Hickox Ann E, Wong Ann C Y, Pak Kwang, Strojny Chelsee, Ramirez Miguel, Yates John R, Ryan Allen F, Savas Jeffrey N
Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
Departments of Surgery and Neuroscience, University of California, San Diego and Veterans Administration Medical Center, La Jolla, California, 92093.
J Neurosci. 2017 Feb 1;37(5):1320-1339. doi: 10.1523/JNEUROSCI.2267-16.2016. Epub 2016 Dec 30.
The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.
Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.
哺乳动物内耳（IE）通过高度特化的细胞和蛋白质来实现听觉和前庭感觉。感觉受体毛细胞（HCs）对于通过一系列已知和未知的蛋白质机制转导机械输入并刺激感觉神经元是必需的。为了了解这些独特的有丝分裂后细胞的蛋白质组成，其中不可逆的蛋白质降解或损伤会导致听力和平衡受损，我们通过串联质谱分析IE样本，以生成关于蛋白质身份和丰度的无偏倚鸟枪法蛋白质组学视图。通过使用Pou4f3/eGFP转基因小鼠，其中HCs表达由Pou4f3驱动的GFP，我们通过荧光激活细胞分选（FACS）纯化了一群HCs，以分析并将HC蛋白质组与来自感觉上皮和整个IE的其他IE亚蛋白质组进行比较。我们表明，哺乳动物HC蛋白质组包含数百种独特或高表达的蛋白质。我们对纯化的HCs进行的全局蛋白质组学分析扩展了现有的HC转录组，揭示了以前未检测到的基因产物和异构体特异性蛋白质表达。将我们的蛋白质组学数据与小鼠和人类遗传性听觉/前庭损伤数据库进行比较，证实了HC蛋白质组对正常IE功能的关键作用，为新的重要HC基因提供了细胞特异性候选池。通过蛋白质组学专门在HCs中鉴定并经免疫组织化学验证的几种蛋白质映射到人类遗传性耳聋位点，可能代表新的耳聋基因。
听力和平衡依赖于内耳（IE）中的特化感觉毛细胞（HCs）将有关声音、加速度和方向的信息传递给大脑。遗传和环境诱导的对HC蛋白质的扰动可导致耳聋和严重失衡。我们使用具有表达GFP的HCs的转基因小鼠，结合FACS分选和串联质谱，来定义迄今为止最完整的HC和IE蛋白质组。我们表明，数百种蛋白质在HCs中被独特鉴定或富集，扩展了先前的基因表达分析以揭示新的HC蛋白质和异构体。重要的是，与耳聋相关的蛋白质在HCs中显著富集，表明这种对IE感觉细胞的深入蛋白质组学分析可能具有发现耳聋基因的潜力。
