A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in aging in Physcomitrella and Arabidopsis

Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is mostly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation ultimately leads to cytotoxic aggregates. In mammals, this correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. AARE enzymes are ATP-independent serine proteases presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests conservation of AARE function in age-related development.

蛋白质氧化是氨基酸侧链与活性氧（reactive oxygen species, ROS）发生反应的结果，且该过程大多不可逆。在非光合组织中，线粒体是活性氧的主要来源；而在光合组织中，质体则是活性氧的主要来源。氧化蛋白质会出现结构完整性降低、甚至功能丧失的情况，其积累最终会形成细胞毒性聚集物。在哺乳动物体内，这一过程与衰老密切相关，并与多种年龄相关性病理状态存在关联。目前学界正对用于清除氧化蛋白质的哺乳动物蛋白水解通路展开深入研究，而学界对植物中该过程的机制认知仍较为匮乏。AARE酶是不依赖ATP的丝氨酸蛋白酶，推测可作用于氧化蛋白质，并以兼具外肽酶与内肽酶的双重模式发挥功能。该类酶广泛存在于所有生命域中。本研究对苔藓小立碗藓（Physcomitrella）和被子植物拟南芥中的AARE酶进行了探究，在小立碗藓中鉴定出3个同源核基因（PpAARE1-3），在拟南芥中鉴定出1个核基因（AtAARE）。令人意外的是，我们在活体中观察到AtAARE与PpAARE1这两种蛋白质具有三重定位特性，可分别靶向质体、线粒体与细胞质，该特性大概率在植物谱系中保守存在。这为植物体内活性氧主要来源细胞器中氧化蛋白质的降解提供了一种不依赖ATP的途径，该途径与哺乳动物的相关机制截然不同。组合敲除植株与蛋白质相互作用分析结果显示，苔藓AARE同工酶存在特异性相互作用，并在渐进式衰老过程中发挥功能。对AtAARE的T-DNA插入突变体的分析进一步表明，AARE的功能在年龄相关的发育进程中具有保守性。