Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.
Mol Cell Proteomics. 2010 Jun;9(6):1243-59. doi: 10.1074/mcp.M900387-MCP200. Epub 2010 Feb 2.
Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a chi(2) analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, alpha-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that alpha-actinin and densin directly interact with residues 151-300 and 446-817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.
螺旋肌联蛋白通过与丝状肌动蛋白、蛋白磷酸酶 1 以及大量其他信号蛋白的相互作用，调节发育过程中兴奋性突触后的功能和形态。为了深入了解螺旋肌联蛋白在成熟大脑中的作用，我们采用一种蛋白质组学的方法，利用亚细胞级分从成年啮齿动物纹状体中提取的螺旋肌联蛋白免疫复合物，来鉴定蛋白质组学图谱，从而鉴定螺旋肌联蛋白的相互作用组。使用一种鼠源螺旋肌联蛋白抗体进行初始分析，在免疫沉淀复合物中检测到 23 种未在 IgG 对照样本中出现的蛋白质，但其中 12 种蛋白质存在于螺旋肌联蛋白敲除组织中。使用两种不同的螺旋肌联蛋白抗体和敲除或 IgG 对照进行的第二轮筛选共鉴定出 125 种蛋白质。每种蛋白质与每种样本中螺旋肌联蛋白特异性相关的概率进行了计算，并根据来自多个样本分析的概率的卡方分析对蛋白质进行了排序。检测到了螺旋肌联蛋白和已知相关蛋白神经钙蛋白和多种蛋白磷酸酶 1 同工型。随后证实，多个新的螺旋肌联蛋白相关蛋白（肌球蛋白 Va、钙/钙调蛋白依赖性蛋白激酶 II、神经丝轻链、突触后密度蛋白 95、α-辅肌动蛋白和 densin）与含有螺旋肌联蛋白片段的 GST 融合蛋白相互作用。进一步的生化和转染细胞成像研究表明，α-辅肌动蛋白和 densin 分别与螺旋肌联蛋白的残基 151-300 和 446-817 直接相互作用。综上所述，我们开发了一种多抗体、蛋白质组学方法，用于鉴定天然组织中的蛋白质相互作用组，阐明了敲除组织对照的重要性，并为成熟纹状体中螺旋肌联蛋白相互作用组的性质和功能提供了新的见解。
