Notes and tips for improving quality of lipid-protein overlay assays

Shirey, Carolyn M.; Scott, Jordan L.; Stahelin, Robert V.

doi:10.1016/j.ab.2016.10.009

Cited by 16 publications

(12 citation statements)

References 10 publications

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“…Lipid overlay assay was carried out using PIP Strips™ (Echelon Biosciences), and the protein–lipid binding experiment was performed according to the manufacturer's instructions . Briefly, the PIP strips were blocked overnight with PBS buffer containing 1% milk and 0.1% Tween‐20 at 4°C.…”

Section: Methodsmentioning

confidence: 99%

A pan‐apicomplexan phosphoinositide‐binding protein acts in malarial microneme exocytosis

et al. 2019

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Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is an essential step in the development of the disease. Consequently, the molecular players involved in host cell invasion represent important targets for inhibitor design and vaccine development. The process of merozoite invasion is a succession of steps underlined by the sequential secretion of the organelles of the apical complex. However, little is known with regard to how their contents are exocytosed. Here, we identify a phosphoinositide‐binding protein conserved in apicomplexan parasites and show that it is important for the attachment and subsequent invasion of the erythrocyte by the merozoite. Critically, removing the protein from its site of action by knock sideways preferentially prevents the secretion of certain types of micronemes. Our results therefore provide evidence for a role of phosphoinositide lipids in the malaria invasion process and provide further insight into the secretion of microneme organelle populations, which is potentially applicable to diverse apicomplexan parasites.

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Section: Methodsmentioning

confidence: 99%

A pan‐apicomplexan phosphoinositide‐binding protein acts in malarial microneme exocytosis

et al. 2019

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“…Vitro. To test whether purified RCD-1 has an affinity for negatively charged lipids, we performed lipid-protein overlay assays using lipid strips (28). We found that RCD-1 showed in vitro affinity for negatively charged phospholipids, notably, cardiolipin (CL) and phosphatidylserine (PS) (Fig.…”

Section: Rcd-1 Interacts With Negatively Charged Phospholipids and Liposomes Inmentioning

confidence: 99%

Molecular characterization of a fungal gasdermin-like protein

Daskalov

Mitchell

Sandstrom

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulatorof cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa. rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1–1/rcd-1–2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1. This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

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“…The predicted binding of PIP 2 to PC2 was tested biochemically using PIP-strips (Shirey et al, 2017), nitrocellulose membranes with lipids including phosphoinositides and other anionic and zwitterionic phospholipids spotted onto their surface (Eshelon Biosciences Ltd). Protein binding to relevant lipids can be detected with antibodies to the tagged protein.…”

Section: Free Energy Landscapes For Pc2/lipid Interactionsmentioning

confidence: 99%

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

Wang

Hedger

Aryal

et al. 2019

Preprint

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Polycystin-2 (PC2) is a member of the TRPP subfamily of TRP channels and is present in ciliary membranes of the kidney. PC2 can be either homo-tetrameric, or heterotetrameric with PC1. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a novel extracellular domain. Several TRP channels have been suggested to be regulated by lipids, including phosphatidylinositol phosphates (PIPs). We have combined molecular dynamics simulations with cryoelectron microscopy to explore possible lipid interactions sites on PC2. We propose that PC2 has a PIP-binding site close to the equivalent vanilloid/lipid-binding site in the TRPV1 channel. A 3.0 Å cryoelectron microscopy map reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are further characterized by MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organization of ciliary membranes.

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Notes and tips for improving quality of lipid-protein overlay assays

Cited by 16 publications

References 10 publications

A pan‐apicomplexan phosphoinositide‐binding protein acts in malarial microneme exocytosis

A pan‐apicomplexan phosphoinositide‐binding protein acts in malarial microneme exocytosis

Molecular characterization of a fungal gasdermin-like protein

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2 (PC2)

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