N-TERMINAL PROCESSING OF IPLA2B AND OF POT CLEAVAGE SITES OF APP CONSTRUCTS

IPLA2B 和 APP 构建体的 Pot 裂解位点的 N 端处理

• 批准号: 7355244
• 负责人: HAOWEI SONG
• 金额: $ 0.85万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355244
• 关键词: TERMINAL; PROCESSING; IPLA2B; POT; CLEAVAGE

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. Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A2 (iPLA2B) is the dominant PLA2 enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA2B and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA2B, and such applications demonstrate the utility of this approach.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。淀粉样蛋白前体蛋白（APP）蛋白水解加工的失调有助于阿尔茨海默氏病的发病机理，并且通过磷脂酶A2（IPLA2B）组的组是中枢神经中的主要PLA2酶，并且是调节性蛋白质解体的调节性蛋白质流程过程。我们已经通过自动鉴定蛋白水解消化物中的LC/MS/MS分析中自动鉴定来鉴定了C-末端6-MYC TAG的APP构建体中IPLA2B的新型N末端变异和以前未识别的蛋白水解位点。我们已经开发了一个签名 - 发现程序（SD）程序来通过鉴定由体内蛋白水解加工引起的特征肽来表征蛋白质同工型。该程序分析了从LC分析的蛋白质混合物的LC分析中的MS/MS数据，这些数据可能包括生物样品中未完全分辨的成分。这减少了纯化的要求，从而最大程度地减少了样品处理过程中的人为修改。一种新算法生成理论特征肽集并计算预测质谱和观察到的质谱之间的相似性得分，已通过模型蛋白进行了测试和优化。该程序已应用于鉴定生物学兴趣蛋白质的变体，包括应用裂解产品和IPLA2B，此类应用证明了这种方法的实用性。