RESUMEN
Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.
Asunto(s)
Carcinoma de Células Escamosas , Diseño de Fármacos , Factor 2 de Crecimiento de Fibroblastos , Neoplasias Pulmonares , Péptidos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Humanos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/metabolismo , Animales , Péptidos/farmacología , Péptidos/química , Péptidos/síntesis química , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Línea Celular Tumoral , Simulación de Dinámica Molecular , Ratones DesnudosRESUMEN
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
Asunto(s)
Adenosina Trifosfato , ADN Helicasas , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Imagen Individual de Molécula , Terminación de la Transcripción Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa II/metabolismo , Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Imagen Individual de Molécula/métodos , ARN Helicasas/metabolismo , ARN Helicasas/genética , Transcripción Genética , ARN de Hongos/metabolismo , ARN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Hongos/genética , HidrólisisRESUMEN
Recent studies have demonstrated the crucial role of cholesterol (Chol) in regulating the mechanical properties and biological functions of cell membranes. Methyl-ß-cyclodextrin (MeßCD) is commonly utilized to modulate the Chol content in cell membranes, but there remains a lack of a comprehensive understanding. In this study, using a range of different techniques, we find that the optimal ratio of MeßCD to Chol for complete removal of Chol from a phosphocholine (PC)/Chol mixed membrane with a 1:1 mol ratio is 4.5:1, while the critical MeßCD-to-Chol ratio for membrane permeation falls within the range between 1.5 and 2.4. MeßCD at elevated concentrations induces the formation of fibrils or tubes from a PC membrane. Single lipid tracking reveals that removing Chol restores the diffusion of lipid molecules in the PC/Chol membrane to levels observed in pure PC membranes. Exposure to 5 mM MeßCD for 30 min effectively eliminates Chol from various cell lines, leading to an up to 8-fold enhancement in melittin cytotoxicity over Hela cells and an up to 3.5-fold augmentation of T cell cytotoxicity against B16F10-OVA cells. This study presents a diagram that delineates the concentration- and time-dependent distribution of MeßCD-induced Chol depletion and membrane deformation, which holds significant potential for modulating the mechanical properties of cellular membranes in prospective biomedical applications.