Dataset Information

Nanodiscs-based proteomics to identify lipid-interacting proteins in yeast

ABSTRACT: Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids’ poor solubility in aqueous solution. Here, we employ nanodiscs – small, water-soluble patches of lipid bilayer encircled with amphipathic scaffold proteins – along with quantitative proteomics to identify lipid-binding proteins in S. cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids, and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of the nanodisc system to identify and characterize protein-lipid interactions that may not be evident using other methods.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: John Young

LAB HEAD: Franck Duong

PROVIDER: PXD027992 | Pride | 2021-12-30

REPOSITORIES: Pride

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Publications

Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Zhang Xiao X XX Young John William JW Foster Leonard J LJ Duong Franck F

Journal of proteome research 20210914 10

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantit ...[more]

PMID: 34519218

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Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.

Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. Bone marrow-derived macrophages were pooled from four individual WT or TLR4-deficient mice and stimulated with either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 for 2 hours and compared to PBS-stimulated control cells. We also compared PBS-stimulated WT cells directly to PBS-stimulated TLR4-deficient cells to compare the basal expression of genes in the two genotypes. This experiment was repeated once in its entirety.

			Action	DRS
	Caj1_Supp_File_1_June_17_FINAL.xlsx	Xlsx
	Nd-PEPA_rep2.RAW	Raw
	Nd-PE_rep2.RAW	Raw
	Nd-Yeast_rep1.RAW	Raw
	Nd-Yeast_rep2.RAW	Raw