Chlorogenic acid, an important active component of coffee with anti-tumor activities, has been found for a hundred years. However, the lack of understanding about its target proteins greatly limits the exploration of its anti-tumor molecular mechanisms and clinical applications. Here, in vitro and animal experiments showed that chlorogenic acid had a significant inhibitory effect on the proliferation of A549 cells. The ability of chlorogenic acid to naturally emit fluorescence was exploited to screen its target proteins while avoiding false positives brought about by chemical modifications when using fluorescent tags. Consequently, we identified and verified annexin A2 as a covalent binding target of chlorogenic acid in A549 cells. We also discovered that chlorogenic acid inhibits the binding of annexin A2 to p50 subunit thereby inhibiting the expression of downstream anti-apoptotic genes cIAP1 and cIAP2 of the NF-κB signaling pathway in A549 cells in vitro and in vivo. Moreover, we found that chlorogenic acid hindered the binding of annexin A2 to actin possibly causing inhibition of tumor cell cycle and migration. Thus, this work demonstrates that chlorogenic acid binds annexin A2, causing a decrease in the expression of NF-κB downstream anti-apoptotic genes, and inhibiting the proliferation of A549 cells in vivo and in vitro.

The autoantibodies profile of Behçet’s disease (BD) is yet incompletely understood. Annexins are a family of highly conserved proteins which are involved in some human autoimmune diseases. Autoantibodies directed toward Annexin A1 and A2 are involved in BD pathology, but correlation in their clinical role is controversial. The aim of our study is to estimate and evaluate the expression correlation between Annexin A1 and A2 autoantibodies in BD patients. We have designed and implemented different technical approaches to prove the hypothesis. First, bioinformatics tools such as amino acid sequence alignment, epitope prediction analysis, and 3D structural comparison were performed to find out the correlation between Annexin A1 and A2. Second, amplification of the corresponding gene by RT-PCR, then cloning, and purification techniques were applied to acquire the recombinant Annexin A1. Third, the target protein band was excised from gel electrophoresis, digested with trypsin, and analyzed by MALDI-TOF/TOF. Finally, in-house ELISA was developed to determine the induced anti-Annexin A1 autoantibodies in BD patients. Obtained results demonstrated that the BD serum reactivity against recombinant Annexin A1 was significantly higher as compared with healthy control (HC) (P<0.001). Moreover, bioassay results of Annexin A1 and A2 also showed the presence, absence, and independent coexistence of autoantibodies, when reacted with BD sera. In conclusion, Annexin A1 has a similar immunogenic expression and correlation with its analog Annexin A2 and their association may be a novel immune target of BD in Han Chinese population.

The aim of this study was to verify whether prohibitin is a novel autoantigen in rheumatoid arthritis. Material and methods: First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified. Then the anti-prohibitin autoantibodies were detected by western blotting by using rhPHB protein to incubate sera from patients with rheumatoid arthritis (RA). Next, immunoprecipitation was employed to further illustrate whether anti-prohibitin antibodies exist in RA patients. And finally, autoantibodies against the rhPHB protein were investigated using a homemade ELISA kit through the assessment of 258 real clinical samples. Results: It was revealed that anti-prohibitin antibodies existed in the sera of patients with RA. Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%). Conclusions: Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients’ blood circulation.

The aim of this study was to verify the hypothesis originated from bioinformatics and literature reviews that hnNRP A1 may be a new immune target of Behçet's disease (BD). First, bioinformatics was used to show the correlation between hnRNP A1 and A2/B1 in amino acid sequences and three dimensional structures. Second, hnRNP A1 was expressed, purified, and immunologically confirmed by systematic immunology methods including: Western blotting, immunoprecipitation and Dot-ELISA. Then, ELISA was used to screen the anti-hnRNP A1 autoantibodies in newly confirmed clinical samples and the clinical significance was compared between anti-hnRNP A1 antibody positive and negative groups. Finally, the endothelial cells antigen profile of one anti-hnRNP A1 antibody positive BD patient was detected using immunoprecipitation with liquid chromatography tandem mass spectrometry (LC-TMS). In total 720 subjects enrolled and tested in this study. Our results demonstrated hnRNP A1 as a new immune target of BD. The reactivity of BD serum IgG antibodies against hnRNP A1 was significantly higher than healthy controls (P<0.0001), and deep vein thrombosis (DVT) showed a significant higher in the anti-hnRNP A1 antibodies positive group (P<0.05).