Lehman Norman L, van de Rijn Matt, Jackson Peter K
Department of Pathology, Stanford University School of Medicine, Palo Alto, California, USA.
Methods Enzymol. 2005;399:334-55. doi: 10.1016/S0076-6879(05)99023-X.
The turnover of key proteins that mediate development, cellular proliferation, and a host of essential biological processes is controlled by the ubiquitin proteasome system (UPS). In several well-studied examples, notably in the cell cycle, regulatory proteins that control ubiquitin-dependent destruction are themselves substrates of the UPS, creating a multilayered system to ensure precise and dynamic control of protein stability. UPS regulators controlled at the level of protein stability--including the F-box protein Skp2 and the VHL protein (substrate adapter proteins for multicomponent E3 ubiquitin ligases)-- seem to be misregulated in tumors. In these cases, especially, measuring levels of critical regulatory and target proteins will often present a more biologically meaningful picture than examining relative mRNA levels, which do not always reflect corresponding protein levels. Tissue microarrays (TMAs) allow simultaneous screening of large numbers of tumors for expression of specific proteins by immunohistochemical staining of a single microscope slide prepared from a TMA paraffin block. Replicate slides prepared from the same block can be immunostained for multiple proteins functioning in a related pathway, and a semiquantitative protein expression profile for a given subset of UPS pathway components, or other subsets of proteins of interest, can be assembled. Protein expression profiles of individual tumors or tissue types can be compared and visualized by hierarchical clustering methods. These expression profiles may be used as screening tools to investigate the relative abundance of components of a biochemical pathway in tumors or other tissues. TMAs have an exciting future as tools for basic research, diagnostic pathology, and drug targeting. In this article, we provide an introduction to the use of TMAs to study the expression of UPS component proteins and substrates in tumors by immunohistochemistry.
介导发育、细胞增殖及许多重要生物学过程的关键蛋白质的周转由泛素蛋白酶体系统（UPS）控制。在几个深入研究的例子中，尤其是在细胞周期中，控制泛素依赖性降解的调节蛋白本身就是UPS的底物，从而形成了一个多层系统，以确保对蛋白质稳定性进行精确而动态的控制。在蛋白质稳定性水平受到调控的UPS调节因子——包括F-box蛋白Skp2和VHL蛋白（多组分E3泛素连接酶的底物衔接蛋白）——在肿瘤中似乎存在调控异常。在这些情况下，特别是测量关键调节蛋白和靶蛋白的水平，往往比检测相对mRNA水平更能呈现出更具生物学意义的情况，因为mRNA水平并不总是反映相应的蛋白质水平。组织微阵列（TMA）允许通过对由TMA石蜡块制备的单个显微镜载玻片进行免疫组织化学染色，同时筛选大量肿瘤中特定蛋白质的表达情况。从同一石蜡块制备的重复载玻片可以针对在相关途径中发挥作用的多种蛋白质进行免疫染色，并且可以汇总给定UPS途径组分子集或其他感兴趣蛋白质子集的半定量蛋白质表达谱。可以通过层次聚类方法比较和可视化单个肿瘤或组织类型的蛋白质表达谱。这些表达谱可作为筛选工具，用于研究肿瘤或其他组织中生化途径组分的相对丰度。作为基础研究、诊断病理学和药物靶向的工具，TMA有着令人兴奋的前景。在本文中，我们介绍了使用TMA通过免疫组织化学研究肿瘤中UPS组分蛋白和底物表达的方法。
