Fusser Markus, Kernstock Stefan, Aileni Vinay Kumar, Egge-Jacobsen Wolfgang, Falnes Pål Ø, Klungland Arne
Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway.
Department of Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway.
PLoS One. 2015 Nov 6;10(11):e0141472. doi: 10.1371/journal.pone.0141472. eCollection 2015.
Valosin-containing protein (VCP) is a homohexameric ATPase involved in a multitude cellular processes and it was recently shown that VCP is trimethylated at lysine 315 by the VCP lysine methyltransferase (VCPKMT). Here, we generated and validated a constitutive knockout mouse by targeting exon 1-4 of the Vcpkmt gene. We show that Vcpkmt is ubiquitously expressed in all tissues examined and confirm the sub-cellular localization to the cytoplasm. We show by (I) mass spectrometric analysis, (II) VCPKMT-mediated in vitro methylation of VCP in cell extracts and (III) immunostaining with a methylation specific antibody, that in Vcpkmt-/- mice the methylation of lysine 315 in VCP is completely abolished. In contrast, VCP is almost exclusively trimethylated in wild-type mice. Furthermore, we investigated the specificity of VCPKMT with in vitro methylation assays using as source of substrate protein extracts from Vcpkmt-/- mouse organs or three human Vcpkmt-/- cell lines. The results show that VCPKMT is a highly specific enzyme, and suggest that VCP is its sole substrate. The Vcpkmt-/- mice were viable, fertile and had no obvious pathological phenotype. Their body weight, life span and acute endurance capacity were comparable to wild-type controls. Overall the results show that VCPKMT is an enzyme required for methylation of K315 of VCP in vivo, but VCPKMT is not essential for development or survival under unstressed conditions.
含缬酪肽蛋白（VCP）是一种同六聚体ATP酶，参与多种细胞过程，最近研究表明VCP在赖氨酸315位点被VCP赖氨酸甲基转移酶（VCPKMT）三甲基化。在此，我们通过靶向Vcpkmt基因的外显子1 - 4构建并验证了一种组成型敲除小鼠。我们发现Vcpkmt在所有检测的组织中均有广泛表达，并证实其亚细胞定位在细胞质中。我们通过（I）质谱分析、（II）细胞提取物中VCPKMT介导的VCP体外甲基化以及（III）用甲基化特异性抗体进行免疫染色，发现Vcpkmt基因敲除小鼠中VCP赖氨酸315位点的甲基化完全消失。相比之下，野生型小鼠中VCP几乎完全是三甲基化的。此外，我们使用来自Vcpkmt基因敲除小鼠器官的底物蛋白提取物或三种人Vcpkmt基因敲除细胞系，通过体外甲基化试验研究了VCPKMT的特异性。结果表明VCPKMT是一种高度特异性的酶，提示VCP是其唯一底物。Vcpkmt基因敲除小鼠存活、可育，无明显病理表型。它们的体重、寿命和急性耐力与野生型对照相当。总体而言，结果表明VCPKMT是体内VCP赖氨酸315位点甲基化所必需的酶，但在无应激条件下，VCPKMT对发育或生存并非必不可少。
