GABAB receptor subunit 1 binds to proteins affected in 22q11 deletion syndrome

GABA_B receptors mediate slow inhibitory effects of the neurotransmitter γ-aminobutyric acid (GABA) on synaptic transmission in the central nervous system. They function as heterodimeric G-protein-coupled receptors composed of the seven-transmembrane domain proteins GABA_B1 and GABA_B2, which are linked through a coiled-coil interaction. The ligand-binding subunit GABA_B1 is at first retained in the endoplasmic reticulum and is transported to the cell surface only upon assembly with GABA_B2. Here, we report that GABA_B1, via the coiled-coil domain, can also bind to soluble proteins of unknown function, that are affected in 22q11 deletion/DiGeorge syndrome and are therefore referred to as DiGeorge critical region 6 (DGCR6). In transfected neurons the GABA_B1-DGCR6 association resulted in a redistribution of both proteins into intracellular clusters. Furthermore, the C-terminus of GABA_B2 interfered with the novel interaction, consistent with heterodimer formation overriding transient DGCR6-binding to GABA_B1. Thus, sequential coiled-coil interactions may direct GABA_B1 into functional receptors.     

Decreased DGCR8 Expression and miRNA Dysregulation in Individuals with 22q11.2 Deletion Syndrome

Deletion of the 1.5–3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome (22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder in humans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broad phenotype, whose characterization has expanded considerably within the last decade and includes many associated findings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70–80%), hypocalcemia (20–60%), and a range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. These phenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in the deleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypic variations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here, we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, in peripheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we found dysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this to be related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level of some miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAs may contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS.     

Human B cells immortalized with Epstein-Barr virus upregulate CCR6 and CCR10 and downregulate CXCR4 and CXCR5

Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.     

Retracted:Propofol exerts anticancer activity on hepatocellular carcinoma cells by raising lncRNA DGCR5

Hepatocellular carcinoma is one of the most fatal cancers worldwide. Propofol is an intravenous anesthetic extensively used in clinical. Herein, we tested the anticancer activity of propofol on hepatocellular carcinoma, along with the internal molecular mechanism related to lncRNA DiGeorge syndrome critical region gene 5 (DGCR5). Followed by propofol stimulation, hepatocellular carcinoma Huh-7 and HepG2 cell viability, proliferation, migration, invasion, and apoptosis were tested, respectively. Then, DGCR5 expression levels in hepatocellular carcinoma tissues and cells were measured. sh-DGCR5 was transfected to silence DGCR5 expression. Subsequently, the influence of DGCR5 silence on propofol caused Huh-7 and HepG2 cell viability loss, proliferation inhibition, migration and invasion suppression, apoptosis induction, as well as Raf1/ERK1/2 and Wnt/β-catenin pathways inactivation were assessed, respectively. We discovered that propofol declined Huh-7 and HepG2 cell viability, proliferation, migration and invasion, but increased cell apoptosis. DGCR5 had a relatively lower expression level in hepatocellular carcinoma tissues and cells. Propofol elevated DGCR5 expression in Huh-7 and HepG2 cells. Increased expression of DGCR5 was connected with the anticancer activity of propofol on Huh-7 and HepG2 cells. Besides, propofol repressed Raf1/ERK1/2 and Wnt/β-catenin pathways through elevating DGCR5 expression. In conclusion, the anticancer activity of propofol on hepatocellular carcinoma was verified in this study. Propofol repressed hepatocellular carcinoma Huh-7 and HepG2 cell growth and metastasis at least by elevating DGCR5 and hereafter inactivating Raf1/ERK1/2 and Wnt/β-catenin pathways.     

CD4 and CD8 T cell responses to tumour-associated Epstein–Barr virus antigens in nasopharyngeal carcinoma patients

Nasopharyngeal carcinoma (NPC), an Epstein–Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-γ release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Backbone 1HN, 13C, and 15N resonance assignments of the tandem RNA-binding domains of human DGCR8

Double-stranded RNA binding domain (dsRBD) containing proteins are critical components of the microRNA (miRNA) pathway, with key roles in small RNA biogenesis, modification, and regulation. DiGeorge Critical Region 8 (DGCR8) is a 773 amino acid, dsRBD-containing protein that was originally identified in humans as a protein encoded in the region of chromosome 22 that is deleted in patients with DiGeorge syndrome. Now, it is realized that DGCR8 complements the nuclear RNase III Drosha to initiate miRNA biogenesis by promoting efficient recognition and cleavage of primary miRNAs (pri-miRNA). A pair of C-terminal tandem dsRBDs separated by a flexible linker are required for pri-miRNA substrate binding and recognition. The crystal structure of the DGCR8 core region comprising residues 493–720 revealed that each dsRBD adopts the canonical αβββα fold. However, several residues located in important flexible regions including the β1-β2-loop implicated in canonical dsRNA recognition are absent in the crystal structure and no RNA-bound structure of DGCR8 has been reported. Here we report the ¹H^N, ¹³C, and ¹⁵N backbone resonance assignments of the 24 kDa, 214 amino acid human DGCR8^core (residues 493–706) by heteronuclear NMR spectroscopy. Our assignments lay the foundation for a detailed solution state characterization of the dynamical and RNA-binding properties of this protein in solution.     

Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses

Human CD4(+) T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8(+) memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4(+) epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4(+) T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4(+) T-helper 1 cells (EBNA1, EBNA3C > LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8(+)-T-cell responses (EBNA3C > EBNA1 > LMP2 > LMP1). Furthermore, the range of CD4(+) memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8(+)-T-cell frequencies.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells.

The Epstein-Barr virus in the Burkitt lymphoma-derived cell line Raji has a deletion in the EBNA3C gene. When Raji cells are allowed to grow to high density and most of the cells become growth arrested in the G1 phase of the cell cycle, the level of detectable latent membrane protein 1 (LMP1) is substantially reduced. After dilution of the cells with fresh growth medium, within 8 h, there is a large increase in LMP1 mRNA, and by 12 h, LMP1 is expressed to a high level (H. Boos, M. Stoehr, M. Sauter, and N. Mueller-Lantzch, J. Gen. Virol. 71:1811-1815, 1990). Here we show that in Raji cells which constitutively express a transfected EBNA3C gene, the down-regulation of LMP1 in growth-arrested cells does not take place. Furthermore, we show that in wild-type Raji cells, low-level LMP1 expression occurs when most of the cells are arrested at a point(s) early in G1 (or G0) when the product of the retinoblastoma gene, pRb, is hypophosphorylated. The dramatic synthesis of LMP1 coincides with the progression of these cells to late G1 when pRb becomes hyperphosphorylated. Thus, in Raji cells, the LMP1 gene is apparently regulated in a cell cycle- or proliferation-dependent manner, but when EBNA3C is present, sustained LMP1 expression occurs as it does in a lymphoblastoid cell line. EBNA3C appears to either relieve the apparent repression of LMP1 in cells progressing through early G1 or possibly alter the stage at which the cells growth arrest to one where they are permissive for LMP1 expression.     

Combined trisomy 9P and Shprintzen syndrome resulting from a paternal t(9;22)

The authors report on a female infant with partial trisomy 9 (pter-->q12) together with partial monosomy 22 (pter-->q11.23) that included DiGeorge critical region (DGCR), as a result of adjacent-2 disjunction. In addition to the clinical features characteristic of trisomy 9p syndrome, the patient had Truncus arteriosus type A2, bilateral hydronephrosis, palatal anomaly, retrognathia, and laryngeal hypotonia, which are likely to be attributed to 22q11.2 deletion. This patient appears to be the first reported case with such unbalanced translocation resulting from a paternal reciprocal translocation. For live birth, the risk for male carrier is 8.7-17.4%. It is important to consider this higher risk when counseling. Precise study concerning the presence of the DGCR can facilitate in the better understanding of the condition.     

Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter

The Epstein-Barr virus (EBV) BGLF4 gene encodes a serine/threonine protein kinase (PK) that is expressed in the cytolytic cycle. EBV nuclear antigen 2 (EBNA2) is a key latency gene essential for immortalization of B lymphocytes and transactivation of viral and cellular promoters. Here we report that EBV PK phosphorylates EBNA2 at Ser-243 and that these two proteins physically associate. PK suppresses EBNA2's ability to transactivate the LMP1 promoter, and Ser-243 of EBNA2 is involved in this suppression. Moreover, EBNA2 is hyperphosphorylated during EBV reactivation in latently infected B cells, which is associated with decreased LMP1 protein levels. This is the first report about the effect of EBV PK on the function of one of its target proteins and regulation of EBNA2 phosphorylation during the EBV lytic cycle.     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.