RESUMO
In order to deepen the understanding of the role and regulation mechanisms of prokaryotic global transcription regulators in complex processes, including virulence, the associations between the affinity and binding sequences of Mycobacterium tuberculosis MtrA have been explored extensively. Analysis of MtrA 294 diversified 26 bp binding sequences revealed that the sequence similarity of fragments was not simply associated with affinity. The unique variation patterns of GC content and periodical and sequential fluctuation of affinity contribution curves were observed along the sequence in this study. Furthermore, docking analysis demonstrated that the structure of the dimer MtrA-DNA (high affinity) was generally consistent with other OmpR family members, while Arg 219 and Gly 220 of the wing domain interacted with the minor groove. The results of the binding box replacement experiment proved that box 2 was essential for binding, which implied the differential roles of the two boxes in the binding process. Furthermore, the results of the substitution of the nucleotide at the 20th and/or 21st positions indicated that the affinity was negatively associated with the value of minor groove width precisely at the 21st position. The dimerization of the unphosphorylated MtrA facilitated by a low-affinity DNA fragment was observed for the first time. However, the proportion of the dimer was associated with the affinity of substrate DNA, which further suggested that the affinity was actually one characteristic of the stability of dimers. Based on the finding of 17 inter-molecule hydrogen bonds identified in the interface of the MtrA dimer, including 8 symmetric complementary ones in the conserved α4-ß5-α5 face, we propose that hydrogen bonds should be considered just as important as salt bridges and the hydrophobic patch in the dimerization. Our comprehensive study on a large number of binding fragments with quantitative affinity values provided new insight into the molecular mechanism of dimerization, binding specificity and affinity determination of MtrA and clues for solving the puzzle of how global transcription factors regulate a large quantity of target genes.
RESUMO
Recently, tyrosine kinase inhibitors (TKIs) have been recommended as a first-line treatment for advanced non-small cell lung cancer (NSCLC), significantly improving the treatment outcomes of lung adenocarcinoma patients with the EGFR mutation. However, the application of TKIs for lung squamous cell carcinoma (SCC), the second largest pathological subtype of NSCLC, remains controversial because available data for the EGFR mutation profile and frequency in SCC patients are limited. In this study, 89 bronchoscopic-biopsy specimens from Chinese SCC male patients were assayed for EGFR exon 19 mutation, using improved polymerase chain reaction-denature gel gradient electrophoresis. EGFR exon 19 mutations were detected in 77 of 89 (86.5%) patients, and included six kinds of point mutations (11.6%) and two deletions (Del_747-751 [64.9%] and Del_746-751 [23.3%]). We found that the proportion of mutated EGFR varied from 0.98% to 100% in positive specimens and increased with the development of the disease. The difference of proportion between Stage IV patients and Stage II patients or Stage III patients was significant (P<0.001). These results provided valuable clues to explain the reason why patients harboring the same mutation responded distinctly to TKI treatment. Del_747-751 and Del_746-751 were the dominant mutations in the assayed SCC patients (76.4%), and both belong to the EGFR-TKI-sensitive mutation. Recently research demonstrated that Del_746-751 patients have better response to EGFR-TKI than Del_L747-751 patients. However, our study indicated that majority of SCC patients (55.5%) carried Del_ L747-751. We suggest that the unique clinic features of SCC should be further studied to reveal the mechanism of poorer treatment outcome of EGFR-TKI therapy, and that a better treatment plan and more specific, potent targeted drugs for lung SCC need to be developed.
