Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism, deficiency of which is associated with the most common metabolic phenotype of phenylketonuria (PKU) and hyperphenylalaninemia (HPA). A bulk of PKU disease-associated missense mutations in the PAH gene have been studied, and the consequence of each PAH variant vary immensely. Prior research established that PKU-associated variants possess defects in protein folding with reduced cellular stability leading to rapid degradation. However, recent evidence revealed that PAH tetramers exist as a mixture of resting state and activated state whose transition depends upon the phenylalanine concentration and certain PAH variants that fail to modulate the structural equilibrium are associated with PKU disease. Collectively, these findings framed our understanding of the complex genotype–phenotype correlation in PKU. In the current study, we substantiate a link between PAH protein stability and its degradation by the ubiquitin-mediated proteasomal degradation system. Here, we provide an evidence that PAH protein undergoes ubiquitination and proteasomal degradation, which can be reversed by deubiquitinating enzymes (DUBs). We identified USP19 as a novel DUB that regulates PAH protein stability. We found that ectopic expression of USP19 increased PAH protein level, whereas depletion of USP19 promoted PAH protein degradation. Our study indicates that USP19 interacts with PAH and prevents polyubiquitination of PAH subsequently extending the half-life of PAH protein. Finally, the increase in the level of PAH protein by the deubiquitinating activity of USP19 resulted in enhanced metabolic function of PAH. In summary, our study identifies the role of USP19 in regulating PAH protein stability and promotes its metabolic activity.

Graphical abstract

Graphical highlights

1. E3 ligase Cdh1 promotes PAH protein degradation leading to insufficient cellular amount of PAH causing PKU.

2. A balance between E3 ligase and DUB is important to regulate the proteostasis of PAH.

3. USP19 deubiquitinates and stabilizes PAH further protecting it from rapid degradation.

4. USP19 increases the enzymatic activity of PAH, thus maintaining normal Phe levels.

中文翻译：

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去泛素酶 USP19 增强苯丙氨酸羟化酶蛋白稳定性及其酶活性

苯丙氨酸羟化酶（PAH）是苯丙氨酸代谢的关键酶，其缺乏与苯丙酮尿症（PKU）和高苯丙氨酸血症（HPA）最常见的代谢表型有关。大量与 PKU 疾病相关的PAH基因错义突变已被研究，每种 PAH 变异的后果差异很大。先前的研究表明，PKU 相关变体具有蛋白质折叠缺陷，细胞稳定性降低，导致快速降解。然而，最近的证据表明，PAH 四聚体以静息状态和激活状态的混合物形式存在，其转变取决于苯丙氨酸浓度，并且某些无法调节结构平衡的 PAH 变体与 PKU 疾病相关。总的来说，这些发现构建了我们对 PKU 复杂基因型-表型相关性的理解。在当前的研究中，我们证实了 PAH 蛋白稳定性与其泛素介导的蛋白酶体降解系统降解之间的联系。在这里，我们提供了证据表明 PAH 蛋白经历泛素化和蛋白酶体降解，这可以通过去泛素化酶 (DUB) 逆转。我们确定 USP19 是一种调节 PAH 蛋白稳定性的新型 DUB。我们发现USP19的异位表达增加了PAH蛋白水平，而USP19的缺失促进了PAH蛋白降解。我们的研究表明，USP19 与 PAH 相互作用并阻止 PAH 的多泛素化，从而延长 PAH 蛋白的半衰期。最后，USP19 的去泛素化活性增加了 PAH 蛋白水平，从而增强了 PAH 的代谢功能。总之，我们的研究确定了 USP19 在调节 PAH 蛋白稳定性并促进其代谢活性中的作用。

图形概要

图形亮点

1. E3连接酶Cdh1促进PAH蛋白降解，导致细胞内PAH量不足，引起PKU。

2. E3连接酶和DUB之间的平衡对于调节PAH的蛋白质稳态非常重要。

3. USP19 去泛素化并稳定 PAH，进一步保护其免于快速降解。

4. USP19 增加 PAH 的酶活性，从而维持正常的 Phe 水平。