ORP8 Dotaz Zobrazit nápovědu
ORPs are lipid-transport proteins belonging to the oxysterol-binding protein family. They facilitate the transfer of lipids between different intracellular membranes, such as the ER and plasma membrane. We have solved the crystal structure of the ORP8 lipid transport domain (ORD8). The ORD8 exhibited a β-barrel fold composed of anti-parallel β-strands, with three α-helices replacing β-strands on one side. This mixed alpha-beta structure was consistent with previously solved structures of ORP2 and ORP3. A large cavity (≈1860 Å3) within the barrel was identified as the lipid-binding site. Although we were not able to obtain a lipid-bound structure, we used computer simulations based on our crystal structure to dock PS and PI4P molecules into the putative lipid-binding site of the ORD8. Comparative experiments between the short ORD8ΔLid (used for crystallography) and the full-length ORD8 (lid containing) revealed the lid's importance for stable lipid binding. Fluorescence assays revealed different transport efficiencies for PS and PI4P, with the lid slowing down transport and stabilizing cargo. Coarse-grained simulations highlighted surface-exposed regions and hydrophobic interactions facilitating lipid bilayer insertion. These findings enhance our comprehension of ORD8, its structure, and lipid transport mechanisms, as well as provide a structural basis for the design of potential inhibitors.
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.
- MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fosfatidylinositolfosfáty metabolismus MeSH
- fosfatidylseriny metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus MeSH
- HEK293 buňky MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- proteinové domény MeSH
- steroidní receptory chemie metabolismus MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
