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.
Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized ...importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.
Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the ...Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.
There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied ...worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.
Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic ...cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.
Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments ...known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3D
enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3D
.
Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to ...life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.
Osh6, a member of the oxysterol-binding protein-related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic ...reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.
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 Å
) 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 ORD8
(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.
The lateral diffusion coefficients of a BODIPY tail-labeled lipid in two model systems, namely, free-standing giant unilamellar vesicles (GUVs) and supported phospholipid bilayers (SPBs), were ...determined by fluorescence correlation spectroscopy (FCS) using the Z-scan approach. For the first time, the performed measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers maintain exactly the same experimental conditions for both systems, which allows for a quantitative comparison of lipid diffusion in these two commonly used model membranes. The results obtained revealed that the lipid mobility in free-standing bilayers (D = 7.8 ± 0.8 μm2 s-1) is significantly higher than in the bilayer created on the solid support (mica) (D = 3.1 ± 0.3 μm2 s-1).
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