Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

Sabapathy, Thiru; Helmerhorst, Erik; Bottomley, Steven; Babaeff, Sharon; Munyard, Kylie; Newsholme, Philip; Mamotte, Cyril

doi:10.1016/j.bbamem.2019.03.021

Cited by 4 publications

(2 citation statements)

References 32 publications

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“…The MAGEA4 protein is able to associate with EVs as well as MLV Gag-induced VLPs in vitro. VLPs serve as an adequate model for EV research [ 53 , 82 ] as they bud from the cellular membrane similar to endogenous EVs but are more homogeneous in nature and can be produced in relatively high quantities [ 83 ]. Rather surprisingly, we discovered that MAGEA4 was able to bind to both vesicles as a result of robust passive incubation.…”

Section: Discussionmentioning

confidence: 99%

MAGEA4 Coated Extracellular Vesicles Are Stable and Can Be Assembled In Vitro

Reinsalu

Samel

Niemeister

et al. 2021

IJMS

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Extracellular vesicles (EVs) are valued candidates for the development of new tools for medical applications. Vesicles carrying melanoma-associated antigen A (MAGEA) proteins, a subfamily of cancer-testis antigens, are particularly promising tools in the fight against cancer. Here, we have studied the biophysical and chemical properties of MAGEA4-EVs and show that they are stable under common storage conditions such as keeping at +4 °C and −80 °C for at least 3 weeks after purification. The MAGEA4-EVs can be freeze-thawed two times without losing MAGEA4 in detectable quantities. The attachment of MAGEA4 to the surface of EVs cannot be disrupted by high salt concentrations or chelators, but the vesicles are sensitive to high pH. The MAGEA4 protein can bind to the surface of EVs in vitro, using robust passive incubation. In addition, EVs can be loaded with recombinant proteins fused to the MAGEA4 open reading frame within the cells and also in vitro. The high stability of MAGEA4-EVs ensures their potential for the development of EV-based anti-cancer applications.

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Section: Discussionmentioning

confidence: 99%

MAGEA4 Coated Extracellular Vesicles Are Stable and Can Be Assembled In Vitro

Reinsalu

Samel

Niemeister

et al. 2021

IJMS

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“…The rapid and reproducible method was able to quantify CXCL12 from urine sample (without any prior treatment) in the physiological range, suggesting the value of using VLPs as the possibility for the rapid diagnosis in autoimmune disorders. Using VLPs as viable membrane model, Sabapathy et al 56 have determined the high affinity binding of insulin receptors (IR) (K D : 2.3 ± 0.4 nM, n = 3) to insulin. The results were similar as obtained from the parental cell (K D : 2.1 ± 0.4 nM, n = 3).…”

Section: ■ Introductionmentioning

confidence: 99%

Direct Binding Methods to Measure Receptor–Ligand Interactions

Ray,

Sayeed,

Ganshert

et al. 2023

J. Phys. Chem. B

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G-protein-coupled receptors (GPCRs) contribute to numerous physiological processes via complex network mechanisms. While indirect signaling assays (Ca 2+ mobilization, cAMP production, and GTP γ S binding) have been useful in identifying and characterizing downstream signaling mechanisms of GPCRs, these methods lack measurements of direct binding affinities, kinetics, binding specificity, and selectivity that are important parameters in GPCR drug discovery. In comparison to existing direct methods that use radio-or fluorescent labels, labelfree techniques can closely emulate the native interactions around binding partners. Surface plasmon resonance (SPR) is a label-free technique that utilizes the refractive index (RI) property and is applied widely in quantitative GPCR−ligand binding kinetics measurement including small molecules screening. However, purified GPCRs are further embedded in a synthetic lipid environment which is immobilized through different tags to the SPR sensor surface, resulting in a non-native environment. Here, we introduced a methodology that also uses the RI property to measure binding interactions in a label-free, immobilization-free arrangement. The free-solution technique is successfully applied in quantifying the interaction of bioactive lipids to cognate lipid GPCRs, which is not purified but rather present in near-native conditions, i.e., in milieu of other cytoplasmic lipids and proteins. To further consider the wide applicability of these free-solution approaches in biomolecular interaction research, additional applications on a variety of receptor−ligand pairs are imperative.

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