ABSTRACT
AIMS: Ubiquitin specific peptidase 5 (USP5), a member of deubiquitinating enzymes, has garnered significant attention for its crucial role in cancer progression. This study aims to explore the role of USP5 and its potential molecular mechanisms in cholangiocarcinoma (CCA). MAIN METHODS: To explore the effect of USP5 on CCA, gain-of-function and loss-of-function assays were conducted in human CCA cell lines RBE and HCCC9810. The CCK8, colony-forming assay, EDU, flow cytometry, transwell assay and xenografts were used to assess cell proliferation, migration and tumorigenesis. Western blot and immunohistochemistry were performed to measure the expression of related proteins. Immunoprecipitation and immunofluorescence were applied to identify the interaction between USP5 and Y box-binding protein 1 (YBX1). Ubiquitination assays and cycloheximide chase assays were carried out to confirm the effect of USP5 on YBX1. KEY FINDINGS: We found USP5 is highly expressed in CCA tissues, and upregulated USP5 is required for the cancer progression. Knockdown of USP5 inhibited cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, along with suppressed xenograft tumor growth and metastasis in vivo. Mechanistically, USP5 could interact with YBX1 and stabilize YBX1 by deubiquitination in CCA cells. Additionally, silencing of USP5 hindered the phosphorylation of YBX1 at serine 102 and its subsequent translocation to the nucleus. Notably, the effect induced by USP5 overexpression in CCA cells was reversed by YBX1 silencing. SIGNIFICANCE: Our findings reveal that USP5 is required for cell proliferation, migration and EMT in CCA by stabilizing YBX1, suggesting USP5-YBX1 axis as a promising therapeutic target for CCA.
Subject(s)
Bile Duct Neoplasms , Cell Movement , Cell Proliferation , Cholangiocarcinoma , Disease Progression , Epithelial-Mesenchymal Transition , Mice, Nude , Y-Box-Binding Protein 1 , Humans , Cholangiocarcinoma/pathology , Cholangiocarcinoma/genetics , Cholangiocarcinoma/metabolism , Bile Duct Neoplasms/pathology , Bile Duct Neoplasms/metabolism , Bile Duct Neoplasms/genetics , Animals , Mice , Cell Line, Tumor , Y-Box-Binding Protein 1/metabolism , Y-Box-Binding Protein 1/genetics , Ubiquitination , Mice, Inbred BALB C , Male , Endopeptidases/metabolism , Endopeptidases/genetics , Gene Expression Regulation, Neoplastic , FemaleABSTRACT
Sulfonamides are widely used antibiotics in agricultural production. However, the potential threat of these drugs to human health has increased global concern. Human serum albumin (HSA) is the main reservoir and transporter of exogenous small molecules in humans. In this study, the interaction between sulfadimethoxine (SMT) and human serum albumin (HSA) was studied using spectroscopy and computer simulation. Our results showed that the hydrogen bonding and van der Waals forces drove SMT to enter the binding site I of HSA spontaneously and resulted in the fluorescence quenching of HSA. The stability of the HSA-SMT complex decreased with an increase in temperature. The binding of SMT to HSA induced alterations in the secondary structure of HSA, where the content of α-helix decreased from 61.0% of the free state to 59.0% of the compound state. The π-π, π-σ, and π-alkyl interactions between HSA and SMT were found to play important roles in maintaining the stability of the complex.
Subject(s)
Serum Albumin, Human , Sulfadimethoxine , Binding Sites , Circular Dichroism , Computer Simulation , Humans , Molecular Docking Simulation , Protein Binding , Serum Albumin, Human/chemistry , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
Sulfonamides are a kind of antibiotics which have been widely used as feed additives for livestock and poultry. However, sulfa drugs have raised worldwide concerns because of their adverse impact on human health. In this study, two sulfonamides, sulfametoxydiazine (SMD) and sulfamonomethoxine (SMM), were selected to explore the binding modes with human serum albumin (HSA). The spectroscopic approaches revealed that SMD or SMM could spontaneously enter into the binding site I of HSA through hydrogen bond interactions and van der Waals forces, and that SMD exhibited much stronger binding affinity toward HSA than SMM at different temperatures (p < 0.01, n = 3). The binding constants for SMD-HSA and SMM-HSA were determined to be (8.297 ± 0.010) × 104 L·mol-1 and (1.178 ± 0.008) × 104 L·mol-1 at 298 K, respectively. The interaction of SMD or SMM to HSA induced microenvironmental and conformational changes in HSA, where SMD had a greater effect on the α-helix content of HSA. Results from molecular docking implied that the amino acid residues of HSA, such as Arg222, Ala291 and Leu238, played key roles in the sulfonamide-HSA binding process. Meanwhile, hydrogen bonds might be a key factor contributing to the binding affinity of sulfa drugs and HSA. Additionally, the combined use of SMD and SMM led to an obvious variation in Ka values of binary systems (p < 0.01, n = 3). These findings might be helpful to understand the biological effects of sulfonamides in humans.
