The motif of SPARC that inhibits DNA synthesis is not a nuclear localization signal.
The motif of SPARC that inhibits DNA synthesis is not a nuclear localization signal.
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DOI：
10.1016/j.jmb.2007.04.088
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发表时间：
2007-08
影响因子：
5.6
通讯作者：
Jeffrey Kosman;Nicole Carmean;Elizabeth M. Leaf;K. Dyamenahalli;J. Bassuk
中科院分区：
文献类型：
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作者：
Jeffrey Kosman;Nicole Carmean;Elizabeth M. Leaf;K. Dyamenahalli;J. Bassuk
尽管最初被认为是一种分泌的基质细胞蛋白，但它也在尿路上皮细胞核中被鉴定出。许多生物学活性，包括抑制细胞粘附和抑制DNA合成，已被归因于Escherichia coli，但其细胞内定位对这些活动的影响是未知的。当暴露的抗原表位检索和核基质解蔽技术，内源性的ESTA被发现强烈本地化培养的尿路上皮细胞的细胞核和核基质。活细胞延时成像显示，外源性荧光标记的重组（r）腺苷酸从培养基中超过16小时的时间内，并积累在细胞内。产生了两种在其假定的核定位信号（NLS）中具有改变的rfos变体，以研究NLS的存在和影响。这些变体表现出与野生型蛋白相似的生物物理特征。可视化的各种技术，包括活细胞成像，去卷积显微镜，和细胞分级分离，都同意外源性rfos是不能本地化的细胞核，而是积累为核周簇。定位的rnostrum NLS变体是没有什么不同的野生型，反对在rnostrum中的活性NLS的存在。成像实验表明，只有透化的死细胞才能将放射性摄取到细胞核中。证明了rDNAs（无NLS）变体在抑制DNA合成方面是无效的，而rDNAs（强NLS）变体是比野生型rDNAs更有效的DNA合成抑制剂。因此，抑制尿路上皮细胞DNA的合成的p53基序不是核定位信号，但其操作具有产生可使增殖组织静止的“超级p53”的治疗潜力。
SPARC (secreted protein acidic and rich in cysteine), although primarily known as a secreted, matricellular protein, has also been identified in urothelial cell nuclei. Many biological activities, including inhibition of cell adhesion and repression of DNA synthesis, have been ascribed to SPARC, but the influence of its intracellular localization on each of these activities is unknown. When exposed by epitope retrieval and nuclear matrix unmasking techniques, endogenous SPARC was found to localize strongly to the nuclei and the nuclear matrix of cultured urothelial cells. Live-cell time-lapse imaging revealed that exogenous fluorescently labeled recombinant (r) SPARC was taken up from medium over a 16 h period and accumulated inside cells. Two variants of rSPARC with alterations in its putative nuclear localization signal (NLS) were generated to investigate the existence and effects of the NLS. These variants demonstrated similar biophysical characteristics as the wild-type protein. Visualization by a variety of techniques, including live-cell imaging, deconvolution microscopy, and cell fractionation, all concurred that exogenous rSPARC was not able to localize to cell nuclei, but instead accumulated as perinuclear clusters. Localization of the rSPARC NLS variants was no different than wild-type, arguing against the presence of an active NLS in rSPARC. Imaging experiments showed that only permeabilized, dead cells avidly took up rSPARC into their nuclei. The rSPARC(no NLS) variant proved ineffective at inhibiting DNA synthesis, whereas the rSPARC(strong NLS) variant was a more potent inhibitor of DNA synthesis than was wild-type rSPARC. The motif of SPARC that inhibits the synthesis of urothelial cell DNA is therefore not a nuclear localization signal, but its manipulation holds therapeutic potential to generate a “Super-SPARC” that can quiesce proliferative tissues.