Biochemical Characterization of P4-ATPase Mutations Identified in Patients with Progressive Familial Intrahepatic Cholestasis

Stone, Alex; Chau, Christopher; Eaton, Christian; Foran, Emily; Kapur, Mridu; Prevatt, Edward; Belkin, Nathan; Kerr, David L.; Kohlin, Torvald Kohlin; Williamson, Patrick

doi:10.1074/jbc.m112.413039

Cited by 19 publications

(25 citation statements)

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“…We suggest that an "asparagine clamp," formed by N220 and N550, positions the glycerol backbone and acyl chains in the appropriate restricted orientation for substrate selection. Although this hypothesis is based on substrate transport data and not on binding or crystallography experiments, our data and interpretations are compatible with previous yeast and mammalian computational simulations and molecular studies (37,40,41).…”

Section: Discussionsupporting

confidence: 77%

“…P4-ATPase substrate selection and translocation are coordinated entirely by the TM domain, which is composed of 10 membrane-spanning helices and several short perimembranous loops (36). The yeast P4-ATPases Dnf1, Dnf2, and Drs2 and the mammalian P4-ATPase ATP8A2 use conserved residues within TM segments 1, 2, 3, 4, and 5 to determine substrate selection and transport (36)(37)(38)(39)(40)(41) , have the ability to change PL selection and transport. For example, Dnf1 normally transports PC and PE, but gain-of-function mutations have been isolated that allow Dnf1 to transport PS (36,38,39).…”

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confidence: 99%

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Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

Roland

Graham

2016

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

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Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-offunction mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.sphingomyelin | P4-ATPase | membrane asymmetry | directed evolution T he asymmetry of the membrane bilayer is a fundamental feature of the eukaryotic plasma membrane (1). Phospholipid (PL) species such as phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) dominate the cytofacial leaflet, whereas phosphatidylcholine (PC) and various sphingolipids (SLs) populate the exofacial leaflet (2, 3). Organelle membrane asymmetry has proven more difficult to study, and the precise distribution of PLs is still unclear; however, evidence suggests that the organelles of the secretory and endocytic pathways also exhibit asymmetric membranes. For example, PS is initially enriched in the luminal leaflet of the endoplasmic reticulum (ER) but flips to the cytofacial leaflet at the trans-Golgi network (4). This asymmetric membrane architecture has been demonstrated to affect membrane curvature (5-8), secretory function (6, 9-16), membrane polarization (17), and intra-and intercellular signaling (17)(18)(19)(20).PL flippases in the type IV P-type ATPase family (P4-ATPases) help establish membrane asymmetry by using ATP to transport specific glycerophospholipids (GPLs) from the luminal/exofacial to the cytofacial leaflet of the membrane (21, 22). P4-ATPases have been implicated in a host of diverse diseases such as hepatic cholestasis (23-25), aberrant lymphocyte development (26, 27), anemia (27, 28), hearing loss (29), neurologic disease (30-32), and diabetes (33). Eukaryotic organisms express several P4-ATPases that have different subcellular localizations, tissue specificities, and substrate preferences (34). Understanding the substrate preferences of these enzymes and the physical means of substrate discrimination is important for understanding their role in membrane biology, development, and disease.The P-type ATPase family shares a conserved enzyme architecture that can be separated into four primary domains: the actuator (A), the nucleotide-binding (N), the phosphorylation (P), and the transmembrane (TM) domains (35). P4-ATPase substrate selection a...

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Section: Discussionsupporting

confidence: 77%

mentioning

confidence: 99%

Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

Roland

Graham

2016

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

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“…In human, intrahepatic cholestasis is one of the most common and devastating manifestations in many hereditary and acquired liver diseases, such as progressive familial intrahepatic cholestasis (Diao et al, 2013;Stone et al, 2012), hepatitis (Ji et al, 2014), drug-induced liver injury (Regev, 2014;Regev and Bjornsson, 2014), pregnancy (Bull and Vargas, 2014;Geenes et al, 2014) and primary biliary cirrhosis (PBC) (Wang et al, 2013). Though liver transplantation is appropriate for some patients with cholestasis, therapeutic options for treating intrahepatic cholestasis are limited (Parrilla et al, 2014).…”

Section: Introductionmentioning

confidence: 98%

Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

Meng

Chen

Wang

et al. 2015

Toxicology and Applied Pharmacology

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“…In addition, in these assays, substrate is presented in detergent-solubilized form, making it difficult to interpret effective substrate concentrations. The third assay is to measure internalization of fluorescent phospholipid analogues in living cells [28,29]. This assay permits kinetic measurements and can be used to make measurements of substrate affinity [29], but it has the problem that the effects of cellular factors, including regulation and cellular distribution, may be indistinguishable from effects on enzyme activity itself.…”

Section: P4-atpase Reaction Cyclementioning

confidence: 99%

Substrate trajectory through phospholipid-transporting P4-ATPases

Williamson

2014

Biochemical Society Transactions

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A difference in the lipid composition between the two leaflets of the same membrane is a relatively simple instance of lipid compositional heterogeneity. The large activation energy barrier for transbilayer movement for some (but not all) membrane lipids creates a regime governed by active transport processes. An early step in eukaryote evolution was the development of a capacity for generating transbilayer compositional heterogeneity far from equilibrium by directly tapping energy from the ATP pool. The mechanism of the P-type ATPases that create lipid asymmetry is well understood in terms of ATP hydrolysis, but the trajectory taken by the phospholipid substrate through the enzyme is a matter of current active research. There are currently three different models for this trajectory, all with support by mutation/activity measurements and analogies with known atomic structures.

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Biochemical Characterization of P4-ATPase Mutations Identified in Patients with Progressive Familial Intrahepatic Cholestasis

Cited by 19 publications

References 50 publications

Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

Substrate trajectory through phospholipid-transporting P4-ATPases

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