Hao-Jen Hsu

Two-pore channel 2 (TPC2) is a lysosomal cation channel involved in calcium and sodium signaling and membrane remodeling. Its dysfunction has been linked to diseases including viral infections, neurodegenerative disorders, and cancer. Here, we identify mucin-type O-linked glycosylation at two conserved luminal residues of TPC2, Ser612 and Ser613, as a structural gating brake. Combining structure-guided mutagenesis, lysosomal patch-clamp recordings, calcium nanodomain imaging, molecular dynamics simulations, and small-molecule modulation, we show that genetic, enzymatic, or pharmacological glycan removal enhances basal and ligand-evoked TPC2 activity. This hyperactivation induces lysosomal tubulation, increases vesicle mobility, and promotes TPC2 clustering. In cancer-related models, glycan-deficient TPC2 also increases cell migration, which can be reversed by inhibitors such as tetrandrine and SC-3. These findings establish luminal glycosylation as a key post-translational regulator of TPC2 gating and reveal a mechanism by which ion channel activity controls lysosomal architecture and disease-relevant cell behavior.

Human β-defensin-3 (hBD3), an epithelial-derived antimicrobial peptide, was recently identified as a regulator of oral squamous cell carcinoma (OSCC) progression through its interaction with the chemokine receptor CCR7a key mediator of immune cell trafficking and tumor metastasis. In this study, we aimed to clarify the structural and mechanistic basis of hBD3-induced CCR7 activation by employing molecular docking combined with atomic-level molecular dynamics simulations. Binding of hBD3 induced marked conformational rearrangements in CCR7, enhancing β-arrestin-2 recruitment and initiating noncanonical intracellular signaling cascades. Comparative studies with the endogenous CCR7 ligands CCL19 and CCL21 revealed that hBD3 exhibited dual functionality: intact disulfide bonds confer agonistic properties by promoting receptor activation, whereas the disruption of these bonds converts the peptide into an antagonist. Moreover, distinct phosphorylation signatures within the serine/threonine clusters of the C-terminal tail of CCR7 were found to govern downstream signaling outcomes. Collectively, these results reveal a mechanism reliant on both disulfide bond stability and phosphorylation patterns through which hBD3 differentially regulates CCR7 function, providing novel insights into ligand-specific receptor modulation and potential therapeutic strategies for CCR7-mediated malignancies.

Therapeutic peptides have become an intensively anticipated research field for novel drug discovery and design owing to their high specificity, efficacy, and biocompatibility. The advances in computer technology and structural biology, together with the invention of chemical peptide synthesis methods, have led to tremendous progress in this research field. Over the years, more than 100 peptide-based therapeutics have been approved for clinical use, and many others are currently under clinical trials. However, the in vivo application of therapeutic peptides is hindered by intrinsic disadvantages of peptides, such as poor stability against enzymatic degradations, short in vivo half-life, and low oral bioavailability. Therefore, strategies for efficiently protecting the peptides inside the body and facilitating the delivery of peptides to their targets are required. Lipid-based nanoparticles are considered a versatile class of carriers for drug delivery. Their biocompatibility, biodegradability, and ability to interact with biological membranes make them ideal platforms for in vivo delivery of peptides. Here, by leveraging examples, we aim to provide a comprehensive review of the current status of therapeutic peptide developments and lipid-based nanoparticles as drug carriers. Recent attempts to utilize lipid-based nanoparticles as platforms for the oral delivery of therapeutic peptides are also discussed.

Human interleukin-10 (IL-10) is an immunosuppressive and anti-inflammatory cytokine, and its expression is upregulated in tumor tissues and serum samples of patients with various cancers. Because of its immunosuppressive nature, IL-10 has also been suggested to be a factor leading to tumor cells’ evasion of immune surveillance and clearance by the host immune system. In this study, we refined a peptide with 20 amino acids, named NK20a, derived from the binding region of IL-10 on the basis of in silico analysis of the complex structure of IL-10 with IL-10Ra, the ligand binding subunit of the IL-10 receptor. The binding ability of the peptide was confirmed through in vitro biophysical biolayer interferometry and cellular experiments. The IL-10 inhibitory peptide exerted anticancer effects on lymphoma B cells and could abolish the suppression effect of IL-10 on macrophages. NK20a was also conjugated with gold nanoparticles to target the chemotherapeutic 5-fluorouracil (5-FU)-loaded nanoparticles to enhance the anticancer efficacy of 5-FU against the breast cancer cell line BT-474. Our study demonstrated that NK20a designed in silico with improved binding affinity to the IL-10 receptor can be used as a tool in developing anticancer strategies.

Background: 8-hydroxydaidzein (8-OHD) is a compound derived from daidzein, known for its anti-inflammatory and anti-proliferative properties in K562 human chronic myeloid leukemia (CML) cells. However, its effects on acute myeloid leukemia (AML) cells have not been fully understood. Method: To investigate its potential anti-AML mechanism, we employed an integrated in vitro–in silico approach. Results: Our findings demonstrate that 8-OHD suppresses the expression of CDK6 and CCND2 proteins and induces cell apoptosis in U-937 cells by activating Caspase-7 and cleaving PARP-1. Microarray analysis revealed that 8-OHD downregulates differentially expressed genes (DEGs) associated with rRNA processing and ribosome biogenesis pathways. Moreover, AML-target genes, including CCND2, MYC, NPM1, FLT3, and TERT, were downregulated by 8-OHD. Additionally, molecular docking software predicted that 8-OHD has the potential to interact with CDK6, FLT3, and TERT proteins, thereby reducing their activity and inhibiting cell proliferation. Notably, we discovered a synergic pharmacological interaction between 8-OHD and cytarabine (Ara-C). Conclusions: Overall, this study provides insights into the therapeutic applications of 8-OHD in treating AML and elucidates its underlying mechanisms of action.

Interleukin (IL)-8 plays a vital role in regulating inflammation and breast cancer formation by activating CXCR1/2. We previously designed an antagonist peptide, (RF16), to inhibits the activation of downstream signaling pathways by competing with IL-8 in binding to CXCR1/2, thereby inhibiting IL-8-induced chemoattractant monocyte binding. To evaluate the effect of the RF16 peptide on breast cancer progression, triple-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells were used to investigate whether RF16 can inhibit the IL-8-induced breast cancer metastasis. Using growth, proliferation, and invasiveness assays, the results revealed that RF16 reduced cell proliferation, migration, and invasiveness in MDA-MB-231 cells. The RF16 peptide also regulated the protein and mRNA expressions of epithelial–mesenchymal transition (EMT) markers in IL-8-stimulated MDA-MB-231 cells. It also inhibited downstream IL-8 signaling and the IL-8-induced inflammatory response via the mitogen-activated protein kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) pathways. In the xenograft tumor mouse model, RF16 synergistically reinforces the antitumor efficacy of docetaxel by improving mouse survival and retarding tumor growth. Our results indicate that RF16 significantly inhibited IL-8-stimulated cell growth, migration, and invasion in MDA-MB-231 breast cancer cells by blocking the activation of p38 and AKT cascades. It indicated that the RF16 peptide may serve as a new supplementary drug for breast cancer.

The structural analysis of proteins is a major domain of biomedical research. Such analysis requires resolved three-dimensional structures of proteins. Advancements in computer technology have led to progress in biomedical research. In silico prediction and modeling approaches have facilitated the construction of protein structures, with or without structural templates. In this study, we used three neural network-based de novo modeling approaches—AlphaFold2 (AF2), Robetta-RoseTTAFold (Robetta), and transform-restrained Rosetta (trRosetta)—and two template-based tools—the Molecular Operating Environment (MOE) and iterative threading assembly refinement (I-TASSER)—to construct the structure of a viral capsid protein, hepatitis C virus core protein (HCVcp), whose structure have not been fully resolved by laboratory techniques. Templates with sufficient sequence identity for the homology modeling of complete HCVcp are currently unavailable. Therefore, we performed domain-based homology modeling for MOE simulations. The templates for each domain were obtained through sequence-based searches on NCBI and the Protein Data Bank. Then, the modeled domains were assembled to construct the complete structure of HCVcp. The full-length structure and two truncated forms modeled using various computational tools were compared. Molecular dynamics (MD) simulations were performed to refine the structures. The root mean square deviation of backbone atoms, root mean square fluctuation of Cα atoms, and radius of gyration were calculated to monitor structural changes and convergence in the simulations. The model quality was evaluated through ERRAT and phi–psi plot analysis. In terms of the initial prediction for protein modeling, Robetta and trRosetta outperformed AF2. Regarding template-based tools, MOE outperformed I-TASSER. MD simulations resulted in compactly folded protein structures, which were of good quality and theoretically accurate. Thus, the predicted structures of certain proteins must be refined to obtain reliable structural models. MD simulation is a promising tool for this purpose.