Hougland James L, Hicks Katherine A, Hartman Heather L, Kelly Rebekah A, Watt Terry J, Fierke Carol A
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
J Mol Biol. 2010 Jan 8;395(1):176-90. doi: 10.1016/j.jmb.2009.10.038. Epub 2009 Oct 28.
Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a(2) position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca(1)a(2)X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a(2) and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.
异戊二烯化是一种翻译后修饰，对许多蛋白质的正确定位和功能至关重要。法尼基化，即在蛋白质底物的C末端附近连接一个15碳的法尼基基团，由蛋白质法尼基转移酶（FTase）催化。法尼基化作为药物开发的靶点受到了广泛关注，法尼基转移酶抑制剂正在作为癌症治疗药物进行临床试验。然而，由于异戊二烯化蛋白质的完整组成尚不清楚，负责法尼基转移酶抑制剂疗效的FTase底物尚未明确。在人类蛋白质组中鉴定新的异戊二烯化蛋白质是理解异戊二烯化依赖性细胞过程的重要一步。基于对已知法尼基化蛋白质的分析得出的FTase序列偏好，我们选择并筛选了一个代表213种人类蛋白质C末端的小肽文库，以检测其与FTase的活性。我们在该文库中鉴定出77种新的FTase底物，它们表现出多轮反应（MTO）活性；我们的文库还包含85种仅在单轮反应（STO）条件下可被FTase法尼基化的肽。基于这些结果，设计了第二个文库，又产生了29种新的MTO FTase底物和45种STO底物。这两类底物表现出不同的特异性要求。高效的MTO活性与α(2)位置存在非极性氨基酸以及末端X残基为苯丙氨酸、甲硫氨酸或谷氨酰胺相关，这与提出的Ca(1)α(2)X序列模型一致。相比之下，STO底物的序列在α(2)和X残基处的变化要大得多，目前的法尼基化算法无法很好地描述。这些结果改进了异戊二烯基转移酶底物特异性的定义，测试了底物算法的有效性，并提供了有关治疗靶点的有价值信息。最后，这些数据揭示了通过调节STO与MTO肽与FTase的反应性在体内调节异戊二烯化的潜力。
