Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, 390002, India.
Appl Microbiol Biotechnol. 2017 Apr;101(7):2905-2917. doi: 10.1007/s00253-016-8023-5. Epub 2016 Dec 24.
Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal.
真核生物中多胺水平的升高导致鸟氨酸脱羧酶（ODC）的快速降解，ODC 是多胺生物合成途径的第一个酶。酵母中的 ODC（yODC）有两个结构域，Nα/β结构域由α/β桶结构域（α/β）组成，其 N 端有 50 个残基的突出（N50），C 端有β片层结构域。yODC 序列中有两个降解决定信号或降解元件（degrons），即 N50 和位于α/β结构域中的抗酶结合元件（AzBE），负责其被蛋白酶体降解。多胺过剩时诱导的抗酶（Az）与 AzBE 结合，并将 ODC 递送到蛋白酶体，而 N50 将蛋白穿入蛋白酶体。我们之前曾报道过，yODC 的肽 Nα/β 作为一个独立的可移植降解元件，其作用可以通过改变多胺水平来调节抗酶的作用来调节。尽管哺乳动物 ODC（mODC）与 yODC 有 40%的序列同源性，但它缺乏 yODC 的 N50，而是有一个 37 个残基的 C 端尾巴（CmODC）。CmODC 作为一个独立的可移植降解元件，在 yODC 中没有对应物。本研究通过与绿色荧光蛋白（GFP）融合表达这两个降解元件 Nα/β和 CmODC，研究了它们一起用于靶向蛋白降解的优点。我们的结果表明，在抗酶的调节下，与单独作用的信号 Nα/β和 CmODC 相比，这两个信号一起作用可以更好地增强降解。可以通过快速去除 Nα/β 和 CmODC 的组合来研究新型蛋白质的功能。
