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An improved protein lipid overlay assay for studying lipid-protein interactions.

ABSTRACT: Background:Lipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues. Results:Herein, we found that blocking proteins may interfere with interactions between lipids and their binding proteins if a separate blocking step is carried out before the incubation step in the PLO assay. To overcome this, we modified the PLO assay by combining an incubation step alongside the blocking step. Verification experiments included phosphatidylinositol-3-phosphate (PI3P) and its commercially available interacting protein G302, C18:1, C18:2, C18:3 and the Arabidopsis plasma membrane H+-ATPase (PM H+-ATPase) AHA2 C-terminus, phosphatidylglycerol (PG) and AtROP6, and phosphatidylserine (PS) and the AHA2 C-terminus. The lipid-protein binding signal in the classical PLO (CPLO) assay was weak and not reproducible, but the modified PLO (MPLO) assay displayed significantly improved sensitivity and reproducibility. Conclusions:This work identified a limitation of the CPLO assay, and both sensitivity and reproducibility were improved in the modified assay, which could prove to be more effective for investigating lipid-protein interactions.

SUBMITTER: Han X

PROVIDER: S-EPMC7060618 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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An improved protein lipid overlay assay for studying lipid-protein interactions.

Han Xiuli X Yang Yongqing Y Zhao Fengyun F Zhang Tianren T Yu Xiang X

Plant methods 20200306

<h4>Background</h4>Lipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues.<h4>Results</h4>Herein, we found that blocking proteins may inte ...[more]

PMID: 32165912

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Project description:A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Project description:IntroductionCardiovascular magnetic resonance (CMR)-guided cardiac catheterization is becoming more widespread due to the ability to acquire both functional CMR measurements and diagnostic catheterization data without exposing patients to ionizing radiation. However, the real-time imaging sequences used for catheter guidance during these procedures are limited in resolution and the anatomical detail they can provide. In this study, we propose a passive catheter tracking approach which simultaneously improves catheter tracking and visualization of the anatomy.Methods60 patients with congenital heart disease underwent CMR-guided cardiac catheterization on a 1.5T CMR scanner (Ingenia, Philips Healthcare, Best the Netherlands) using the Philips iSuite system. The proposed T1-overlay technique uses a commercially available heavily T1-weighted sequence to image the catheter, and overlays it on a high-resolution 3D dataset within iSuite in real-time. Suppressed tissue in the real-time images enables the use of a thick imaging slab to assist in tracking of the catheter. Improvement in catheter visualization time was compared between T1-overlay and the conventional invasive CMR (iCMR) balanced steady state free precession (bSSFP) sequence. This technique also enabled selective angiography visualization for real-time evaluation of blood flow dynamics (such as pulmonary transit time), similar to direct contrast injection under standard fluoroscopy. Estimates of pulmonary transit time using iCMR were validated using x-ray fluoroscopy in 16 patients.ResultsThe T1-overlay approach significantly increased the time that the catheter tip was kept in view by the technologist compared to the bSSFP sequence conventionally used for iCMR. The resulting images received higher ratings for blood/balloon contrast, anatomy visualization, and overall suitability for iCMR guidance by three cardiologists. iCMR selective angiography using T1-overlay also provided accurate estimates of pulmonary transit time that agreed well with x-ray fluoroscopy.ConclusionWe demonstrate a new passive catheter tracking technique using the iSuite platform that improves visualization of the catheter and cardiac anatomy. These improvements significantly increase the time that the catheter tip is seen throughout the procedure. We also demonstrate the feasibility of iCMR selective angiography for the measurement of pulmonary transit time.

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An improved protein lipid overlay assay for studying lipid-protein interactions.

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An improved protein lipid overlay assay for studying lipid-protein interactions.

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