Riehl Jana, Rijal Ramesh, Nitz Leonie, Clemen Christoph S, Hofmann Andreas, Eichinger Ludwig
Medical Faculty, Center for Biochemistry, Institute of Biochemistry I, University of Cologne, Cologne, Germany.
Department of Biology, College Station, Texas A&M University, Texas, TX, United States.
Front Cell Dev Biol. 2021 Sep 23;9:748860. doi: 10.3389/fcell.2021.748860. eCollection 2021.
The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in . Pull-down assays revealed that the UBX domain (amino acids 384-466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β-α -α lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1' intermolecular interface. We thus propose that UBXD9 regulates p97 activity by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.
丰富的同型六聚体AAA + ATP酶p97（也称为含缬酪肽蛋白，VCP）从酵母到人类高度保守，是细胞蛋白质稳态的关键因子，因为它催化蛋白质的解折叠。由于其在蛋白质质量控制途径中的基本功能，它受到30多种辅助因子的调节，包括UBXD蛋白家族，其成员都携带一个泛素调节X（UBX）结构域，能够与p97结合。后一个蛋白家族的一个成员是基本未被表征的含UBX结构域蛋白9（UBXD9）。在这里，我们使用一系列N端和C端截短构建体分析了UBXD9与p97的蛋白质-蛋白质相互作用，并在细胞中探究了UBXD9相互作用组。下拉实验表明，UBX结构域（氨基酸384 - 466）对于p97相互作用是必要且充分的，并且UBX结构域的N端延伸折叠成β-α-α套索结构，是p97六聚体解离所必需的。在功能上，这一发现表现为加入全长UBXD9或UBXD9后p97的ATP酶活性大幅降低。蓝色非变性聚丙烯酰胺凝胶电泳结果以及结构模型预测表明，UBXD9或UBXD9的六聚体与p97六聚体相互作用，并通过插入螺旋套索结构破坏p97亚基相互作用，推测是通过破坏p97 D1:D1'分子间界面的稳定性。因此，我们提出UBXD9通过将四级结构平衡从六聚体转变为单体来调节p97活性。使用三种独立的方法，我们进一步鉴定了UBXD9的新型相互作用伙伴，包括III型谷氨酰胺合成酶以及几种肌动蛋白结合蛋白。这些发现表明UBXD9在肌动蛋白细胞骨架的组织中起作用，并且与假设的p97调节的寡聚化依赖性机制一致。
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