Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Mol Cell Proteomics. 2010 Mar;9(3):565-78. doi: 10.1074/mcp.M900286-MCP200. Epub 2009 Dec 10.
Protein expression profiles in rat corporal smooth muscle tissue were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls (AMCs) at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four AMC rat corpora tissues were prepared independently and analyzed together across multiple quantitative two-dimensional gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 170 spots were differential expressed among the four experimental groups. A subsequent mass spectrometry analysis of the 170 spots identified a total of 57 unique proteins. Network analysis of these proteins using MetaCore suggested altered activity of transcriptional factors that are of too low abundance to be detected by the two-dimensional gel method. The proteins that were down-regulated with diabetes include isoforms of collagen that are precursors to fibril-forming collagen type 1; Hsp47, which assists and mediates the proper folding of procollagen; and several proteins whose abundance is controlled by sex hormones (e.g. CRP1 and A2U). On the other hand, proteins seen or predicted to be up-regulated include proteins involved in cell apoptosis (e.g. p53, 14-3-3-gamma, Serpinf1, Cct4, Cct5, and Sepina3n), proteins that neutralize the biological activity of nerve growth factor (e.g. anti-NGF 30), and proteins involved in lipid metabolism (e.g. apoA-I and apoA-IV). Subsequent Western blot validation analysis of p53, 14-3-3-gamma, and Hsp47 confirmed increased p53 and 14-3-3-gamma and decreased Hsp47 levels in separate samples. According to the results from the Western blot analysis, Hsp47 protein showed a approximately 3-fold decrease at 1 week and was virtually undetectable at 2 months in diabetic versus control. Taken together, our results identify novel candidate proteins playing a role in erectile dysfunction in diabetes resulting from STZ treatment.
采用 STZ 处理诱导高血糖，分别在诱导后 1 周和 2 个月时，比较链脲佐菌素诱导的糖尿病（STZ-DM）动物模型和年龄匹配对照（AMC）的大鼠 corporal 平滑肌组织中的蛋白质表达谱。在每个时间点，独立制备来自 4 只 STZ-DM 和 4 只 AMC 大鼠 corpora 组织的蛋白质样品，并使用混合内部标准样品在多个定量二维凝胶上进行分析，以具有统计学置信度的定量表达变化。在四个实验组之间，共有 170 个斑点差异表达。随后对这 170 个斑点进行的质谱分析总共鉴定出 57 种独特的蛋白质。使用 MetaCore 对这些蛋白质进行网络分析表明，转录因子的活性发生改变，但由于二维凝胶方法的检测限制，其丰度太低而无法检测到。糖尿病下调的蛋白质包括前胶原 1 纤维形成胶原的同种型；Hsp47，其协助并介导前胶原的正确折叠；以及几种丰度受性激素（如 CRP1 和 A2U）控制的蛋白质。另一方面，看到或预测上调的蛋白质包括参与细胞凋亡的蛋白质（如 p53、14-3-3-γ、Serpinf1、Cct4、Cct5 和 Sepina3n）、中和神经生长因子生物学活性的蛋白质（如抗-NGF 30）和参与脂质代谢的蛋白质（如 apoA-I 和 apoA-IV）。p53、14-3-3-γ 和 Hsp47 的随后 Western blot 验证分析证实，在单独的样本中，p53 和 14-3-3-γ 增加，Hsp47 水平降低。根据 Western blot 分析的结果，Hsp47 蛋白在糖尿病中大约 3 倍减少，在 2 个月时几乎无法检测到。总的来说，我们的结果确定了在 STZ 处理导致的糖尿病引起的勃起功能障碍中起作用的新型候选蛋白质。
