lipid flippase
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2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesca Mattioli ◽  
Hossein Darvish ◽  
Sohail Aziz Paracha ◽  
Abbas Tafakhori ◽  
Saghar Ghasemi Firouzabadi ◽  
...  

AbstractIntellectual disability (ID) is a highly heterogeneous disorder with hundreds of associated genes. Despite progress in the identification of the genetic causes of ID following the introduction of high-throughput sequencing, about half of affected individuals still remain without a molecular diagnosis. Consanguineous families with affected individuals provide a unique opportunity to identify novel recessive causative genes. In this report, we describe a novel autosomal recessive neurodevelopmental disorder. We identified two consanguineous families with homozygous variants predicted to alter the splicing of ATP9A which encodes a transmembrane lipid flippase of the class II P4-ATPases. The three individuals homozygous for these putatively truncating variants presented with severe ID, motor and speech impairment, and behavioral anomalies. Consistent with a causative role of ATP9A in these patients, a previously described Atp9a−/− mouse model showed behavioral changes.


2021 ◽  
Author(s):  
Meng-Ting Cheng ◽  
Yu Chen ◽  
Zhi-Peng Chen ◽  
Cong-Zhao Zhou ◽  
Wen-Tao Hou ◽  
...  

The human P4-type ATPase ATP8B1 in complex with the auxiliary noncatalytic protein CDC50A or CDC50B mediates the transport of cell membrane lipids from the outer to the inner membrane leaflet, which is crucial to maintain the asymmetry of membrane lipid. Its dysfunction usually leads to imbalance of bile acid circulation, and eventually causing intrahepatic cholestasis diseases. Here we found that both ATP8B1-CDC50A and ATP8B1-CDC50B possess a higher ATPase activity in the presence of the most favored substrate phosphatidylserine (PS); and moreover, the PS-stimulated activity could be augmented upon the addition of bile acids. The cryo-electron microscopy structures of ATP8B1-CDC50A at 3.36 angstrom and ATP8B1-CDC50B at 3.39 angstrom enabled us to capture an unprecedented phosphorylated and autoinhibited state, with the N- and C-terminal tails separately inserting into the cytoplasmic inter-domain clefts of ATP8B1. The PS-bound ATP8B1-CDC50A structure at 3.98 angstrom indicated the autoinhibited state could be released upon PS binding. Structural analysis combined with mutagenesis revealed the residues that determine the substrate specificity, and a unique positively charged loop in the phosphorylated domain of ATP8B1 for the recruitment of bile acids. Altogether, we updated the Post-Albers transport cycle, with an extra autoinhibited state of ATP8B1, which could be activated upon substrate binding. These findings not only provide structural insights into the ATP8B1-mediated restoration of human membrane lipid asymmetry during bile acid circulation, but also advance our understanding on the molecular mechanism of P-type ATPases.


2021 ◽  
Author(s):  
Thibaud Dieudonne ◽  
Sara Abad Herrera ◽  
Michelle Juknaviciute Laursen ◽  
Maylis Lejeune ◽  
Charlott Stock ◽  
...  

P-type ATPases from the P4 subfamily (P4-ATPases) are primary active transporters that maintain lipid asymmetry in eukaryotic cell membranes by flipping lipids from the exoplasmic to the cytosolic leaflet. Mutations in several human P4-ATPase genes are associated with severe diseases. For instance, mutations in the ATP8B1 gene result in progressive familial intrahepatic cholestasis, a rare inherited disorder that usually progresses toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 angstrom resolution. The lipid flippase complex is autoinhibited by the N- and C-termini of ATP8B1, which in concert form extensive interactions with the catalytic sites and flexible domain interfaces of ATP8B1. Consistently, ATP hydrolysis by the ATP8B1-CDC50A complex requires truncation of its C-terminus as well as the presence of phosphoinositides, with a marked preference for phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminus further suggests molecular communication between N- and C-termini in the autoinhibition process and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A conserved (G/A)(Y/F)AFS motif in the C-termini of several P4-ATPase subfamilies suggests that this mechanism is employed widely across P4-ATPase lipid flippases, including both plasma membrane and endomembrane P4-ATPases.


2021 ◽  
Vol 7 (10) ◽  
pp. 843
Author(s):  
Lyubomir Dimitrov Stanchev ◽  
Juliana Rizzo ◽  
Rebecca Peschel ◽  
Lilli A. Pazurek ◽  
Lasse Bredegaard ◽  
...  

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.


2021 ◽  
Author(s):  
NANAKA KANESHIRO ◽  
KOMAI MASATO ◽  
RYOSUKE IMAOKA ◽  
ATSUYA IKEDA ◽  
YUJI KAMIKUBO ◽  
...  

β-amyloid precursor protein (APP) and their metabolites are deeply involved in the development of Alzheimer's disease (AD). Upon the upregulation of β-site APP cleaving enzyme 1 (BACE1), its product, the β-carboxyl-terminal fragment of APP (βCTF), is accumulated in the early stage of sporadic AD brains. βCTF accumulation is currently considered the trigger for endosomal anomalies to form enlarged endosomes, one of the earliest pathologies in AD. However, the details of the underlying mechanism remain largely unclear. In this study, using BACE1 stably-overexpressing cells, we describe that lipid flippase subcomponent TMEM30A interacts with accumulated βCTF. Among the lipid flippases in endosomes, those composed of TMEM30A and active subcomponent ATP8A1 transports phospholipid, phosphatidylserine (PS), to the cytosolic side of the endosomes. The lipid flippase activity and cytosolic PS distribution are critical for membrane fission and vesicle transport. Intriguingly, accumulated βCTF in model cells impaired lipid flippase physiological formation and activity, along with endosome enlargement. Moreover, in the brains of AD model mice before the amyloid-β (Aβ) deposition, the TMEM30A/βCTF complex formation occurred, followed by lipid flippase dysfunction. Importantly, our novel Aβ/βCTF interacting TMEM30A-derived peptide "T-RAP" improved endosome enlargement and reduced βCTF levels. These T-RAP effects could result from the recovery of lipid flippase activity. Therefore, we propose lipid flippase dysfunction as a key pathogenic event and a novel therapeutic target for AD.


2021 ◽  
Author(s):  
Francesca Mattioli ◽  
Hossein Darvish ◽  
Sohail Aziz Paracha ◽  
Abbas Tafakhori ◽  
Saghar Ghasemi Firouzabadi ◽  
...  

Intellectual disability (ID) is a highly heterogeneous disorder with hundreds of associated genes. Despite progress in the identification of the genetic causes of ID following the introduction of high-throughput sequencing, about half of affected individuals still remain without a molecular diagnosis. Consanguineous families with affected individuals provide a unique opportunity to identify novel recessive causative genes. In this report we describe a novel autosomal recessive neurodevelopmental disorder. We identified two consanguineous families with homozygous variants predicted to alter the splicing of ATP9A which encodes a transmembrane lipid flippase of the class II P4-ATPases. The three individuals homozygous for these putatively truncating variants presented with severe ID, motor and speech impairment, and behavioral anomalies. Consistent with a causative role of ATP9A in these patients, a previously described Atp9a-/- mouse model showed behavioral changes.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Danfeng Song ◽  
Haizhan Jiao ◽  
Zhenfeng Liu

AbstractAs a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.


2021 ◽  
Author(s):  
Lin Bai ◽  
Bhawik K. Jain ◽  
Qinglong You ◽  
H. Diessel Duan ◽  
Todd R. Graham ◽  
...  

ABSTRACTP4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory β-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a β-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the first structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1’s ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.


2021 ◽  
Author(s):  
Zhenfeng Liu ◽  
Danfeng Song ◽  
Haizhan Jiao

Abstract As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.


2020 ◽  
Author(s):  
Irene Pazos ◽  
Marta Puig-Tintó ◽  
Jorge Cordero ◽  
Nereida Jiménez-Menéndez ◽  
Marc Abella ◽  
...  

AbstractAtg9 is a transmembrane protein essential for selective autophagy, a pathway that mediates the targeted degradation of cellular components to sustain the cell fitness. To preserve the functionality of this pathway, the cell adjusts the transport of vesicles loaded with Atg9 through mechanisms that are not understood. Here we used live-cell imaging to investigate the interactome that regulates Multisubunit Tethering Complexes (MTCs), a set of conserved protein complexes that control vesicle tethering. We found that P4-ATPases, a family of lipid transporters involved in the biogenesis of vesicles, interact with MTCs that participate in the transport of Atg9, such as TRAPPIII. Using the lipid flippase Drs2, we demonstrated that the I(S/R)TTK motif nested in the N-terminal tail cavity of P4-ATPases is necessary for the interaction with MTCs and to maintain the homeostasis of Atg9. At low temperature, the cell enhances the assembly of the Drs2-TRAPPIII module and Drs2 is fundamental for the early stages of selective autophagy, a function that is independent from its activity as lipid flippase and its role in other vesicle transport pathways.


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