Function and Mechanisms of the N-End Rule Pathway

描述（由申请人提供）：泛素依赖性N-末端规则途径将蛋白质的体内半衰期与其N-末端残基的身份联系起来。为了了解N端规则通路的生理功能和潜在的分子机制，我们开始在小鼠中对该通路进行生化和遗传解剖。我们已经展示了N-末端天冬酰胺特异性脱酰胺在社会条件行为中的功能，N-末端乙酰化在心血管发育中的功能，以及N-末端半胱氨酸氧化作为氧传感器的功能。哺乳动物UBR 1/E3 calpha是泛素系统中第一个被鉴定的泛素连接酶（E3），也是唯一一个识别1型和2型蛋白质N端不稳定残基的E3。在过去的二十年中，体外生物化学研究表明UBR 1具有许多生物学功能，包括神经元细胞分化、两栖动物肢体再生、细胞凋亡、肌肉萎缩和各种蛋白质（辛德毕斯病毒RNA聚合酶、HIV整合酶、单核细胞增生李斯特菌p60、RGS 4和RGS 16以及脑心肌炎病毒3C蛋白酶）的降解。然而，令人惊讶的是，缺乏UBR 1的小鼠除了肌肉蛋白质降解和脂肪代谢方面的细微缺陷外，其他都是正常的。我们假设，在哺乳动物的N-末端规则途径的底物识别介导的一组不同的E3的合作活动。事实上，我们鉴定了称为UBR 2和UBR 3的新型UBR 1样E3，并进一步假设UBR 2和/或UBR 3是可能与UBR 1协同发挥功能的E3。作为解决这些问题的初步努力，我们构建了UBR 2-/-、UBR 3-/-、UBR 1-/-UBR 2-/-和UBR 1-/-UBR 3-/-小鼠，发现UBR 2 - 1 oss引起雄性特异性不育和雌性特异性致死。因此，我们假设UBR 2是精子发生所必需的。这项建议的重点是下列目标：（1）表征UBR 2和UBR 3作为候选E3的生化特性，其可能在N-末端规则途径中与UBR 1协同发挥作用，（2）表征UBR 2-/-小鼠以评估UBR 2在精子发生中的体内功能，（3）研究UBR 2依赖的精子发生过程中与UBR 2相互作用的蛋白质或依赖UBR 2的分子通路。

DESCRIPTION (provided by applicant): The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. As an effort to understand the physiological functions and the underlying molecular mechanisms of the N-end rule pathway, we began the biochemical and genetic dissection of this pathway in mice. We have shown the functions of N-terminal asparagine-specific deamidation in socially conditioned behavior, of N-terminal arginylation in cardiovascular development, and of N-terminal cysteine oxidation as an oxygen sensor. Mammalian UBR1/E3calpha is the first identified ubiquitin ligase (E3) of the ubiquitin system, and has been known as the only E3 that recognizes type 1 and type 2 N-terminal destabilizing residues of proteins. For the last two decades, the in vitro biochemical studies have suggested numerous biological functions of UBR1, including neuronal cell differentiation, amphibian limb regeneration, apoptosis, muscle wasting, and the degradation of various proteins (Sindbis virus RNA polymerase, HIV integrase, the Listeria monocytogenes p60, RGS4 and RGS16, and the encephalomyocarditis virus 3C protease). Surprisingly, however, mice lacking UBR1 were apparently normal except for the subtle defects in muscle protein degradation and fat metabolism. We hypothesize that the substrate recognition in the mammalian N-end rule pathway is mediated by the cooperative activity of a set of distinct E3s. Indeed we identified novel UBRl-like E3s termed UBR2 and UBR3, and further hypothesize that UBR2 and/or UBR3 are the E3(s) that may cooperatively function with UBRI. As a preliminary effort to address these issues, we have constructed UBR2 -/-, UBR3 -/-, and UBR1-/-UBR2 -/- and UBR1-/-UBR3 -/- mice, and found that UBR2-1oss caused male-specific infertility and female-specific lethality. Thus, we hypothesize that UBR2 is essential for spermatogenesis. This proposal focuses on the following aims: (1) To characterize the biochemical properties of UBR2 and UBR3 as candidate E3s that may function cooperatively with UBR1 in the N-end rule pathway, (2) To characterize UBR2 -/- mice to assess the in vivo function of UBR2 in spermatogenesis, (3) To identify the UBR2-interacting proteins or UBR2-dependent molecular circuits underlying the UBR2- dependent spermatogenesis.