Zhang Hongjie, Abraham Nessy, Khan Liakot A, Gobel Verena
Mucosal Immunology and Biology Research Center, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Nat Protoc. 2015 May;10(5):681-700. doi: 10.1038/nprot.2015.031. Epub 2015 Apr 2.
The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of nonproteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes of these compounds-findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic acid-based metabolites beyond their metabolic roles. It will typically require several months to identify a metabolic end product by biosynthetic pathway screens, but this time will vary widely depending, among other factors, on the end product's location in the pathway, which determines the number of screens required for its identification.
代谢组学领域不断有新化合物被编入目录，但其功能分析在技术上仍然具有挑战性，而且其代谢以外的作用大多未知。无偏向性的遗传/RNA干扰筛选是识别蛋白质编码基因体内功能的有力工具，但对于脂质等非蛋白质类化合物则不适用。不过，它们可以识别这些化合物的生物合成酶——这些发现通常被忽视，因为这些酶通常会合成多种产物。在这里，我们提供一种方法，利用后续生物合成途径筛选来识别终点生物合成酶，从而确定它们所作用的化合物。该方法基于这样一个原则，即所有随后鉴定出的下游生物合成酶至少都参与了这一种终产物的合成。我们描述了如何系统地靶向脂质生物合成途径；优化靶向条件；利用途径分支点；并通过遗传检测和生化分析验证结果。这种方法扩展了无偏向性遗传/RNA干扰筛选的能力，以识别基于非核酸的代谢物在代谢作用之外的体内功能。通过生物合成途径筛选来识别代谢终产物通常需要几个月时间，但这段时间会因多种因素而有很大差异，其中包括终产物在途径中的位置，这决定了识别它所需的筛选次数。
