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。     

PML and COP1--two proteins with much in common

The recent discovery that the RING-finger domain is involved in mediating ubiquitin transfer from ubiquitin-conjugating enzymes to substrates have highlighted the importance of protein degradation through the ubiquitin-proteasome pathway in the regulation of different cellular processes. Two RING-finger-containing proteins, the promyelocytic leukemia protein (PML) from mammals and the constitutive photomorphogenic protein (COP1) from plants, show conspicuous similarities in their cellular distribution, dynamics and structure, indicating that they share a related function. Comparison of these two proteins suggests that they are involved in regulating the targeting of nuclear proteins to specific nuclear compartments for degradation through the ubiquitin-proteasome pathway.     

Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus.

The PML protein, identified first as part of the oncogenic PML-RARalpha chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML-RARalpha and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1-PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML-RARalpha, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of 'ubiquitin-like' post-translational modification is not limited to a degradation signal.     

The PML and PML/RARα Domains: From Autoimmunity to Molecular Oncology and from Retinoic Acid to Arsenic

Acute promyelocytic leukemia (APL) is specifically associated to a t(15; 17) translocation which fuses a gene encoding a nuclear receptor for retinoic acid, RARα, to a previously unknown gene PML. The PML protein is localized in the nucleus on a specific domain of unknown function (PML nuclear bodies, NB) previously detected with autoimmune sera from patients with primary biliary cirrhosis (PBC). These bodies are nuclear matrix-associated and all of their identified components (PML, Sp100, and NDP52) are sharply upregulated by interferons. We show that autoantibodies against both PML and Sp100 are usually associated in sera with multiple nuclear dot anti-nuclear antibodies and demonstrate that PML is an autoantigen, not only in PBC, but also in other autoimmune diseases. In APL, the PML/RARα fusion interferes with both the retinoic acid (RA) response and PML localization on nuclear bodies, but the respective contribution of each defect to leukemogenesis is unclear. RA induces the terminal differentiation of APL blasts, yielding to complete remissions, and corrects the localization of NB antigens. Arsenic trioxide (As₂O₃) also induces remissions in APL, seemingly through induction of apoptosis. We show that in APL, As₂O₃leads to the rapid reformation of PML bodies. Thus, both agents correct the defect in NB antigen localization, stressing the role of nuclear bodies in the pathogenesis of APL.     

PML contributes to a cellular mechanism of repression of herpes simplex virus type 1 infection that is inactivated by ICP0

Promyelocytic leukemia (PML) nuclear bodies (also known as ND10) are nuclear substructures that contain several proteins, including PML itself, Sp100, and hDaxx. PML has been implicated in many cellular processes, and ND10 are frequently associated with the replicating genomes of DNA viruses. During herpes simplex virus type 1 (HSV-1) infection, the viral regulatory protein ICP0 localizes to ND10 and induces the degradation of PML, thereby disrupting ND10 and dispersing their constituent proteins. ICP0-null mutant viruses are defective in PML degradation and ND10 disruption, and concomitantly they initiate productive infection very inefficiently. Although these data are consistent with a repressive role for PML and/or ND10 during HSV-1 infection, evidence in support of this hypothesis has been inconclusive. By use of short interfering RNA technology, we demonstrate that depletion of PML increases both gene expression and plaque formation by an ICP0-negative HSV-1 mutant, while having no effect on wild-type HSV-1. We conclude that PML contributes to a cellular antiviral repression mechanism that is countered by the activity of ICP0.