Function and Mechanisms of the N-End Rule Pathway

描述(由申请人提供)：泛素依赖的N-末端规则通路将蛋白质在体内的半衰期与其N-末端残基的同一性联系起来。为了了解N末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这一通路进行生化和遗传剖析。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作用，N-末端精氨酸化在心血管发育中的作用，以及N-末端半胱氨酸氧化作为氧感受器的功能。哺乳动物UBR1/E3calpha是第一个被发现的泛素系统泛素连接酶(E3)，被认为是唯一识别蛋白质1和2 N末端不稳定残基的E3。在过去的二十年里，体外生化研究表明UBR1具有多种生物学功能，包括神经细胞分化、两栖动物肢体再生、细胞凋亡、肌肉萎缩和各种蛋白质的降解(Sindbis病毒RNA聚合酶、HIV整合酶、单核细胞增生性李斯特氏菌p60、RGS4和Rgs16，以及脑心肌炎病毒3C酶)。然而，令人惊讶的是，缺乏UBR1的小鼠显然是正常的，除了肌肉蛋白质降解和脂肪代谢方面的细微缺陷。我们假设，哺乳动物N-端规则通路中的底物识别是由一组不同的E3的协同活动介导的。事实上，我们发现了新的与Ubr1类似的E3，称为Ubr2和Ubr3，并进一步假设Ubr2和/或Ubr3是可能与Ubr1协同作用的E3(S)。作为解决这些问题的初步努力，我们构建了UBR2-/-、UBR3-/-、UBR1-/-UBR2-/-和UBR1-/-UBR3-/-小鼠，并发现UBR2-1oss导致男性特异性不育和女性特异性死亡。因此，我们假设UBR2在精子发生中是必不可少的。本研究的目的在于：(1)鉴定UBR2和UBR3作为候选E3在N端规则通路中可能与UBR1协同作用的生化特性；(2)鉴定UBR2-/-小鼠以评估UBR2在体内精子发生中的功能；(3)鉴定UBR2相互作用的蛋白质或UBR2依赖的分子回路。

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.