Cellular signal transduction is propagated through protein interaction network from the surface membrane to various sub-cellular locations. Receptor tyrosine kinases (RTKs) are the family of cellular membrane-embedded protein, which receive the extracellular signals and covert them to the intracellular outputs by activating relevant signaling cascades. When RTK lost its regular function, it aberrantly activates its downstream signaling pathways even in the absence of extracellular cues. In diverse human cancers, critical cascades for cell survival such as mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, were incessantly stimulated by this RTK activation. During cancer formation, cancer cells have evolved to depend on this specific RTK-originated signaling for their survival so called oncogene addiction. Thus, RTK family have been focused as a therapeutic target to suppress tumor growth. Small molecule kinase inhibitors are the one kind of cancer drugs which inhibited kinase activity of signaling proteins. Especially, tyrosine kinase inhibitors (TKIs) abrogate RTK-mediated cellular signaling by suppressing the kinase activity of RTK family in cancer treatment. Current gold standard to determine TKI treatment is based on the gene sequencing results, identifying genetic lesions in a patient genome. However, this approach infers the status of the translated protein from sequence and structural information, but do not directly measure the activation of the signaling protein. Furthermore, the activation of proteins is regulated by post-translational modification, the result of complex interaction network. The lack of protein-protein interaction information, the main communication system for cellular activities, in cancer drug treatment has yielded only subsets of patients who were predicted to be beneficial upon cancer drug treatment. Thus, profiling of protein interaction network quantitatively has become to be necessary. In this work, I developed protein-protein interaction measurement platform based on single-molecule technique, so called single-molecule co-immunoprecipitation (SM co-IP). SM co-IP enabled to measure protein-protein interaction at single molecule resolution with 50 ms real time recording, which was applicable to observe transient interaction. In the first part of this work, I demonstrated the dynamic features of Ras and Raf protein interaction, which showed rapid turnover and transient interaction. Then, it was applied to reveal the active fraction of oncogenic Ras protein in tumor xenograft and cancer cell lines, validating that oncogenic Ras increased the active fraction of Ras protein rather than altering the dynamics of interaction with its downstream protein. In the second part, I focused this technique to RTK family and its targeted drug response prediction. SM co-IP measured PPI of RTK with the relevant downstream signaling proteins showing oncogenic EGFR mutation preferentially recruited the specific downstream protein, Grb2, than others. Then, I profiled the signaling network of 4 representative RTKs in lung adenocarcinoma cell line. The PPI score, developed to predict the targeted drug response, showed high correlation to the EGFR-targeted drug, suggesting that this PPI based prediction can provide hidden information from gene sequencing for cancer drug treatment. It was confirmed in breast cancer cell lines and patient derived tumor xeongrafts in mice indicating that PPI score system do not limit to the specific types of cancer or cancer drugs. This approach allows to probe protein-protein interaction more quantitatively than the conventional version of co-IP. It also provide quantitative prediction of targeted drugs. By collaborating with modern microfluidic system, I anticipate that this technique suggests an advanced concept of precision medicine in the future.
본 연구는 전반사 형광 현미경에 기반하여 단분자 수준의 정밀도로 두 단백질 사이의 상호작용을 실시간으로 측정하는 방법에 대한 연구 결과이다. 단분자 면역침강법으로 명명된 본 기술은 세포나 인체 조직에서 추출된 생체 내 단백질을 기본으로 이용한다. 해당 단백질의 하위 신호전달 단백질은 형광단백질로 표지되어 전반사 형광 현미경을 통해 관측토록 하는 것이 주요 골자이다. 기존에 사용되던 면역침강법은 두 단백질 사이의 상호작용을 실시간으로 관측하는 것이 불가능했다. 해당 기술은 이러한 단점을 극복하여 실시간으로 단백질 상호작용을 수 십 밀리 초 단위에서 측정할 수 있게 만들었다. 또한 단분자 수준의 정밀도와 시료 요구량은 소량의 시료가 채집되는 인체 수술 조직 및 생체검사 조직에도 이용할 수 있는 가능성을 보여주었다. 더불어 시료 준비 후 수 십 분 이내에 신속하게 결과를 분석할 수 있다.
본 논문의 3장에서는 Ras 단백질의 동역학적 신호전달 특성과 유전자변이가 발생된 Ras가 세포의 정상적인 신호전달을 파괴하여 암세포의 특징을 지니게 되는 기작을 세포 신호전달 측면에서 설명하였다. 후속 연구로 진행된 4장에서는 표적항암제의 주요 목표로 설정되는 EGFR 단백질과 소속된 단백질군을 주요 연구 요소로 설정하였다. 유전자변이로 인해 아미노산 염기 서열이 바뀐 EGFR단백질에서 발생된 세포신호 전달이 정상 단백질에서 발생한 신호와 어떻게 다른지를 단백질 상호작용 관측을 통해 보일 수 있었다. 나아가 표적항암제의 목표단백질 활성화 정도에 따라 같은 유전자변이를 가지고 있더라도 표적항암제에 대한 반응성이 달라지는 것을 입증하여 표적항암제 처방에 있어 새로운 판단요소를 제공할 제반을 마련하였다. 이는 기존 유전자 서열에만 의존하던 방식에서는 입증할 수 없는 새로운 정보로 미래의 개인 맞춤형 진단에 있어 필수적인 정보가 될 것으로 기대된다.
