Department of Biochemistry, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5G 1M1, Canada.
Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 1L7, Canada.
Proc Natl Acad Sci U S A. 2024 Sep 10;121(37):e2408104121. doi: 10.1073/pnas.2408104121. Epub 2024 Sep 4.
Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.
脯氨酰羟化作用是一种氧依赖性的翻译后修饰（PTM），已知其可以调节胶原蛋白纤维的形成，并调节低氧诱导因子（HIF）α亚基的细胞表达。然而，由于缺乏能够直接测量内在无序蛋白质中多个脯氨酰羟化事件的生物物理方法，我们对这一重要但相对罕见的 PTM 的理解仍然不完整。在这里，我们描述了一种实时 C-直接检测 NMR 基测定法，用于通过利用脯氨酸的“无质子”性质来研究低氧诱导因子 1α的氧依赖性降解结构域中两个进化保守脯氨酸（P402 和 P564）的同时羟化。我们明确地表明，P564 以时间分辨的方式迅速羟化，而 P402 羟化明显滞后于 P564。这种差异羟化速率与脯氨酰羟化酶的结合亲和力几乎没有影响，而是受周围氨基酸组成的影响，特别是 P564 位置+1 的保守酪氨酸残基。这些发现支持了一个意想不到的观点，即进化保守的 P402 似乎在正常氧感应途径中几乎没有影响。
