Braganza Andrea, Li Jianfeng, Zeng Xuemei, Yates Nathan A, Dey Nupur B, Andrews Joel, Clark Jennifer, Zamani Leila, Wang Xiao-Hong, St Croix Claudette, O'Sullivan Roderick, Garcia-Exposito Laura, Brodsky Jeffrey L, Sobol Robert W
From the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
the Hillman Cancer Center, University of Pittsburgh Cancer Institute.
J Biol Chem. 2017 Feb 10;292(6):2470-2484. doi: 10.1074/jbc.M116.766824. Epub 2016 Dec 21.
Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer from aufman culocerebrofacial yndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene However, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein with omologous to the 6-AP erminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758-1068) results in loss of UBE3B's ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29-58) results in loss of calmodulin binding and a significant increase in the ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels disrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.
最近的全基因组研究发现，患有肌张力减退、发育迟缓、智力残疾、先天性异常、特征性面部畸形特征和低胆固醇水平的患者患有考夫曼颅脑面部综合征（KOS，也被报道为睑裂狭小-上睑下垂-智力残疾综合征）。KOS的主要病因是该基因的常染色体隐性突变。然而，迄今为止，尚无研究确定UBE3B的细胞或酶功能。在此，我们报告UBE3B是一种与线粒体相关的蛋白质，具有与6-氨基嘌呤末端（HECT）E3泛素连接酶活性同源的活性。催化半胱氨酸突变（C1036A）或删除整个HECT结构域（氨基酸758-1068）会导致UBE3B泛素化活性丧失。在人类细胞中敲低UBE3B会诱导线粒体形态和生理变化、线粒体体积减小以及细胞增殖的严重抑制。我们还发现UBE3B通过其N端异亮氨酸-谷氨酰胺（IQ）基序与钙调蛋白相互作用。删除IQ基序（氨基酸29-58）会导致钙调蛋白结合丧失以及UBE3B泛素化活性显著增加。此外，我们发现钙水平的变化会破坏钙调蛋白-UBE3B相互作用。这些研究表明UBE3B是一种E3泛素连接酶，并揭示该酶受钙调蛋白调节。此外，通过钙调蛋白和钙对UBE3B的调节暗示了钙信号在线粒体蛋白泛素化、蛋白周转和疾病中的作用。
