Homeostasis of cellular membranes is maintained by fine-tuning their lipid composition. Yeast lipid transporter Osh6, belonging to the oxysterol-binding protein-related proteins family, was found to participate in the transport of phosphatidylserine (PS). PS synthesized in the endoplasmic reticulum is delivered to the plasma membrane, where it is exchanged for phosphatidylinositol 4-phosphate (PI4P). PI4P provides the driving force for the directed PS transport against its concentration gradient. In this study, we employed an in vitro approach to reconstitute the transport process into the minimalistic system of large unilamellar vesicles to reveal its fundamental biophysical determinants. Our study draws a comprehensive portrait of the interplay between the structure and dynamics of Osh6, the carried cargo lipid, and the physical properties of the involved membranes, with particular attention to the presence of charged lipids and to membrane fluidity. Specifically, we address the role of the cargo lipid, which, by occupying the transporter, imposes changes in its dynamics and, consequently, predisposes the cargo to disembark in the correct target membrane.

• MeSH
• Biological Transport MeSH
• Cell Membrane * metabolism   MeSH
• Membrane Fluidity MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Phosphatidylserines metabolism   MeSH
• Oxysterol Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Receptors, Steroid metabolism   MeSH
• Unilamellar Liposomes metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphatidylinositol Phosphates MeSH
• Phosphatidylserines MeSH
• phosphatidylinositol 4-phosphate MeSH Browser
• Oxysterol Binding Proteins MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Receptors, Steroid MeSH
• Unilamellar Liposomes MeSH

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.

• Keywords
• OSH6, SAC1, lipid transport, oxysterol-binding protein–related proteins, phosphatidylinositol (4, 5)-bisphosphate, phosphatidylinositol 4-phosphate, phosphatidylserine,
• Publication type
• Journal Article MeSH

Neutral sphingomyelinase 2 (nSMase2) catalyzes the cleavage of sphingomyelin to phosphorylcholine and ceramide, an essential step in the formation and release of exosomes from cells that is critical for intracellular communication. Chronic increase of brain nSMase2 activity and related exosome release have been implicated in various pathological processes, including the progression of Alzheimer's disease (AD), making nSMase2 a viable therapeutic target. Recently, we identified phenyl (R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate 1 (PDDC), the first nSMase2 inhibitor that possesses both favorable pharmacodynamics and pharmacokinetic (PK) parameters, including substantial oral bioavailability, brain penetration, and significant inhibition of exosome release from the brain in vivo. Herein we demonstrate the efficacy of 1 (PDDC) in a mouse model of AD and detail extensive structure-activity relationship (SAR) studies with 70 analogues, unveiling several that exert similar or higher activity against nSMase2 with favorable pharmacokinetic properties.

• MeSH
• Alzheimer Disease drug therapy   pathology   MeSH
• Exosomes metabolism   MeSH
• Enzyme Inhibitors chemistry   metabolism   pharmacology   therapeutic use   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Brain metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Pyridazines chemistry   metabolism   therapeutic use   MeSH
• Sphingomyelin Phosphodiesterase antagonists & inhibitors   metabolism   pharmacology   MeSH
• Body Weight drug effects   MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Enzyme Inhibitors MeSH
• Pyridazines MeSH
• Sphingomyelin Phosphodiesterase MeSH
• SMPD3 protein, human MeSH Browser

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Humans MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Protein Multimerization MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane 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

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

• MeSH
• HIV Infections drug therapy   MeSH
• HIV-1 drug effects   physiology   MeSH
• Anti-HIV Agents chemistry   isolation & purification   pharmacology   MeSH
• Humans MeSH
• Nucleocapsid analysis   drug effects   MeSH
• Viral Core Proteins chemistry   genetics   metabolism   MeSH
• RNA, Viral genetics   MeSH
• High-Throughput Screening Assays MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Virus Uncoating drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-HIV Agents MeSH
• Viral Core Proteins MeSH
• RNA, Viral 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

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.

• Keywords
• M-PMV, RNA packaging, assembly, basic residues, human immunodeficiency virus, infectivity, nucleocapsid, retroviruses, reverse transcription,
• MeSH
• Cell Line MeSH
• Gene Products, gag genetics   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mason-Pfizer monkey virus genetics   physiology   MeSH
• Mutation genetics   MeSH
• Nucleocapsid Proteins genetics   MeSH
• Reverse Transcription genetics   MeSH
• RNA, Viral genetics   MeSH
• Amino Acid Sequence genetics   MeSH
• Virus Assembly genetics   MeSH
• Zinc Fingers genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, gag MeSH
• Nucleocapsid Proteins MeSH
• RNA, Viral 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