Computational design and experimental confirmation of a disulfide-stapled YAP helixα1-trap derived from TEAD4 helical hairpin to selectively capture YAP α1-helix with potent antitumor activity.

Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.

Cell-permeable fluorescent indicator for imaging formaldehyde activity in living systems.

Formaldehyde (FA), as a reactive signaling molecule, plays an important role in living systems through a diverse array of cellular pathways. However, no systematic investigation for detection and imaging of FA by rendering cells transiently permeable has been reported yet. Specifically, we developed a new cell-permeable fluorescence probe functionality that was enhanced cellular entry efficiency and well retained intracellularly after activation for visualizing endogenous FA changes. Moreover, a smart "multi-lock system -key-and-lock" strategy，which have provoked a starting point for the use of probe and related biochemical tools to monitor FA in lysosomes. The versatile "latent" fluorophore that can undergo a subsequent self-immolative spacer for interrogating the roles and functions of FA in living systems as well as related biomedical applications.

Construction of synergistic pH/H2O2-responsive prodrug for prolonging blood circulation and accelerating cellular internalization.

We have developed a real-time and multifunctional doxifluridine-conjugate prodrug (LYX), which involved the preliminary methylfluorescein with 5-fluorouracil linker as protecting group, the targeting biotin unit, and a model therapeutic drug (doxifluridine). The shielding group (5'-DFUR) was found to be effective in prolonging circulation at physiological pH 7.4 and improving accumulation in the acidic microenvironment of the tumor. Based on this strategy, the stability and stimulus responsive properties of prodrug could enhance drug release efficiency and exhibit fewer side effects, thereby providing a unique opportunity for diagnosis and imaging additional analytes or enzymatic activities.

Upregulation of CPNE3 suppresses invasion, migration and proliferation of glioblastoma cells through FAK pathway inactivation.

Glioblastoma (GBM) is a deadly brain tumor with a bleak prognosis. In recent years, the copine III (CPNE3) protein was discovered to be associated to metastasis across various types of malignancies. Nevertheless, its function has not been well documented in glioma. This study characterizes CPNE3 expression in GBM along with its impact and underlying molecular mechanism with regards to cellular migration, invasion and proliferation. Immunohistochemistry was used to characterizes CPNE3 expression in the glioma tissues. Then, knockdown of CPNE3 expression was used to analyze the role of CPNE3 in GBM cell viability, migration, invasion. Western blot analysis was performed to measure the protein levels of FAK signaling pathway. We found that GBM tissues had higher CPNE3 expressions as compared to those in normal brain tissues. CPNE3 silencing in GBM cells impaired the migratory, invasive and proliferative abilities of GBM cells that can be attributed to inactivation of the FAK signaling pathway. Collectively, these findings highlight the role of CPNE3 as a new biomarker, offering deeper insights into its carcinogenic role in GBM.

Serine/arginine rich splicing factor 2 expression and clinic pathological features indicating a prognostic factor in human hepatocellular carcinoma patients.

BACKGROUND: This research was aimed to study the expression of Serine/arginine rich splicing factor 2 (SRSF2) in tissues of hepatocellular carcinoma, and explore the relationship between the expression and the clinic pathological and prognosis of human hepatocellular carcinoma (HCC). METHODS: One hundred and fifty-three pairs HCC tissues and adjacent normal tissue were collected from January 2010 to March 2013. The expression of SRSF2 gene was detected by immunohistochemistry, western blotting and real-time quantitative polymerase chain reaction (PCR), and the relationship between the expression and the clinic pathological and prognosis of HCC being analyzed. RESULTS: In 153 cases of hepatocellular carcinoma, SRSF2 was highly expressed in 93 cases, low expression of 60 cases, immunohistochemistry score (6.50 ± 2.82), which was significantly higher than that in adjacent normal tissues (2.94 ± 1.23) (P< 0.05). The expression of SRSF2 in HCC was not associated with gender (χ2= 0.014, P= 0.906), age (χ2= 0.007, P= 0.931), tumor size (χ2= 3.566, P= 0.059) and T stage (χ2= 2.708, P= 0.100), and was significantly correlated with tumor differentiation (χ2= 9.687, P= 0.007), lymph node metastasis (χ2= 4.827, P= 0.028), distal metastasis (χ2= 9.235, P= 0.002), tumor, node, metastasis (TNM) stage (χ2= 3.978, P= 0.046), portal vein invasion and serum alpha-fetoprotein (χ2= 14.919, P= 0.000). The expression of SRSF2 protein in hepatocellular carcinoma was positively correlated (r = 0.704, P< 0.05) with serum alpha-fetoprotein through Pearson analysis. The survival rates of SRSF2 overexpressing hepatocellular carcinoma were 74.19%, 44.09%, 26.88%, 24.73% and 21.51% at 1 year, 2 years, 3 years, 4 years and 5 years respectively, which were lower than those of SRSF2 low expression group (93.33%, 71.67%, 56.67%, 51.67% and 50.00%). CONCLUSION: SRSF2 is highly expressed in hepatocellular carcinoma and its expression increases with the degree of tumor differentiation and TNM staging. It is related to lymph node metastasis and metastasis of tumor cells, and is positively related to serum alpha fetoprotein content, and affects the postoperative survival time of HCC patients.