scholarly journals Latency-Associated Nuclear Antigen E3 Ubiquitin Ligase Activity Impacts Gammaherpesvirus-Driven Germinal Center B Cell Proliferation

2016 ◽  
Vol 90 (17) ◽  
pp. 7667-7683 ◽  
Author(s):  
Sofia A. Cerqueira ◽  
Min Tan ◽  
Shijun Li ◽  
Franceline Juillard ◽  
Colin E. McVey ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Ubiquitin Ligase ◽  
Germinal Center ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Nuclear Antigen ◽  
Ubiquitin Ligase Activity ◽  
Socs Box

ABSTRACTViruses have evolved mechanisms to hijack components of cellular E3 ubiquitin ligases, thus modulating the ubiquitination pathway. However, the biological relevance of such mechanisms for viral pathogenesisin vivoremains largely unknown. Here, we utilized murid herpesvirus 4 (MuHV-4) infection of mice as a model system to address the role of MuHV-4 latency-associated nuclear antigen (mLANA) E3 ligase activity in gammaherpesvirus latent infection. We show that specific mutations in the mLANA SOCS box (V199A, V199A/L202A, or P203A/P206A) disrupted mLANA's ability to recruit Elongin C and Cullin 5, thereby impairing the formation of the Elongin BC/Cullin 5/SOCS (EC5SmLANA) complex and mLANA's E3 ligase activity on host NF-κB and Myc. Although these mutations resulted in considerably reduced mLANA binding to viral terminal repeat DNA as assessed by electrophoretic mobility shift assay (EMSA), the mutations did not disrupt mLANA's ability to mediate episome persistence.In vivo, MuHV-4 recombinant viruses bearing these mLANA SOCS box mutations exhibited a deficit in latency amplification in germinal center (GC) B cells. These findings demonstrate that the E3 ligase activity of mLANA contributes to gammaherpesvirus-driven GC B cell proliferation. Hence, pharmacological inhibition of viral E3 ligase activity through targeting SOCS box motifs is a putative strategy to control gammaherpesvirus-driven lymphoproliferation and associated disease.IMPORTANCEThe gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) cause lifelong persistent infection and play causative roles in several human malignancies. Colonization of B cells is crucial for virus persistence, and access to the B cell compartment is gained by virus-driven proliferation in germinal center (GC) B cells. Infection of B cells is predominantly latent, with the viral genome persisting as a multicopy episome and expressing only a small subset of viral genes. Here, we focused on latency-associated nuclear antigen (mLANA) encoded by murid herpesvirus-4 (MuHV-4), which exhibits homology in sequence, structure, and function to KSHV LANA (kLANA), thereby allowing the study of LANA-mediated pathogenesis in mice. Our experiments show that mLANA's E3 ubiquitin ligase activity is necessary for efficient expansion of latency in GC B cells, suggesting that the development of pharmacological inhibitors of LANA E3 ubiquitin ligase activity may allow strategies to interfere with gammaherpesvirus-driven lymphoproliferation and associated disease.

scholarly journals The E3 ubiquitin ligase Itch restricts antigen-driven B cell responses

2019 ◽  
Vol 216 (9) ◽  
pp. 2170-2183 ◽  
Author(s):  
Emily K. Moser ◽  
Jennifer Roof ◽  
Joseph M. Dybas ◽  
Lynn A. Spruce ◽  
Steven H. Seeholzer ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Ubiquitin Ligase ◽  
Cell Activity ◽  
E3 Ubiquitin Ligase ◽  
Normal Numbers ◽  
Cell Responses ◽  
B Cell Responses

The E3 ubiquitin ligase Itch regulates antibody levels and prevents autoimmune disease in humans and mice, yet how Itch regulates B cell fate or function is unknown. We now show that Itch directly limits B cell activity. While Itch-deficient mice displayed normal numbers of preimmune B cell populations, they showed elevated numbers of antigen-experienced B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit naive and, to a greater extent, germinal center (GC) B cell numbers. B cells lacking Itch exhibited increased proliferation, glycolytic capacity, and mTORC1 activation. Moreover, stimulation of these cells in vivo by WT T cells resulted in elevated numbers of GC B cells, PCs, and serum IgG. These results support a novel role for Itch in limiting B cell metabolism and proliferation to suppress antigen-driven B cell responses.

scholarly journals Inhibition of cIAP1 as a strategy for targeting c-MYC–driven oncogenic activity

2018 ◽  
Vol 115 (40) ◽  
pp. E9317-E9324 ◽  
Author(s):  
Haoyan Li ◽  
Yanjia Fang ◽  
Chunyi Niu ◽  
Hengyi Cao ◽  
Ting Mi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Pharmacological Agents ◽  
Protein Levels ◽  
Ubiquitin Ligase Activity ◽  
Molecule Inhibitor ◽  
Smac Mimetics ◽  
High Throughput Assay ◽  
In Cells

Protooncogenec-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.

scholarly journals Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 222-229 ◽  
Author(s):  
KF Norrback ◽  
K Dahlenborg ◽  
R Carlsson ◽  
G Roos
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Telomerase Activity ◽  
Lymphoid Tissues ◽  
Low Grade ◽  
Malignant Lymphomas ◽  
Hodgkin's Lymphomas ◽  
Germinal Center B Cells

Abstract Activation of telomerase seems to be a prerequisite for immortalization and is found in permanent cell lines and most malignant tumors. Normal somatic cells are generally telomerase negative, except for bone marrow stem cells. Weak activity is also present in peripheral blood cells. In the present study strong telomerase activity was demonstrated in vivo in normal mature cells of the immune system, as well as in malignant lymphomas. Benign lymph nodes had lower telomerase activity than benign tonsils, which exhibited intermediate to high activity comparable with findings in malignant lymphomas. In benign tonsils the activity seemed to be restricted to germinal center B cells. In benign lymphoid tissues telomerase activity correlated with B-cell numbers and cell proliferation, but this was not observed in the lymphoma group. High- grade lymphomas exhibited higher levels of telomerase compared with low- grade cases. The data showed that in vivo activation of telomerase is a characteristic feature of germinal center B cells. Different signals for activation of telomerase are likely to exist, one of them being immune stimulation. The data suggest that telomerase activity in malignant lymphomas can be explained by an “induction and retention” model, ie, transformation occurs in a normal, mature B cell with reactivated telomerase, which is retained in the neoplastic clone.

scholarly journals Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

2017 ◽  
Vol 114 (18) ◽  
pp. 4751-4756 ◽  
Author(s):  
Takeharu Minamitani ◽  
Yijie Ma ◽  
Hufeng Zhou ◽  
Hiroshi Kida ◽  
Chao-Yuan Tsai ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Germinal Center ◽  
Epstein Barr Virus ◽  
Target Genes ◽  
Nk Cell ◽  
Barr Virus ◽  
Epstein Barr

Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

scholarly journals Covalent Linkage of HIV-1 Trimers to Synthetic Liposomes Elicits Improved B Cell and Antibody Responses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Shridhar Bale ◽  
Geraldine Goebrecht ◽  
Armando Stano ◽  
Richard Wilson ◽  
Takayuki Ota ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Neutralizing Antibodies ◽  
Tier 2 ◽  
Covalent Linkage ◽  
His Tag ◽  
Hiv 1

ABSTRACT We have demonstrated that a liposomal array of well-ordered trimers enhances B cell activation, germinal center formation, and the elicitation of tier-2 autologous neutralizing antibodies. Previously, we coupled well-ordered cleavage-independent NFL trimers via their C-terminal polyhistidine tails to nickel lipids integrated into the lipid bilayer. Despite favorable in vivo effects, concern remained over the potentially longer-term in vivo instability of noncovalent linkage of the trimers to the liposomes. Accordingly, we tested both cobalt coupling and covalent linkage of the trimers to the liposomes by reengineering the polyhistidine tail to include a free cysteine on each protomer of model BG505 NFL trimers to allow covalent linkage. Both cobalt and cysteine coupling resulted in a high-density array of NFL trimers that was stable in both 20% mouse serum and 100 mM EDTA, whereas the nickel-conjugated trimers were not stable under these conditions. Binding analysis and calcium flux with anti-Env-specific B cells confirmed that the trimers maintained conformational integrity following coupling. Following immunization of mice, serologic analysis demonstrated that the covalently coupled trimers elicited Env-directed antibodies in a manner statistically significantly improved compared to soluble trimers and nickel-conjugated trimers. Importantly, the covalent coupling not only enhanced gp120-directed responses compared to soluble trimers, it also completely eliminated antibodies directed to the C-terminal His tag located at the “bottom” of the spike. In contrast, soluble and noncovalent formats efficiently elicited anti-His tag antibodies. These data indicate that covalent linkage of well-ordered trimers to liposomes in high-density array displays multiple advantages in vitro and in vivo. IMPORTANCE Enveloped viruses typically encode a surface-bound glycoprotein that mediates viral entry into host cells and is a primary target for vaccine design. Liposomes with modified lipid head groups have a unique feature of capturing and displaying antigens on their surfaces, mimicking the native pathogens. Our first-generation nickel-based liposomes captured HIV-1 Env glycoprotein trimers via a noncovalent linkage with improved efficacy over soluble glycoprotein in activating germinal center B cells and eliciting tier-2 autologous neutralizing antibodies. In this study, we report the development of second-generation cobalt- and maleimide-based liposomes that have improved in vitro stability over nickel-based liposomes. In particular, the maleimide liposomes captured HIV-1 Env trimers via a more stable covalent bond, resulting in enhanced germinal center B cell responses that generated higher antibody titers than the soluble trimers and liposome-bearing trimers via noncovalent linkages. We further demonstrate that covalent coupling prevents release of the trimers prior to recognition by B cells and masks a nonneutralizing determinant located at the bottom of the trimer.

scholarly journals Deletion of Murine Gammaherpesvirus Gene M2 in AID-Expressing B Cells Impairs Host Colonization and Viral Reactivation

2020 ◽  
Author(s):  
Shana M. Owens ◽  
Darby G. Oldenburg ◽  
Douglas W. White ◽  
J. Craig Forrest
Keyword(s):  
Immune System ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Viral Reactivation ◽  
Viral Gene ◽  
Gene Products ◽  
Chronic Infections ◽  
Murine Gammaherpesvirus

ABSTRACTGammaherpesviruses (GHVs) are DNA tumor viruses that establish life-long, chronic infections in lymphocytes of humans and other mammals. GHV infections are associated with numerous cancers, especially in immune compromised hosts. While it is known that GHVs utilize host germinal center (GC) B cell responses during latency establishment, an understanding of how viral gene products function in specific B cell subsets to regulate this process is incomplete. Using murine gammaherpesvirus 68 (MHV68) as a small-animal model to define mechanisms of GHV pathogenesis in vivo, we generated a virus in which the M2 gene was flanked by loxP sites (M2.loxP), enabling the use of Cre-lox technology to define M2 function in specific cell types in infection and disease. The M2 gene encodes a protein that is highly expressed in GC B cells that promotes plasma cell differentiation and viral reactivation. M2 was efficiently deleted in Cre-expressing cells, and the presence of loxP sites flanking M2 did not alter viral replication or latency in mice that do not express Cre. In contrast, M2.loxP MHV68 exhibited a deficit in latency establishment and reactivation that resembled M2-null virus, following intranasal (IN) infection of mice that express Cre in all B cells (CD19-Cre). Nearly identical phenotypes were observed for M2.loxP MHV68 in mice that express Cre in germinal center (GC) B cells (AID-Cre). However, neither colonization of draining lymph nodes after IN infection nor the spleen after intraperitoneal (IP) infection required M2, although the reactivation defect was retained. Together, these data confirm that M2 function is B cell-specific and demonstrate that M2 primarily functions in AID-expressing cells to facilitate MHV68 dissemination to distal latency reservoirs within the host and reactivation from latency. Our study reveals that a viral latency gene functions within a distinct subset of cells to facilitate host colonization.IMPORTANCEGammaherpesviruses establish life-long chronic infections in cells of the immune system that can lead to lymphomas and other diseases. To facilitate colonization of a host, gammaherpesviruses encode gene products that manipulate processes involved in cellular proliferation and differentiation. Whether and how these viral gene products function in specific cells of the immune system is poorly defined. We report here the use of a viral genetic system that allows for deletion of specific viral genes in discrete populations of cells. We employ this system in an in vivo model to demonstrate cell-type-specific requirements for a particular viral gene. Our findings reveal that a viral gene product can function in distinct cellular subsets to direct gammaherpesvirus pathogenesis.

Enforced BCL6 Expression Inhibits B Cell Development in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1535-1535
Author(s):  
Davide F. Robbiani ◽  
Kaity Colon ◽  
Kruti Naik ◽  
Helen Nickerson ◽  
Maurizio Affer ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
Light Chain ◽  
Germinal Center ◽  
Regulatory Elements ◽  
Cell Development ◽  
B Cell Development ◽  
Kappa Light Chain

Abstract The B-Cell Lymphoma 6 (BCL6) gene encodes for a zinc finger motifs containing transcriptional repressor that is frequently dysregulated by chromosomal translocations in germinal center lymphomas. A putative protooncogene, its transforming ability in vivo was reported in I-mu-HA-BCL6 knock-in mice by Cattoretti et al last year. We also tested this assumption in transgenic mice expressing BCL6 in B cells under the control of kappa light chain regulatory elements. We replaced the murine C-kappa locus with the 16kb human BCL6 genomic locus in a construct containing the murine kappa light chain regulatory elements (Vk, EiK, 3′RR). While control transgenics were readily obtained (5/32 founders), only 3/68 founders were positive for the BCL6 transgene, of which only one (bearing a single copy of the transgene) was able to transmit the transgene to its progeny, thus suggesting embryonal toxicity of exogenous BCL6. In the bone marrow, flow cytometry revealed a nearly complete block of B cell development at the pro-B to pre-B transition. This was also the stage at which we first detected expression of EGFP in control reporter mice that were generated in parallel. Spleens of transgenic mice weighed about 50% of control spleens and less than 5% of splenocytes were CD19+ B cells. These were IgM high, IgD intermediate, corresponding to an immature B cell phenotype. Lymph nodes were smaller and B cells barely detected. Peyers’ patches were not visible. Combined, our analysis of 6–8 weeks old VkHABCL6 transgenic mice reveals that enforced expression of BCL6 early in development results in a profound block of B lymphocyte differentiation. How transgenic BCL6 modulates this effect at the transcriptional level remains to be investigated. To test the oncogenic potential of BCL6 in B cells, it will be interesting to precisely turn on this gene in the germinal center.

SCF-β-TrCP E3 Ubiquitin Ligase-Mediated Proteolysis Of EZH2 Regulates H3K27 Trimethylation Activity In Germinal Center-Derived B-Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 244-244
Author(s):  
Anagh A Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Fuzon Chung ◽  
Thirunavukkarasu Velusamy ◽  
Megan S Lim ◽  
...  
Keyword(s):  
B Cell ◽  
Ubiquitin Ligase ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
Histone H3 ◽  
E3 Ubiquitin Ligase ◽  
B Cell Lymphoma ◽  
Ubiquitin Ligases ◽  
Dependent Manner ◽  
H3k27 Trimethylation

Abstract EZH2 is a critical enzymatic subunit of the PRC2 which trimethylates histone H3 (H3K27) to mediate gene repression. While overexpression of EZH2 and its increased H3K27 methylation have generally been associated with hematologic malignancies and solid tumors, recurrent gain of function somatic mutations in EZH2 have been recently reported in germinal center B-cell (GCB) derived lymphomas. These mutations, which replace a single tyrosine in the SET domain of the EZH2 protein (Y641), occur in 7.2% of FLs (follicular lymphoma) and 21.7% of GCB DLBCL (diffuse large B-cell lymphoma) and are absent from ABC DLBCLs. Consequently, B-cell lymphoma cell lines and lymphoma primary samples harboring heterozygous EZH2Y641mutations exhibit increased levels of histone H3 Lys27 trimethylation (H3K27me3) and promote a lymphoproliferative phenotype with expansion of the GC B cell compartment. Although extensive studies have been conducted to understand the role of polycomb group of proteins in cancer pathogenesis, few studies have been aimed at understanding the post-translational regulation of these proteins. Skp1/Cullin1/F- box protein (SCF) ubiquitin ligase complexes are the largest family of multicomponent ring E3 ubiquitin ligases that control the degradation of many important regulatory proteins and have been implicated in the regulation of epigenetic regulators. However, the mechanism of EZH2 degradation and its implication in PRC2 mediated methyl transferase activity in a cancer-specific context is not fully understood. Cullin-ring ubiquitin ligases (CRLs) form the largest known class of multicomponent E3 ubiqutin ligase family in eukaryotes and target a wide array of substrates involved in several biological processes. To investigate a CRL-mediated basis for post-translational EZH2 regulation, we screened a panel of cullin family E3 ligases for interaction with EZH2 and discovered that EZH2 was immunoprecipitated by cullin1 exclusively among all other cullins (cul2,3,4a,4b,5 and 7). We engineered and expressed a cullin1 dominant negative protein which resulted in EZH2 accumulation in a dose dependent manner. Using co-immunoprecipitation approach, we screened 8 different F box proteins and observed that SCF E3 ubiquitin ligase β-TrCP (FBXW1) uniquely and specifically interacted with EZH2. Further, RNAi-mediated silencing of β-TrCP resulted in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the Y641 mutants recurrently identified and relevant in lymphoma pathogenesis were unable to bind β-TrCP. Further, cycloheximide chase experiments showed that Y641 mutants endogenously expressed in lymphoma-derived cells exhibit increased EZH2 stability as well as enhanced H3K27 trimethylation activity. Our studies demonstrate that β-TrCP plays an important role in controlling H3K27 trimethylation activity via an unusual recognition mechanism targeting EZH2 for proteasomal degradation. Our findings delineate a novel β-TrCP/EZH2 axis requiring an intact Y641 residue for EZH2 regulation by β-TrCP. Oncogenic mutations in Y641 in GCB-cell derived lymphomas confer increased stability to EZH2 and render it resistant to β-TrCP-mediated degradation. This newly identified mechanism might help in design potential novel therapeutic approaches for clinical management of cancer potentially driven by aberrant accumulation/function of EZH2. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Use of a Virus-Encoded Enzymatic Marker Reveals that a Stable Fraction of Memory B Cells Expresses Latency-Associated Nuclear Antigen throughout Chronic Gammaherpesvirus Infection

2010 ◽  
Vol 84 (15) ◽  
pp. 7523-7534 ◽  
Author(s):  
Michael S. Nealy ◽  
Carrie B. Coleman ◽  
Haiyan Li ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Germinal Center ◽  
Maintenance Phase ◽  
Memory B Cells ◽  
Nuclear Antigen ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Enzymatic Marker ◽  
Epstein Barr

ABSTRACT An integral feature of gammaherpesvirus infections is the ability to establish lifelong latency in B cells. During latency, the viral genome is maintained as an extrachomosomal episome, with stable maintenance in dividing cells mediated by the viral proteins Epstein-Barr nuclear antigen 1 (EBNA-1) for Epstein-Barr virus and latency-associated nuclear antigen (LANA) for Kaposi's sarcoma-associated herpesvirus. It is believed that the expression of episome maintenance proteins is turned off in the predominant long-term latency reservoir of resting memory B cells, suggesting that chronic gammaherpesvirus infection is primarily dormant. However, the kinetics of LANA/EBNA-1 expression in individual B-cell subsets throughout a course of infection has not been examined. The infection of mice with murine gammaherpesvirus 68 (MHV68, γHV68) provides a model to determine the specific cellular and molecular events that occur in vivo during lifelong gammaherpesvirus latency. In work described here, we make use of a heterologously expressed enzymatic marker to define the types of B cells that express the LANA homolog (mLANA) during chronic MHV68 infection. Our data demonstrate that mLANA is expressed in a stable fraction of B cells throughout chronic infection, with a prominent peak at 28 days. The expression of mLANA was detected in naïve follicular B cells, germinal-center B cells, and memory B cells throughout infection, with germinal-center and memory B cells accounting for more than 80% of the mLANA-expressing cells during the maintenance phase of latency. These findings suggest that the maintenance phase of latency is an active process that involves the ongoing proliferation or reseeding of latently infected memory B cells.

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