Enguo Fan

Development of a DNA-Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins

Histone post-translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA-templated technology and a photo-crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications.

DNA-Templated Aptamer Probe for Identification of Target Proteins

Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

Reactive oxygen species inhibits Listeria monocytogenes invasion into HepG2 epithelial cells

Abstract Listeria monocytogenes (Lm) can colonize human gastrointestinal tract and subsequently cross the intestinal barrier. Reactive oxygen species (ROS) are produced by NADPH oxidase. However, the role of ROS in bacterial invasion remains to be less understood. Herein, we investigated the impact of ROS on Lm invasion to HepG2 using NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the ROS scavenger, N‐acetyl cysteine (NAC). Our results showed that inhibiting ROS increased the invasive capability of Lm. Moreover, after Lm infection, inflammatory cytokines such as tumor necrosis factor alpha (TNF‐α) and interleukin 1beta (IL‐1β) in HepG2 were significantly upregulated. However, after inhibiting ROS, the expression levels of TNF‐α and IL‐1β were downregulated, indicating a failure of host cells to activate the immune mechanism. Taken together, ROS in Lm might be as a signal for host cells to sense Lm invasion and then stimulate cells to activate the immune mechanism.

Quantitative characterization of histone post-translational modifications using a stable isotope dimethyl-labeling strategy

Histone post-translational modifications (PTMs) have been considered to be a major group of important epigenetic marks and play critical roles in the regulation of chromatin-templated biological processes. To date, novel strategies for the quantification of histone PTMs are still highly desirable. Herein, we present an efficient approach to quantitatively characterize histone PTMs using stable isotope dimethyl labeling coupled with mass spectrometry. At first, all of the e-amino groups of free lysines are derivatized by heavy formaldehyde to enable an easy distinction of free lysines from those of naturally occurring lysine-dimethylation upon MS analysis. After tryptic digestion, a second derivatization was applied with heavy- and light-stable isotope dimethyl labeling to label the N-termini of tryptic peptides from different sample sources. The mixture was further identified and quantified by HPLC-MS/MS. This method enables the comparison of histone PTMs from multiple sample sources and the quantification of different PTMs at certain amino-acid residues of histones in one single experiment. Thus, it is highly attractive for the identification of epigenetic histone marks.