Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mit...Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B （LMB）, a specific inhibitor of Crml, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals （NESs） in pericentrin with diverse export activities. All of the five NESs could bind to Crml in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal （NLS） consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.展开更多
Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that ca...Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20(H4K20me1)to promote chromosome condensation and prevent DNA damage.Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage.This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage.Methods We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo.With this model,staining of PR-Set7 in the preneoplastic and tumor lesions in Adeno Cre-induced mouse lungs was performed.Meanwhile we identified higher protein level changes ofγ-tubulin and pericentrin by IHC.Results The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A.We also identified higher levels of the proteins pericentrin andγ-tubulin in Cul4A mouse lungs induced by Adeno Cre.Conclusion PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.展开更多
小头畸形-骨发育不良-原基性侏儒综合征Ⅱ型（Microcephalic or Majewski′s osteodysplastic primordial dwarfism type Ⅱ，MOPD Ⅱ）主要由中心体粒周蛋白（Pericentrin，PCNT）基因功能性突变所引起，临床上极其罕见。本文报道1例1...小头畸形-骨发育不良-原基性侏儒综合征Ⅱ型（Microcephalic or Majewski′s osteodysplastic primordial dwarfism type Ⅱ，MOPD Ⅱ）主要由中心体粒周蛋白（Pericentrin，PCNT）基因功能性突变所引起，临床上极其罕见。本文报道1例13岁男孩，因＂身高增长缓慢13年余，肤色加深半年余＂入院，除MOPD Ⅱ表现外还合并完全性生长激素缺乏症、2型糖尿病、高血压和黑棘皮病，多发牛奶咖啡斑。但头颅磁共振无动脉瘤等血管畸形。二代测序显示患者PCNT基因存在无义突变c.502C〉T（p.Gln168＊杂合突变）和c.3103C〉T（p.Arg1035＊杂合突变），均为新突变，其父携带无义突变c.3103C〉T（p.Arg1035＊杂合突变），其母携带无义突变c.502C〉T（p.Gln168＊杂合突变）。给予患儿二甲双胍片等治疗后，其血糖趋于正常，黑棘皮较治疗前减轻。针对PCNT基因突变的MOPD Ⅱ患者，需要定期评估血管异常情况。展开更多
目的:探讨中心粒周蛋白(pericentrin,PCNT)对胰岛素双相分泌的调节作用及其机制。方法:构建在小鼠胰岛β细胞中PCNT表达减少的转基因鼠(△PCNTβ小鼠),检测△PCNTβ小鼠与正常对照小鼠在给予糖耐量试验后第一时相和第二时相血糖和胰岛...目的:探讨中心粒周蛋白(pericentrin,PCNT)对胰岛素双相分泌的调节作用及其机制。方法:构建在小鼠胰岛β细胞中PCNT表达减少的转基因鼠(△PCNTβ小鼠),检测△PCNTβ小鼠与正常对照小鼠在给予糖耐量试验后第一时相和第二时相血糖和胰岛素分泌情况。检测两组小鼠胰岛内PCNT、胰岛素、纤维肌动蛋白(F-actin)变化情况,以及相关蛋白表达情况。结果:Western blot和RT-PCR显示△PCNTβ小鼠胰尾组织PCNT表达较对照组明显减低,免疫荧光提示△PCNTβ小鼠胰岛内PCNT、胰岛素表达较对照组明显减低。行腹腔注射葡萄糖耐量试验(Intraperitoneal glucose tolerance tests,IPGTT)△PCNTβ小鼠第一时相血糖曲线下面积(Quantification of area under the curve,AUC)显著高于对照组,第二时相血糖AUC两组小鼠间无统计学差异。△PCNTβ小鼠空腹胰岛素水平与对照组比较明显升高,葡萄糖刺激胰岛素分泌(Glucose stimulated insulin secretion,GSIS)后15min胰岛素增加值显著低于对照组,30 min和120 min时胰岛素水平与对照组无显著差异。Western blot显示△PCNTβ小鼠与对照组比较F-actin表达明显减低,ERK、p-ERK表达明显升高。RT-PCR测定△PCNTβ小鼠与对照组比较ETV4表达显著升高。免疫荧光提示△PCNTβ小鼠胰岛内F-actin和突触融合蛋白4(Syntaxin4,Syn-4)表达较对照组明显减低。结论:抑制小鼠胰岛β细胞内PCNT表达后,其通过抑制F-actin和Syn-4表达影响胰岛素分泌,导致空腹时胰岛素过度分泌和第一时相胰岛素分泌受损。展开更多
文摘Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B （LMB）, a specific inhibitor of Crml, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals （NESs） in pericentrin with diverse export activities. All of the five NESs could bind to Crml in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal （NLS） consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin β directly or via the adaptor importin α to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin β. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.
基金support from the Kazan, Mc Clain, Abrams, Fernandez, Lyons, Greenwood, Harley & Oberman Foundation, Incthe Estate of Robert Griffiths+3 种基金the Jeffrey and Karen Peterson Family FoundationPaul and Michelle Zygielbaumthe Estate of Norman Mancinithe Barbara Isackson Lung Cancer Research Fund
文摘Background and objective Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer.PR-Set7(also known as Set8)is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20(H4K20me1)to promote chromosome condensation and prevent DNA damage.Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage.This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage.Methods We developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A.We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo.With this model,staining of PR-Set7 in the preneoplastic and tumor lesions in Adeno Cre-induced mouse lungs was performed.Meanwhile we identified higher protein level changes ofγ-tubulin and pericentrin by IHC.Results The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A.We also identified higher levels of the proteins pericentrin andγ-tubulin in Cul4A mouse lungs induced by Adeno Cre.Conclusion PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.
文摘目的:探讨中心粒周蛋白(pericentrin,PCNT)对胰岛素双相分泌的调节作用及其机制。方法:构建在小鼠胰岛β细胞中PCNT表达减少的转基因鼠(△PCNTβ小鼠),检测△PCNTβ小鼠与正常对照小鼠在给予糖耐量试验后第一时相和第二时相血糖和胰岛素分泌情况。检测两组小鼠胰岛内PCNT、胰岛素、纤维肌动蛋白(F-actin)变化情况,以及相关蛋白表达情况。结果:Western blot和RT-PCR显示△PCNTβ小鼠胰尾组织PCNT表达较对照组明显减低,免疫荧光提示△PCNTβ小鼠胰岛内PCNT、胰岛素表达较对照组明显减低。行腹腔注射葡萄糖耐量试验(Intraperitoneal glucose tolerance tests,IPGTT)△PCNTβ小鼠第一时相血糖曲线下面积(Quantification of area under the curve,AUC)显著高于对照组,第二时相血糖AUC两组小鼠间无统计学差异。△PCNTβ小鼠空腹胰岛素水平与对照组比较明显升高,葡萄糖刺激胰岛素分泌(Glucose stimulated insulin secretion,GSIS)后15min胰岛素增加值显著低于对照组,30 min和120 min时胰岛素水平与对照组无显著差异。Western blot显示△PCNTβ小鼠与对照组比较F-actin表达明显减低,ERK、p-ERK表达明显升高。RT-PCR测定△PCNTβ小鼠与对照组比较ETV4表达显著升高。免疫荧光提示△PCNTβ小鼠胰岛内F-actin和突触融合蛋白4(Syntaxin4,Syn-4)表达较对照组明显减低。结论:抑制小鼠胰岛β细胞内PCNT表达后,其通过抑制F-actin和Syn-4表达影响胰岛素分泌,导致空腹时胰岛素过度分泌和第一时相胰岛素分泌受损。
