Auger Shelby A, Pedersen Jodi S, Maity Sanjay, Sprague-Getsy Andrea M, Lorimer Ellen L, Koehn Olivia J, Krauklis Steven A, Berns Brenna, Murphy Katherine M, Hussain Jamal, Thao Pa, Bernhagen Kaitlyn, Justyna Katarzyna, Sundaresan Anjana P, McKim Daniel B, Williams Carol L, Hougland James L, Li Ling, Distefano Mark D
Department of Chemistry, University of Minnesota-Twin Cities, Minneapolis, Minnesota 55455, United States.
Department of Chemistry, Syracuse University, Syracuse, New York 13244, United States.
Biochemistry. 2025 Jan 7;64(1):138-155. doi: 10.1021/acs.biochem.4c00558. Epub 2024 Dec 9.
Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed combined with metabolic labeling , resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated , was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.
异戊二烯化是指在靶蛋白C末端附近的半胱氨酸处，用法尼基二磷酸（FPP）或香叶基香叶基二磷酸（GGPP）对蛋白质进行修饰，以生成硫醚连接的脂化蛋白。在最近的研究中，含炔基类异戊二烯类似物（包括C15AlkOPP）的代谢标记已被用于鉴定异戊二烯化蛋白并追踪其在不同疾病中的水平。在此，我们对类异戊二烯长度对用这些探针标记的蛋白质的影响进行了系统研究。通过化学合成生成了两种新的类似物C15hAlkOPP和C20AlkOPP，使本研究中使用的化合物总数达到八种。结合代谢标记进行的酶动力学研究，鉴定出C10AlkOPP标记的蛋白质有8种，C15AlkOPP标记的蛋白质有70种，C15hAlkOPP标记的蛋白质有41种，C20AlkOPP标记的蛋白质有7种。虽然C10AlkOPP对法尼基化蛋白的选择性最高，C20AlkOPP对香叶基香叶基化蛋白的选择性最高，但使用这些探针鉴定出的蛋白质数量相对较少。相比之下，C15AlkOPP标记的蛋白质最多，包括所有类别的异戊二烯化蛋白的代表。对这些类似物的功能分析表明，C15AlkOPP特别适合生物学研究，因为它能有效掺入，能够使H-Ras蛋白正确定位于质膜，并补充巨噬细胞中GGPP消耗的影响，从而产生正确的细胞极化和丝状伪足。总体而言，这些结果表明，C15AlkOPP是一种具有生物学功能的通用代谢标记实验探针，对细胞生理学的影响最小。
