  
pp. e2015794118
Author(s):  
Stephanie L. Sarbanes ◽  
Vincent A. Blomen ◽  
Eric Lam ◽  
Søren Heissel ◽  
Joseph M. Luna ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
E3 Ubiquitin Ligase ◽  
Respiratory Illness ◽  
Host Factors ◽  
Therapeutic Interventions ◽  
Nuclear Pore ◽  
Dna Viruses ◽  
Viral Capsids ◽  
Severe Respiratory Illness

The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.

scholarly journals Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Praveen Bawankar ◽  
Tina Lence ◽  
Chiara Paolantoni ◽  
Irmgard U. Haussmann ◽  
Migle Kazlauskiene ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Sex Determination ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
E3 Ubiquitin Ligase ◽  
Human Cells ◽  
Biological Functions ◽  
Mrna Metabolism ◽  
Mrna Methylation ◽  
Core Components

AbstractN6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

scholarly journals Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

2015 ◽  
Vol 36 (5) ◽  
pp. 794-808 ◽  
Author(s):  
Sarah M. Pedersen ◽  
Waipan Chan ◽  
Rakhi P. Jattani ◽  
deMauri S. Mackie ◽  
Joel L. Pomerantz
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
E3 Ubiquitin Ligase ◽  
Antigen Receptor ◽  
Resonance Energy ◽  
B Cell Lymphoma ◽  
Receptor Signaling ◽  
Antigen Receptor Signaling

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activityin vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.

scholarly journals Wnt Regulation: Exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

2019 ◽  
Author(s):  
Kristina N. Schaefer ◽  
Mira Pronobis ◽  
Clara E. Williams ◽  
Shiping Zhang ◽  
Lauren Bauer ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Human Cancer ◽  
E3 Ubiquitin Ligase ◽  
Adult Stem Cell ◽  
Dynamic Component ◽  
Test Models ◽  
Rgs Domain ◽  
Underlying Mechanisms

AbstractWnt signaling plays key roles in embryonic development and adult stem cell homeostasis, and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin. The multiprotein destruction complex binds and phosphorylates β-catenin, and transfers it to the SCF-TrCP E3-ubiquitin ligase, for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-catenin. Recent work clarified underlying mechanisms, but important questions remain. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin’s RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. SIM imaging in mammalian cells suggests that this may result by promoting Dishevelled: Dishevelled interactions at the expense of Dishevelled:Axin interactions when Dishevelled levels are high.

scholarly journals Immunogenetic Factors Associated with Severe Respiratory Illness Caused by Zoonotic H1N1 and H5N1 Influenza Viruses

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Jennifer Juno ◽  
Keith R. Fowke ◽  
Yoav Keynan
Keyword(s):  
Influenza Infection ◽  
Severe Infection ◽  
Respiratory Illness ◽  
Influenza Viruses ◽  
Therapeutic Interventions ◽  
H1n1 Pandemic ◽  
Factors Associated ◽  
H5n1 Influenza ◽  
At Risk Populations ◽  
Severe Respiratory Illness

Following the 2009 H1N1 pandemic and ongoing sporadic avian-to-human transmission of H5N1 viruses, an emphasis has been placed on better understanding the determinants and pathogenesis of severe influenza infections. Much of the current literature has focused on viral genetics and its impact on host immunity as well as novel risk factors for severe infection (particularly within the H1N1 pandemic). An understanding of the host genetic determinants of susceptibility and severe respiratory illness, however, is currently lacking. By better defining the role of genetic variability in influenza infection and identifying key polymorphisms that impair the host immune response or correlate with protection, we will be able to better identify at-risk populations and new targets for therapeutic interventions and vaccines. This paper will summarize known immunogenetic factors associated with susceptibility or severity of both pH1N1 and H5N1 infections and will also identify genetic pathways and polymorphisms of high relevance for future study.

scholarly journals Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters

2021 ◽  
Author(s):  
Rakesh Kulkarni ◽  
Wen-Ching Chen ◽  
Ying Lee ◽  
Chi-Fei Kao ◽  
Shiu-Lok Hu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Respiratory Illness ◽  
Cold Chain ◽  
Infection Model ◽  
Recombinant Vaccines ◽  
Viral Spread ◽  
Severe Respiratory Illness

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the cold chain transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

scholarly journals Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Ziying Han ◽  
Cari A. Sagum ◽  
Fumio Takizawa ◽  
Gordon Ruthel ◽  
Corbett T. Berry ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
E3 Ubiquitin Ligase ◽  
Hemorrhagic Fever ◽  
Efficient Production ◽  
Ww Domain ◽  
Ww Domains ◽  
E3 Ligases

ABSTRACT Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress. IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

scholarly journals The E3 Ubiquitin Ligase Mind Bomb 1 Controls Adenovirus Genome Release at the Nuclear Pore Complex

Cell Reports ◽  
2019 ◽  
Vol 29 (12) ◽  
pp. 3785-3795.e8 ◽  
Author(s):  
Michael Bauer ◽  
Justin W. Flatt ◽  
Daria Seiler ◽  
Bettina Cardel ◽  
Mario Emmenlauer ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Nuclear Pore Complex ◽  
E3 Ubiquitin Ligase ◽  
Nuclear Pore

scholarly journals E3 Ubiquitin Ligase Activity and Targeting of BAT3 by Multiple Legionella pneumophila Translocated Substrates

2010 ◽  
Vol 78 (9) ◽  
pp. 3905-3919 ◽  
Author(s):  
Alexander W. Ensminger ◽  
Ralph R. Isberg
Keyword(s):  
Ubiquitin Ligase ◽  
Legionella Pneumophila ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
E3 Ubiquitin Ligase ◽  
Bacterial Pathogen ◽  
Type Iv ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitination Machinery

ABSTRACT The intracellular bacterial pathogen Legionella pneumophila modulates a number of host processes during intracellular growth, including the eukaryotic ubiquitination machinery, which dictates the stability, activity, and/or localization of a large number of proteins. A number of L. pneumophila proteins contain eukaryotic-like motifs typically associated with ubiquitination. Central among these is a family of five F-box-domain-containing proteins of Legionella pneumophila. Each of these five proteins is translocated to the host cytosol by the Dot/Icm type IV protein translocation system during infection. We show that three of these proteins, LegU1, LegAU13, and LicA, interact with components of the host ubiquitination machinery in vivo. In addition, LegU1 and LegAU13 are integrated into functional Skp-Cullin-F-box (SCF) complexes that confer E3 ubiquitin ligase activity. LegU1 specifically interacts with and can direct the ubiquitination of the host chaperone protein BAT3. In a screen for additional L. pneumophila proteins that associate with LegU1 in mammalian cells, we identified the bacterial protein Lpg2160. We demonstrate that Lpg2160 also associates with BAT3 independently of LegU1. We show that Lpg2160 is a translocated substrate of the Dot/Icm system and contains a C-terminal translocation signal. We propose a model in which LegU1 and Lpg2160 may function redundantly or in concert to modulate BAT3 activity during the course of infection.

scholarly journals Mammalian SWI/SNF-A Subunit BAF250/ARID1 Is an E3 Ubiquitin Ligase That Targets Histone H2B

2010 ◽  
Vol 30 (7) ◽  
pp. 1673-1688 ◽  
Author(s):  
Xuan Shirley Li ◽  
Patrick Trojer ◽  
Tatsushi Matsumura ◽  
Jessica E. Treisman ◽  
Naoko Tanese
Keyword(s):  
Chromatin Remodeling ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
E3 Ubiquitin Ligase ◽  
Dependent Manner ◽  
Histone H2b ◽  
Enzymatic Function ◽  
Chromatin Remodeling Complex

ABSTRACT The mammalian SWI/SNF chromatin-remodeling complex facilitates DNA access by transcription factors and the transcription machinery. The characteristic member of human SWI/SNF-A is BAF250/ARID1, of which there are two isoforms, BAF250a/ARID1a and BAF250b/ARID1b. Here we report that BAF250b complexes purified from mammalian cells contain elongin C (Elo C), a BC box binding component of an E3 ubiquitin ligase. BAF250b was found to have a BC box motif, associate with Elo C in a BC box-dependent manner, and, together with cullin 2 and Roc1, assemble into an E3 ubiquitin ligase. The BAF250b BC box mutant protein was unstable in vivo and was autoubiquitinated in a manner similar to that for the VHL BC box mutants. The discovery that BAF250 is part of an E3 ubiquitin ligase adds an enzymatic function to the chromatin-remodeling complex SWI/SNF-A. The immunopurified BAF250b E3 ubiquitin ligase was found to target histone H2B at lysine 120 for monoubiquitination in vitro. To date, all H2B monoubiquitination was attributed to the human homolog of yeast Bre1 (RNF20/40). Mutation of Drosophila osa, the homolog of BAF250, or depletion of BAF250 by RNA interference (RNAi) in cultured human cells resulted in global decreases in monoubiquitinated H2B, implicating BAF250 in the cross talk of histone modifications.

scholarly journals Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257191
Author(s):  
Rakesh Kulkarni ◽  
Wen-Ching Chen ◽  
Ying Lee ◽  
Chi-Fei Kao ◽  
Shiu-Lok Hu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Respiratory Illness ◽  
Cold Chain ◽  
Infection Model ◽  
Recombinant Vaccines ◽  
Viral Spread ◽  
Severe Respiratory Illness

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10–15% of infected individuals and mortality in 2–3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the “cold chain” transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS-CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

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