Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

• Keywords
• Golgi Complex, Membrane Contact Sites, Non-Vesicular Lipid Transport, Phosphatidylinositol, Phospholipase C,
• MeSH
• Cell Membrane metabolism   MeSH
• Endosomes metabolism   MeSH
• Phosphatidylinositols * metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Lipid Metabolism MeSH
• Organelles metabolism   MeSH
• Phospholipid Transfer Proteins * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• ER, ORD, ORP8, PI4P, PS, lipid transport, plasma membrane,
• MeSH
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Lipids * chemistry   MeSH
• Carrier Proteins * metabolism   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipids * MeSH
• Carrier Proteins * MeSH

Phosphorylation by kinases governs many key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Unsurprisingly, tight and precise kinase regulation is a prerequisite for normal cell functioning, whereas kinase dysregulation often leads to disease. Moreover, the functions of many kinases are regulated through protein-protein interactions, which in turn are mediated by phosphorylated motifs and often involve associations with the scaffolding and chaperon protein 14-3-3. Therefore, the aim of this review article is to provide an overview of the state of the art on 14-3-3-mediated kinase regulation, focusing on the most recent mechanistic insights into these important protein-protein interactions and discussing in detail both their structural aspects and functional consequences.

• Keywords
• 14-3-3, ASK1, CaMKK2, LRRK2, PI4KB, PKC, RAF kinase, kinase, phosphorylation,
• MeSH
• Allosteric Regulation genetics   MeSH
• Apoptosis genetics   MeSH
• Phosphorylation genetics   MeSH
• Humans MeSH
• p38 Mitogen-Activated Protein Kinases genetics   MeSH
• Protein Kinases genetics   MeSH
• 14-3-3 Proteins genetics   MeSH
• Signal Transduction genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• p38 Mitogen-Activated Protein Kinases MeSH
• Protein Kinases MeSH
• 14-3-3 Proteins MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• Keywords
• ACBD3, RNA virus, coarse-grained simulations, host factor, intrinsically disordered regions, picornavirus, small-angle X-ray scattering (SAXS),
• MeSH
• Acyl Coenzyme A chemistry   metabolism   MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• Humans MeSH
• Membrane Proteins chemistry   metabolism   MeSH
• Picornaviridae chemistry   metabolism   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Acyl Coenzyme A MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Membrane Proteins MeSH

The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIβ (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.

• Keywords
• Hedgehog signaling, PI4KB, biological activity, inhibitors,
• MeSH
• Cell Line MeSH
• Cilia metabolism   MeSH
• Gene Expression drug effects   MeSH
• Phosphotransferases (Alcohol Group Acceptor) antagonists & inhibitors   genetics   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Morpholines pharmacology   MeSH
• Mice MeSH
• Osteogenesis drug effects   MeSH
• Hedgehog Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Purines pharmacology   MeSH
• Smoothened Receptor genetics   metabolism   MeSH
• RNA Interference MeSH
• Signal Transduction drug effects   MeSH
• Thiophenes chemistry   pharmacology   MeSH
• Minor Histocompatibility Antigens genetics   metabolism   MeSH
• Cell Survival drug effects   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• RNA, Small Interfering MeSH
• Morpholines MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Browser
• Hedgehog Proteins MeSH
• Antineoplastic Agents MeSH
• Purines MeSH
• purmorphamine MeSH Browser
• Smoothened Receptor MeSH
• Thiophenes MeSH
• Minor Histocompatibility Antigens MeSH

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• Keywords
• endosome, phosphoinositide, vesicular traffic,
• MeSH
• 1-Phosphatidylinositol 4-Kinase antagonists & inhibitors   chemistry   metabolism   MeSH
• Biological Transport MeSH
• Chlorocebus aethiops MeSH
• COS Cells MeSH
• Endosomes drug effects   metabolism   MeSH
• Golgi Apparatus drug effects   metabolism   MeSH
• HEK293 Cells MeSH
• Enzyme Inhibitors metabolism   pharmacology   MeSH
• Protein Conformation MeSH
• Humans MeSH
• Drug Evaluation, Preclinical MeSH
• High-Throughput Screening Assays * MeSH
• Molecular Docking Simulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• 1-Phosphatidylinositol 4-Kinase MeSH
• Enzyme Inhibitors MeSH

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.

• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• Intracellular Membranes metabolism   MeSH
• Scattering, Small Angle MeSH
• Membrane Proteins metabolism   MeSH
• Protein Multimerization * MeSH
• 14-3-3 Proteins metabolism   MeSH
• rab GTP-Binding Proteins metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Membrane Proteins MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Browser
• 14-3-3 Proteins MeSH
• rab GTP-Binding Proteins MeSH
• rab11 protein MeSH Browser
• Recombinant Proteins MeSH

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
• Biological Transport MeSH
• Cell Membrane metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Phosphatidylserines metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• HEK293 Cells MeSH
• Rats MeSH
• Humans MeSH
• Protein Domains MeSH
• Oxysterol Binding Proteins MeSH
• Receptors, Steroid chemistry   metabolism   MeSH
• Substrate Specificity MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Phosphatidylinositol Phosphates MeSH
• Phosphatidylserines MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• phosphatidylinositol 4-phosphate MeSH Browser
• Oxysterol Binding Proteins MeSH
• Receptors, Steroid MeSH

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 3Dpol 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 3Dpol.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) genetics   metabolism   MeSH
• Kobuvirus enzymology   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Picornaviridae Infections metabolism   virology   MeSH
• Viral Nonstructural Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Phosphatidylinositol Phosphates MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Membrane Proteins MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Browser
• phosphatidylinositol 4-phosphate MeSH Browser
• Viral Nonstructural Proteins MeSH

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.

• Keywords
• crystal structure, endolysin, histidine tag, lysozyme, phage T4, protein purification,
• MeSH
• Bacteriophage T4 * enzymology   genetics   MeSH
• Chromatography, Affinity methods   MeSH
• Crystallography, X-Ray methods   MeSH
• Muramidase * chemistry   genetics   isolation & purification   MeSH
• Mutation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Muramidase * MeSH