PML Suppresses Influenza Virus Replication by Promoting FBXW7 Expression

Virologica Sinica - Influenza A viruses (IAV) are responsible for seasonal flu epidemics, which can lead to high morbidity and mortality each year. Like other viruses, influenza virus can hijack...     

组蛋白乙酰转移酶PCAF通过与PML蛋白的作用而定位于细胞核ND10结构

PCAF(P300/CBP associated factor,P300/CBP相关因子)是一种重要的组蛋白乙酰转移酶,主要通过使核小体组蛋白发生乙酰化而参与基因转录的调控。P300/CBP是一种能够与PCAF相互作用的宿主细胞蛋白,定位于细胞核ND10结构(nuclear dot10)。ND10是由70余种蛋白质分子组成的、以分散的斑点或团块样方式存在于细胞核内的大分子复合物,与基因转录调控过程有着密切关系。早幼粒细胞白血病蛋白(promyelocytic leukemia protein,PML)是ND10的主要组成成分,对于维持ND10的正常结构与功能至关重要。在成功构建pGBK-PML3、pGAD-PCAF、pGEX-PCAF、pFlag-PCAF和pcDNA-PML3重组质粒的基础上,用酵母双杂交证明PCAF与PML3之间存在着相互作用;用GST-pull down技术证明GST-PCAF能够将PML3蛋白捕获并沉降下来,表明在体外条件下两者之间存在直接的相互作用;用免疫共沉淀和荧光共定位证明在体内PCAF能够与PML发生直接的相互作用而定位于ND10。这些结果为进一步深入研究PCAF的生物学功能和作用模式奠定了基础。     

Acetylation of SUMO1 Alters Interactions with the SIMs of PML and Daxx in a Protein-Specific Manner

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ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.     

Contribution of the C-terminal Regions of Promyelocytic Leukemia Protein (PML) Isoforms II and V to PML Nuclear Body Formation

Promyelocytic leukemia protein (PML) nuclear bodies are dynamic and heterogeneous nuclear protein complexes implicated in various important functions, most notably tumor suppression. PML is the structural component of PML nuclear bodies and has several nuclear splice isoforms that share a common N-terminal region but differ in their C termini. Previous studies have suggested that the coiled-coil motif within the N-terminal region is sufficient for PML nuclear body formation by mediating homo/multi-dimerization of PML molecules. However, it has not been investigated whether any of the C-terminal variants of PML may contribute to PML body assembly. Here we report that the unique C-terminal domains of PML-II and PML-V can target to PML-NBs independent of their N-terminal region. Strikingly, both domains can form nuclear bodies in the absence of endogenous PML. The C-terminal domain of PML-II interacts transiently with unknown binding sites at PML nuclear bodies, whereas the C-terminal domain of PML-V exhibits hyperstable binding to PML bodies via homo-dimerization. This strong interaction is mediated by a putative α-helix in the C terminus of PML-V. Moreover, nuclear bodies assembled from the C-terminal domain of PML-V also recruit additional PML body components, including Daxx and Sp100. These observations establish the C-terminal domain of PML-V as an additional important contributor to the assembly mechanism(s) of PML bodies.     

The role of Pdcd4 in tumour suppression and protein translation

Programmed cell death 4 (Pdcd4), a tumour suppressor, is frequently down‐regulated in various types of cancer. Pdcd4 has been demonstrated to efficiently suppress tumour promotion, progression and proliferation. The biochemical function of Pdcd4 is a protein translation inhibitor. Although the fact that Pdcd4 inhibits protein translation has been known for more than a decade, the mechanism by which Pdcd4 controls tumorigenesis through translational regulation of its target genes is still not fully understood. Recent studies show that Pdcd4 inhibits translation of stress‐activated‐protein kinase interacting protein 1 to suppress tumour invasion, depicting a picture of how Pdcd4 inhibits tumorigenesis through translational inhibition. Thus, understanding the mechanism of how Pdcd4 attenuates tumorigenesis by translational control should provide a new strategy for combating cancer.     

In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies

SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. Interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function.     

Nuclear domain 10 (ND10) associated proteins are also present in nuclear bodies and redistribute to hundreds of nuclear sites after stress

The promyelocytic leukemia protien fused to the retinoic acid receptor in t(15;17) acute promyelocytic leukemia, the primary biliary cirrhosis autoantigen, Sp100, as well as the incompletely charterized protien NDP55, are co-localized in specific immunohistochemically defined nuclear domains (ND10), which are potential equyivalents of ultrastructurally defined nuclear bodies. We investigated whether the distribution of these proteins depends on environmental conditions and whether ND10 correlate with nuclear bodies. Certain nuclear bodies and ND10 react in a similar bodies, which herpes simplex virus infection or heat shock modify both. Redistribution of ND10-associated proteins to hundreds of small sites throughout the chromatin was inducible by stress in the form of heat shock and exposure to Cd⁺⁺ ions. The change of distribution was rapid and independent of proteins synthesis, and thus not part of the classical heat shock response. The very rapid redistribution of these proteins after heat shock, together with the development of ND10 upon interferon actication, raises the possibility that ND10 represent storage sites of certain matrix proteins readily accessible throughout the chromatin in response to stress or other effectors that induce global nuclear changes. © 1995 Wiley-Liss, Inc.     

Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11

Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication. Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function. HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II. The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11. Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11. The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML. Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin. These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication.     

Interferon-Induced Antiviral Mx1 GTPase Is Associated with Components of the SUMO-1 System and Promyelocytic Leukemia Protein Nuclear Bodies

Mx proteins are interferon-induced large GTPases, some of which have antiviral activity against a variety of viruses. The murine Mx1 protein accumulates in the nucleus of interferon-treated cells and is active against members of the Orthomyxoviridae family, such as the influenza viruses and Thogoto virus. The mechanism by which Mx1 exerts its antiviral action is still unclear, but an involvement of undefined nuclear factors has been postulated. Using the yeast two-hybrid system, we identified cellular proteins that interact with Mx1 protein. The Mx1 interactors were mainly nuclear proteins. They included Sp100, Daxx, and Bloom's syndrome protein (BLM), all of which are known to localize to specific subnuclear domains called promyelocytic leukemia protein nuclear bodies (PML NBs). In addition, components of the SUMO-1 protein modification system were identified as Mx1-interacting proteins, namely the small ubiquitin-like modifier SUMO-1 and SAE2, which represents subunit 2 of the SUMO-1 activating enzyme. Analysis of the subcellular localization of Mx1 and some of these interacting proteins by confocal microscopy revealed a close spatial association of Mx1 with PML NBs. This suggests a role of PML NBs and SUMO-1 in the antiviral action of Mx1 and may allow us to discover novel functions of this large GTPase.     

p53 and p21(Waf1) Are Recruited to Distinct PML‐Containing Nuclear Foci in Irradiated and Nutlin‐3a‐Treated U2OS Cells

Promyelocytic leukemia nuclear bodies (PML‐NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML‐NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53‐dependent growth inhibition. ALT‐associated PML bodies (APBs) are specialized PML‐NBs that include telomere‐repeat binding‐factor TRF1 and are exclusively in telomerase‐negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell‐cycle and p53 responses in ALT‐positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin‐3a. Both IR and Nutlin‐3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin‐3a. p53 was recruited to PML‐NBs 3–4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML‐NBs marked sites of apparently unrepaired DNA double‐strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin‐3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin‐3a‐treated cells. These data indicate (1) p53 is recruited to PML‐NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53–p21 pathway activation increases the percentage of APB‐positive cells, (3) p21 and p53 are recruited to ALT‐associated PML‐NBs after Nutlin‐3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance. J. Cell. Biochem. 111: 1280–1290, 2010. © 2010 Wiley‐Liss, Inc.     

Approaching tumour therapy beyond platinum drugs: status of the art and perspectives of ruthenium drug candidates

The study of metal complexes for the treatment of cancer diseases has resulted in the identification of some unique properties of ruthenium-based compounds. Among these inorganic-based agents, two of them, namely the ruthenium(III) drugs NAMI-A and KP1019 have undertaken with some success the clinical evaluations of phase I and preliminary phase II trials in patients. Here we highlight the strategies that have led to the discovery of metal-based (NAMI-A and KP1019) and of organometallic (RM175, RAPTA-T, RDC11 and DW1/2) ruthenium-based complexes, and we report their main biological/pharmacological characteristics and expectations for further development.