RESUMO
The syndecans represent an ongoing research field focused on their regulatory roles in normal and pathological conditions. The role of syndecans in cancer progression is well documented, implicating their importance in diagnosis and even proposing various potential cancer treatments. Thus, the characterization of the unbinding properties at the single-molecule level will appeal to their use as targets for therapeutics. In our study, syndecan-1 and syndecan-4 were measured during the interaction with the vitronectin HEP II binding site. Our findings show that syndecans are calcium ion dependent molecules that reveal distinct, unbinding properties indicating the alterations in the structure of heparan sulfate (HS) chains, possibly in the chain sequence or sulfation pattern. In this way, we suppose that HS chain affinity to extracellular matrix proteins may govern cancer invasion by altering the syndecans' ability to interact with cancer-related receptors present in the tumor microenvironment, thereby promoting the activation of various signaling cascades regulating tumor cell behavior.
Assuntos
Heparitina Sulfato , Vitronectina , Transdução de Sinais , Análise EspectralRESUMO
The knowledge on how cells interact with microenvironment is particularly important in understanding the interaction of cancer cells with surrounding stroma, which affects cell migration, adhesion, and metastasis. The main cell surface receptors responsible for the interaction with extracellular matrix (ECM) are integrins, however, they are not the only ones. Integrins are accompanied to other molecules such as syndecans. The role of the latter has not yet been fully established. In our study, we would like to answer the question of whether integrins and syndecans, possessing similar functions, share also similar unbinding properties. By using single molecule force spectroscopy (SMFS), we conducted measurements of the unbinding properties of αVß1 and syndecan-4 in the interaction with vitronectin (VN), which, as each ECM protein, possesses two binding sites specific to integrins and syndecans. The unbinding force and the kinetic off rate constant derived from SMFS describe the stability of single molecular complex. Obtained data show one barrier transition for each complex. The proposed model shows that the unbinding of αVß1 from VN proceeds before the unbinding of SDC-4. However, despite different unbinding kinetics, the access to both receptors is needed for cell growth and proliferation.
Assuntos
Integrina alfa5beta1/química , Integrina alfa5beta1/metabolismo , Imagem Individual de Molécula/métodos , Sindecana-4/química , Sindecana-4/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Matriz Extracelular , Humanos , Integrina alfa5beta1/genética , Sindecana-4/genética , Neoplasias da Bexiga Urinária , Vitronectina/metabolismoRESUMO
Detailed knowledge of intramolecular hydrogen bonds, including their nanomechanics, in a peptide secondary structure is crucial for understanding mechanisms of numerous biochemical processes. Single-molecule force spectroscopy has become a powerful tool to study directly the mechanical properties of single biopolymers and monitoring the hydrogen bonds. However, the interpretation of such experiments, due to their poor temporal resolution relative to the rate of intramolecular dynamics, requires the support of molecular simulations. In this work, we provide a methodology for determining the kinetic and energetic characteristics of hydrogen bonds in a template model of the protein secondary structure. Our approach, based on the steered molecular dynamics method, employs dynamic force spectroscopy calculations and uses two advanced theoretical models of force-induced unbinding. A systematic analysis of the simulated data with these models allowed for quantitative characterization of a single hydrogen bond in the α-helix of the AAKA(AEAAKA)5AC peptide model and detailed explanation of the mechanism of the α-helix unfolding. The methodology proposed here may be extended to other molecular structures stabilized by internal hydrogen bonds.
Assuntos
Química/métodos , Ligação de Hidrogênio , Modelos Químicos , Peptídeos/química , Simulação por Computador , Estrutura Secundária de Proteína , Análise EspectralRESUMO
BACKGROUND: Silk is a biocompatible and biodegradable material, able to self-assemble into different morphological structures. Silk structures may be used for many biomedical applications, including carriers for drug delivery. The authors designed a new bioengineered spider silk protein, EMS2, and examined its property as a carrier of chemotherapeutics. MATERIALS AND METHODS: To obtain EMS protein, the MS2 silk monomer (that was based on the MaSp2 spidroin of Nephila clavipes) was modified by the addition of a glutamic acid residue. Both bioengineered silks were produced in an Escherichia coli expression system and purified by thermal method. The silk spheres were produced by mixing with potassium phosphate buffer. The physical properties of the particles were characterized using scanning electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and zeta potential measurements. The MTT assay was used to examine the cytotoxicity of spheres. The loading and release profiles of drugs were studied spectrophotometrically. RESULTS: The bioengineered silk variant, EMS2, was constructed, produced, and purified. The EMS2 silk retained the self-assembly property and formed spheres. The spheres made of EMS2 and MS2 silks were not cytotoxic and had a similar secondary structure content but differed in morphology and zeta potential values; EMS2 particles were more negatively charged than MS2 particles. Independently of the loading method (pre- or post-loading), the loading of drugs into EMS2 spheres was more efficient than the loading into MS2 spheres. The advantageous loading efficiency and release rate made EMS2 spheres a good choice to deliver neutral etoposide (ETP). Despite the high loading efficiency of positively charged mitoxantrone (MTX) into EMS2 particles, the fast release rate made EMS2 unsuitable for the delivery of this drug. A faster release rate from EMS2 particles compared to MS2 particles was observed for positively charged doxorubicin (DOX). CONCLUSION: By modifying its sequence, silk affinity for drugs can be controlled.