分类: 生物学 >> 生物工程 提交时间: 2024-03-05
摘要: Single-molecule discrimination among amino acids is crucial to the realization of next-generation protein sequencing. Owing to the heterogeneous charge and subtle volume difference of underivatized amino acids, it remains a challenge for single-molecule techniques to recognize each of them. Here, we report the direct detection of twenty proteinogenic amino acids using a copper(II)-functionalized MspA nanopore. The binding sites for copper(II) ion are constructed by introducing histidine mutation (N91H) to M2MspA protein. With copper ion binding to histidine residues, amino acids can reversibly coordinate the copper-histidine complex, generating well-defined current signals. Using this strategy, all twenty amino acids can be detected. Assisted by a machine learning algorithm, we can identify 100% of signals with 70.2% accuracy or 60% of signals with 93.4% accuracy in the validation set. In successively addition experiment, each amino acid in a mixture of 10 amino acids can be identified precisely. Furthermore, we use carboxypeptidase A1 to partly release the C-terminal amino acids of peptides with different lengths (9, 10 and 22 residues). The hydrolysates of peptides can be identified and distinguished. These results demonstrate the feasibility of this system for amino acids detection and peptide identification, shedding new lights on the development of single-molecule protein sequencing.
分类: 生物学 >> 生物物理学 提交时间: 2016-05-12
摘要: The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway has been identified as an important pathway in renal cell carcinoma (RCC). We have reported a nonsense mutation in PIK3R1, which encodes the regulatory subunit of PI3K, in a metastatic RCC (mRCC), while the mutation was absent in the corresponding primary RCC (pRCC). To identify the function of PIK3R1 in RCC, we examined its expression in normal kidney, pRCC and mRCC by immunohistochemistry and real-time polymerase chain reaction. The expression of PIK3R1 significantly decreased in pRCC and was further reduced in mRCC compared with normal tissue. Besides, its expression levels were negatively correlated with T-category of tumor stage. Additionally, 786-O and A-704 cells with PIK3R1 depletion introduced by CRISPR/Cas9 system displayed enhanced proliferation, migration and epithelial-mesenchymal transition (EMT), and acquired a stem-like phenotype. Moreover, the PIK3R1 depletion promoted the phosphorylation of AKT in the cells. The knockdown of AKT by shRNA reduced p-GSK3 beta and CTNNB1 expression in the cells, while the depletion of CTNNB1 impaired stem-like phenotype of the cells. Overall, PIK3R1 down-regulation in RCC promotes propagation, migration, EMT and stem-like phenotype in renal cancer cells through the AKT/GSK3 beta/CTNNB1 pathway, and may contribute to progression and metastasis of RCC.