An improved protein lipid overlay assay for studying lipid–protein interactions

Han, Xiuli; Yang, Yongqing; Zhao, Fangming; Zhang, Tianren; Yu, Xiang

doi:10.1186/s13007-020-00578-5

Cited by 16 publications

(10 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At the same time, no interaction with other membrane lipids also having two palmitoyl chains was found. We assumed that hydrophobic tails of the adsorbed lipid molecules are mainly anchored to the membrane, and hydrophilic heads were placed on the exposed side of hydrophobic membrane strips [ 18 ]. Primarily, surface electrostatic interactions of cationic plant LTPs were able to take place with the polar heads of lipids.…”

Section: Discussionmentioning

confidence: 99%

Interaction between the Lentil Lipid Transfer Protein Lc-LTP2 and Its Novel Signal Ligand PI(4,5)P2

et al. 2020

View full text Add to dashboard Cite

It is known that plant lipid transfer proteins (LTPs) bind a broad spectrum of ligands including fatty acids (FAs), phospho- and glycolipids, acyl-coenzyme A and secondary metabolites. In this work, we used protein−lipid overlay assays to identify new putative LTP ligands. In our experiments, the lentil lipid transfer protein Lc-LTP2 as well as LTPs from other plants were shown to bind phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Molecular modeling, 2-p-toluidinonaphthalene-6-sulphonate (TNS) displacement and liposome leakage experiments with Lc-LTP2 and its mutant analogs (R45A, Y80A, R45A/Y80A) were performed to investigate interactions between the protein and PI(4,5)P2. It was shown that PI(4,5)P2 initially interacted with the “bottom” entrance of the protein cavity and after complex formation the large polar head of this ligand was also oriented towards the same entrance. We also found that two highly conserved residues in plant LTPs, Arg45 and Tyr80, played an important role in protein-ligand interactions. Apparently, Arg45 is a key residue for interaction with PI(4,5)P2 during both initial contacting and holding in the protein cavity, while Tyr80 is probably a key amino acid playing an essential role in Lc-LTP2 docking to the membrane. Thus, we assumed that the ability of Lc-LTP2 to bind PI(4,5)P2 suggests the involvement of this protein in plant signal transduction.

show abstract

Section: Discussionmentioning

confidence: 99%

Interaction between the Lentil Lipid Transfer Protein Lc-LTP2 and Its Novel Signal Ligand PI(4,5)P2

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Lipid‐binding assays were conducted as described previously (Dowler et al, 2002; Han et al, 2020). PIP Strips (Echelon, USA) were blocked in blocking buffer (1× PBS, 0.1% (v/v) Tween 20, and 3% (w/v) bovine serum albumin) for 1 h. The strips were incubated in fresh blocking buffer for 1 h containing 5 nmol/L of recombinant purified XopAP or XopAP truncated variants.…”

Section: Methodsmentioning

confidence: 99%

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification

Liu

et al. 2022

JIPB

View full text Add to dashboard Cite

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid (ABA) or salicylic acid (SA) and after recognition of pathogen‐associated molecular patterns (PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type III effectors (T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas oryzae pv. oryzicola (Xoc), which is the causal agent of rice bacterial leaf streak disease, drastically increased in transgenic rice (Oryza sativa L.) plants overexpressing the Xoc T3E gene XopAP, which encodes a protein annotated as a lipase. We discovered that XopAP binds to phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P2), a membrane phospholipid that functions in pH control in lysosomes, membrane dynamics, and protein trafficking. XopAP inhibited the acidification of vacuoles by competing with vacuolar H+‐pyrophosphatase (V‐PPase) for binding to PtdIns(3,5)P2, leading to stomatal opening. Transgenic rice overexpressing XopAP also showed inhibition of stomatal closure when challenged by Xoc infection and treatment with the PAMP flg22. Moreover, XopAP suppressed flg22‐induced gene expression, reactive oxygen species burst and callose deposition in host plants, demonstrating that XopAP subverts PAMP‐triggered immunity during Xoc infection. Taken together, these findings demonstrate that XopAP overcomes stomatal immunity in plants by binding to lipids.

show abstract

“…The protocol of protein lipid overlay assay for studying lipid-protein interaction was described previously [46]. Briefly, the DOPC, GlcCer and GalCer were dissolved in MeOH at a final concentration of 10 mg/mL.…”

Section: Protein Lipid Overlay (Plo) Assaymentioning

confidence: 99%

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion

Xia

Hong

et al. 2023

PLoS Pathog

View full text Add to dashboard Cite

Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection.

show abstract

An improved protein lipid overlay assay for studying lipid–protein interactions

Cited by 16 publications

References 41 publications

Interaction between the Lentil Lipid Transfer Protein Lc-LTP2 and Its Novel Signal Ligand PI(4,5)P2

Interaction between the Lentil Lipid Transfer Protein Lc-LTP2 and Its Novel Signal Ligand PI(4,5)P2

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification

Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion

Contact Info

Product

Resources

About