Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1.
Characterization of a novel posttranslational modification in neuronal nitric oxide synthase by small ubiquitin-related modifier-1.
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DOI：
10.1016/j.bbapap.2011.04.006
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发表时间：
2011-07
期刊：
影响因子：
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通讯作者：
Masatomo Watanabe;K. Itoh
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文献类型：
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作者：
Masatomo Watanabe;K. Itoh
一氧化氮（NO）在中枢神经系统中的多方面功能由神经元NO合酶（nNOS）的活性定义。nNOS的活性受翻译后修饰的调节，如磷酸化和泛素化，但它是否被小泛素相关修饰物（SUMO）修饰仍不清楚。本研究的目的是阐明nNOS是否被SUMO蛋白修饰。利用SUMOplot和SUMOFI进行的生物信息学分析预测nNOS具有潜在的SUMO修饰位点。当HEK 293 T细胞瞬时共表达nNOS和SUMO-1时，检测到对应于nNOS-SUMO-1缀合物的两条带。此外，两个nNOS-SUMO-1缀合物通过使用重组蛋白的体外sumoylation测定来确认。此外，通过MALDI-QIT/TOF质谱鉴定nNOS-SUMO-1缀合物。这些发现表明nNOS在体外和细胞水平上都被明确定义为SUMO-1的靶蛋白。接下来，我们在细胞水平上表征了nNOS-SUMO-1缀合循环中的特定酶。nNOS的SUMO-1结合依赖于Ubc 9（E2）。PIASxβ（E3）促进nNOS与Ubc 9的相互作用。另一方面，SUMO-1通过SENP 1和SENP 2与nNOS解缀合。总的来说，这项研究新发现，nNOS是由SUMO-1后修饰。
The multifaceted functions of nitric oxide (NO) in the CNS are defined by the activity of neuronal NO synathase (nNOS). The activities of nNOS are modulated by posttranslational modifications, such as phosphorylation and ubiquitination, but whether it is modified by small ubiquitin-related modifier (SUMO) remains unknown. The aim of this study was to elucidate whether nNOS is posttranslationally modified by SUMO proteins. Bioinformatic analyses using SUMOplot and SUMOFI predicted that nNOS had potential SUMO modification sites. When HEK293T cells were transiently co-expressed with nNOS and SUMO-1, two bands corresponding to nNOS-SUMO-1 conjugates were detected. In addition, two nNOS-SUMO-1 conjugates were confirmed by anin vitrosumoylation assay using recombinant proteins. Furthermore, nNOS-SUMO-1 conjugates were identified by MALDI-QIT/TOF mass spectrometry. These findings indicate that nNOS is clearly defined as a SUMO-1 target protein bothin vitroand at the cellular level. We next characterized specific enzymes in the nNOS-SUMO-1 conjugation cycle at the cellular level. SUMO-1 conjugation of nNOS depended on Ubc9 (E2). The interaction between nNOS and Ubc9 was facilitated by PIASxβ (E3). On the other hand, SUMO-1 was deconjugated from nNOS by SENP1 and SENP2. Overall, this study has newly identified that nNOS is posttranslationally modified by SUMO-1.