Characterization of a new monoclonal antibody (PG- M3) directed against the aminoterminal portion of the PML gene product.Immunocytochemical evidence for high PML expression on activated macrophages and endothelial cells
PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).
"At least seven isoforms have been described based on alternative splicing of the C-terminal exons, all of which contain the first three exons that encode the RBCC/TRIM motif (Jensen et al., 2001). The majority of PML isoforms (I-VI) have nuclear localization signal (NLS) that regulate their incorporation into NBs (Jensen et al., 2001; Melnick and Licht 1999); however, PML VII lacks this signal and subsequently, is confined to the cytoplasm (Flenghi et al., 1995; Fagioli et al., 1992). PML-NBs are macromolecular complexes of helicases and transcription factors that are important sites for transcriptional control (Maul et al., 2000; Nefkens et al., 2003). "
[Show abstract][Hide abstract] ABSTRACT: Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.
"The human PML gene contains 9 exons and generates multiple isoforms due to alternative splicing designated as PML I-VII that differ in their C-termini (Jensen et al., 2001). PML expression is dependent on cell type and the stage of differentiation (Cho et al., 1998; Flenghi et al., 1995), as well as extrinsic factors such as infections and stressinduced responses (Maul et al., 2000). IFN, IRF3, and the tumor suppressor p53 have all been found to induce PML expression through distinct mechanisms (Ferbeyre et al., 2000; Kim et al., 2007; Stadler et al., 1995). "
[Show abstract][Hide abstract] ABSTRACT: Bovine herpesvirus 1 (BHV-1) is a significant pathogen of cattle. Following acute infection, BHV-1 establishes a latent infection that persists for the life of the infected host. Stress induced factors cause the virus to reactivate from latency, resulting in virus transmission and transient immune suppression. BHV-1 encoded bICP0 is expressed early and constitutively throughout productive infection. bICP0 is critical for efficient viral replication, virulence, and reactivation in cattle because it stimulates viral transcription and interferes with innate immune responses. bICP0 potentially interacts with a variety of proteins to activate viral gene expression and inhibit innate antiviral defenses. bICP0 localizes to promyelocytic leukemia (PML) protein-containing nuclear domains, which are associated with antiviral activity and commonly targeted for disruption by a wide variety of viruses. The zinc RING finger motif within bICP0 plays a critical role in the biological functions of bICP0, and possesses intrinsic E3 ubiquitin ligase activity that is important for polyubiquitination and subsequent degradation of proteins.
Results from these studies demonstrated mutations within the zinc RING finger increased bICP0 protein levels, presumably due to disruption of the ability of bICP0 to induce its own ubiquitination. Sequences at its C-terminus are also important for regulating the half-life of bICP0. BHV-1 infection and bICP0 expression alone reduced PML protein levels. Unexpectedly, bICP0 mutants that localized primarily in the cytoplasm also induced PML degradation. bICP0 was readily detected in the cytoplasm of low passage bovine cells at later times post infection, suggesting bICP0 induces degradation of cytoplasmic isoforms of PML to promote viral replication. Identification of bICP0-interacting proteins was also investigated to further elucidate the mechanisms underlying bICP0 functions. The ability of BHV-1 and a bICP0 zinc RING finger mutant to grow in oncogenic cells was also examined to determine if defects in viral growth of the mutant could be relieved in cells with potential defects in their interferon response. Collectively, studies presented in this dissertation determine that nuclear and cytoplasmic bICP0 have functions that promote productive infection.
Advisor: Clinton Jones
"PML expression is regulated by the cell or tissue type and the differentiation stage of the cell 27-29. One of the main contributing factors to this varied expression is alternative splicing which results in the expression of at least 11 different isoforms 30. "
[Show abstract][Hide abstract] ABSTRACT: The promyelocytic leukemia protein (PML) is involved in many cellular processes including cell cycle progression, DNA damage response, transcriptional regulation, viral infection, and apoptosis. These cellular activities often rely on the localization of PML to unique subnuclear structures known as PML nuclear bodies (NBs). More than 50 cellular proteins are known to traffic in and out of PML NBs, either transiently or constitutively. In order to understand the dynamics of these NBs, it is important to delineate the regulation of PML itself. PML is subject to extensive regulation at transcriptional, post-transcriptional, and post-translational levels. Many of these modes of regulation depend on the cellular context and the presence of extracellular signals. This review focuses on the current knowledge of regulation of PML under normal cellular conditions as well as the role for regulation of PML in viral infection and cancer.
International journal of biological sciences 02/2009; 5(4):366-76. DOI:10.7150/ijbs.5.366 · 4.51 Impact Factor
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