Cetin Mehmet, Evenson William E, Gross Garrett G, Jalali-Yazdi Farzad, Krieger Daniel, Arnold Don, Takahashi Terry T, Roberts Richard W
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
J Mol Biol. 2017 Feb 17;429(4):562-573. doi: 10.1016/j.jmb.2016.11.008. Epub 2016 Nov 16.
K- and H-Ras are the most commonly mutated genes in human tumors and are critical for conferring and maintaining the oncogenic phenotype in tumors with poor prognoses. Here, we design genetically encoded antibody-like ligands (intrabodies) that recognize active, GTP-bound K- and H-Ras. These ligands, which use the 10th domain of human fibronectin as their scaffold, are stable inside the cells and when fused with a fluorescent protein label, the constitutively active G12V mutant H-Ras. Primary selection of ligands against Ras with mRNA display resulted in an intrabody (termed RasIn1) that binds with a K of 2.1μM to H-Ras(G12V) (GTP), excellent state selectivity, and remarkable specificity for K- and H-Ras. RasIn1 recognizes residues in the Switch I region of Ras, similar to Raf-RBD, and competes with Raf-RBD for binding. An affinity maturation selection based on RasIn1 resulted in RasIn2, which binds with a K of 120nM and also retains excellent state selectivity. Both of these intrabodies colocalize with H-Ras, K-Ras, and G12V mutants inside the cells, providing new potential tools to monitor and modulate Ras-mediated signaling. Finally, RasIn1 and Rasin2 both display selectivity for the G12V mutants as compared with wild-type Ras providing a potential route for mutant selective recognition of Ras.
K-Ras和H-Ras是人类肿瘤中最常见的突变基因，对于预后不良的肿瘤赋予并维持致癌表型至关重要。在此，我们设计了可识别活性、GTP结合型K-Ras和H-Ras的基因编码抗体样配体（胞内抗体）。这些以人纤连蛋白第10结构域为支架的配体，在细胞内稳定，当与荧光蛋白标签融合时，可标记组成型活性G12V突变体H-Ras。通过mRNA展示进行针对Ras的配体初步筛选，得到了一种胞内抗体（称为RasIn1），它以2.1μM的解离常数（K）与H-Ras(G12V)（GTP）结合，具有出色的状态选择性以及对K-Ras和H-Ras的显著特异性。RasIn1识别Ras开关I区域中的残基，类似于Raf-RBD，并与Raf-RBD竞争结合。基于RasIn1进行亲和力成熟筛选，得到了RasIn2，其结合解离常数为120nM，并且同样保留了出色的状态选择性。这两种胞内抗体在细胞内均与H-Ras、K-Ras及G12V突变体共定位，为监测和调节Ras介导的信号传导提供了新的潜在工具。最后，与野生型Ras相比，RasInl和Rasin2对G12V突变体均表现出选择性，为Ras的突变体选择性识别提供了一条潜在途径。
