PEPTIDE:N-GLYCANASE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 内质网相关蛋白降解(ERAD)途径是细胞存活所必需的，内质网中错误折叠的蛋白质通过逆转录转运子运输，被蛋白酶体泛化和降解。在蛋白酶体降解之前，ERAD底物上的N-葡聚糖被多肽：N-聚糖酶(PNGase)处理。我们试图通过对PNGase结构的研究来阐明其功能。我们最近解决了小鼠PNGase(MPNGase)的催化结构域的结构，它显示了与酵母蛋白相似的折叠。除了催化结构域，高等真核生物中的PNGase还具有N-端和C-端延伸。我们对mPNGase C-末端结构域的结构和生化研究证实它是一个糖识别基序。然而，N-末端结构域的功能尚不清楚。N-末端结构域与其他蛋白质没有序列相似性，可能采用新的折叠。 最近有报道称，mPNGase直接与AAA-ATPase家族蛋白P97/VCP相互作用。我们提出了一个护送模型，在该模型中，P97作为蛋白质相互作用的平台，将错误折叠的蛋白质从内质网呈现到mPNGase和泛素化酶。我们的生化数据显示，mPNGase的N-末端区域与P97的C-末端区域结合。这是首次建立了mPNGase N-末端结构域的蛋白质相互作用功能。这是非常有趣的，因为人们普遍认为P97通过其N-末端结构域与其效应蛋白相互作用。全长P97的结构已经解决，但结构中无法显示C-末端区域。为了研究mPNGase的N-末端结构域的功能及其与P97相互作用的细节，人们正在对这些蛋白质及其复合体进行结晶学研究。我们最近获得了N-末端结构域的晶体，处于脱氧态，并与P97片段形成络合物，预计在RapiData课程开始时，它们将准备好进行数据收集和结构解决。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The endoplasmic reticulum-related protein degradation (ERAD) pathway, where misfolded proteins in the ER are transported through the retro-translocon, ubiquinated, and degraded by the proteasome, is essential for cell viability. Before proteasome degradation, the N-glycan on the ERAD substrates is processed by the Peptide:N-glycanase (PNGase). We tried to illustrate the function of PNGase by studying its structure. We recently solved the structure of the catalytic domain of mouse PNGase (mPNGase), which shows a similar fold as the yeast protein. In addition to the catalytic domain, PNGase in higher eukaryotes has both N- and C-terminal extensions. Our structural and biochemical studies on the C-terminal domain of mPNGase identified it as a sugar-recognizing motif. However, the function of the N-terminal domain is unknown. The N-terminal domain has no sequence similarity with other proteins and may adopt a novel fold. Recently it was reported that mPNGase directly interacts with P97/VCP, an AAA ATPase family protein. We proposed an escort model in which P97 functions as a protein interacting platform to present misfolded proteins from the ER to mPNGase and ubiquitinating enzymes. Our biochemical data showed that the N-terminal domain of mPNGase binds to the C-terminal region of P97. This is the first time that a protein-interacting function of the mPNGase N-terminal domain has been established. This is very interesting since it was generally believed that P97 interacts with its effector proteins through its N-terminal domain. The structure of full length P97 has been solved, however, the C-terminal region could not be visualized in the structure. To investigate the function of the N-terminal domain of mPNGase and the details of its interaction with P97, crystallographic studies on these proteins and their complexes are being undertaken. We have recently obtained crystals of the N-terminal domain in its apo-state and in complex with a fragment of P97 and expect them to be ready for data collection and structure solution by the time the RapiData course starts.