Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. 4 Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.     

Structural Analysis of Cholesterol Binding and Sterol Selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.     

Passive Diffusion of Ciprofloxacin and its Metalloantibiotic: A Computational and Experimental study

Fluoroquinolones (FQ) are antibiotics widely used in clinical practise, but the development of bacterial resistance to these drugs is currently a critical public health problem. In this context, ternary copper complexes of FQ (CuFQPhen) have been studied as a potential alternative. In this study, we compared the passive diffusion across the lipid bilayer of one of the most used FQ, ciprofloxacin (Cpx), and its ternary copper complex, CuCpxPhen, that has shown previous promising results regarding antibacterial activity and membrane partition. A combination of spectroscopic studies and molecular dynamics simulations were used and two different model membranes tested: one composed of anionic phospholipids, and the other composed of zwitterionic phospholipids. The obtained results showed a significantly higher membrane permeabilization activity, larger partition, and a more favourable free energy landscape for the permeation of CuCpxPhen across the membrane, when compared to Cpx. Furthermore, the computational results indicated a more favourable translocation of CuCpxPhen across the anionic membrane, when compared to the zwitterionic one, suggesting a higher specificity towards the former. These findings are important to decipher the influx mechanism of CuFQPhen in bacterial cells, which is crucial for the ultimate use of CuFQPhen complexes as an alternative to FQ to tackle multidrug-resistant bacteria.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.     

The SARS-CoV2 envelope differs from host cells,exposes procoagulant lipids,and is disrupted in vivo by oral rinses

The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.     

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization

Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.     

Cholesterol sulfate fluidizes the sterol fraction of the stratum corneum lipid phase and increases its permeability

Desulfation of cholesterol sulfate (CholS) to cholesterol (Chol) is an important event in epidermal homeostasis and necessary for stratum corneum (SC) barrier function. The CholS/Chol ratio decreases during SC maturation but remains high in pathological conditions, such as X-linked ichthyosis, characterized by dry and scaly skin. The aim of this study was to characterize the influence of the CholS/Chol molar ratio on the structure, dynamics, and permeability of SC lipid model mixtures. We synthesized deuterated CholS and investigated lipid models with specifically deuterated components using ²H solid-state NMR spectroscopy at temperatures from 25°C to 80°C. Although the rigid acyl chains in ceramides and fatty acids remained essentially rigid upon variation of the CholS/Chol ratio, both sterols were increasingly fluidized in lipid models containing higher CholS concentrations. We also show the X-ray repeat distance of the lipid lamellar phase (105 Å) and the orthorhombic chain packing of the ceramide’s acyl chains and long free fatty acids did not change upon the variation of the CholS content. However, the Chol phase separation visible in models with high Chol concentration disappeared at the 50:50 CholS/Chol ratio. This increased fluidity resulted in higher permeabilities to model markers of these SC models. These results reveal that a high CholS/Chol ratio fluidizes the sterol fraction and increases the permeability of the SC lipid phase while maintaining the lamellar lipid arrangement with an asymmetric sterol distribution.     

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.     

MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism

The outer membrane (OM) of Gram-negative bacteria acts as a formidable barrier against a plethora of detrimental compounds owing to its asymmetric nature. This is because the OM possesses lipopolysaccharides (LPSs) in the outer leaflet and phospholipids (PLs) in the inner leaflet. The maintenance of lipid asymmetry (Mla) system is involved in preserving the distribution of PLs in OM. The periplasmic component of the system MlaC serves as the substrate-binding protein (SBP) that shuttles PLs between the inner and outer membranes. However, an in-depth report highlighting its mechanism of ligand binding is still lacking. This study reports the crystal structure of MlaC from Escherichia coli (EcMlaC) at a resolution of 2.5 Å in a quasi-open state, complexed with PL. The structural analysis reveals that EcMlaC and orthologs comprise two major domains, viz. nuclear transport factor 2-like (NTF2-like) and phospholipid-binding protein (PBP). Each domain can be further divided into two subdomains arranged in a discontinuous fashion. This study further reveals that EcMlaC is polyspecific in nature and follows a reverse mechanism of the opening of the substrate-binding site during the ligand binding. Furthermore, MlaC can bind two PLs by forming subsites in the binding pocket. These findings, altogether, have led to the proposition of the unique “segmented domain movement” mechanism of PL binding, not reported for any known SBP to date. Further, unlike typical SBPs, MlaC has originated from a cystatin-like fold. Overall, this study establishes MlaC to be a non-canonical SBP with a unique ligand-binding mechanism.     

Impact on S. aureus and E. coli Membranes of Treatment with Chlorhexidine and Alcohol Solutions: Insights from Molecular Simulations and Nuclear Magnetic Resonance

Membranes form the first line of defence of bacteria against potentially harmful molecules in the surrounding environment. Understanding the protective properties of these membranes represents an important step towards development of targeted anti-bacterial agents such as sanitizers. Use of propanol, isopropanol and chlorhexidine can significantly decrease the threat imposed by bacteria in the face of growing anti-bacterial resistance via mechanisms that include membrane disruption. Here we have employed molecular dynamics simulations and nuclear magnetic resonance to explore the impact of chlorhexidine and alcohol on the S. aureus cell membrane, as well as the E. coli inner and outer membranes. We identify how sanitizer components partition into these bacterial membranes, and show that chlorhexidine is instrumental in this process.     

Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

Fusarium oxysporum is one of the most abundant and diverse fungal species found in soils and includes nonpathogenic, endophytic, and pathogenic strains affecting a broad range of plant and animal hosts. Conidiation is the major mode of reproduction in many filamentous fungi, but the regulation of this process is largely unknown. Lysine acetylation (Kac) is an evolutionarily conserved and widespread posttranslational modification implicated in regulation of multiple metabolic processes. A total of 62 upregulated and 49 downregulated Kac proteins were identified in sporulating mycelia versus nonsporulating mycelia of F. oxysporum. Diverse cellular proteins, including glycolytic enzymes, ribosomal proteins, and endoplasmic reticulum–resident molecular chaperones, were differentially acetylated in the sporulation process. Altered Kac levels of three endoplasmic reticulum–resident molecular chaperones, PDI^K70, HSP70^K604, and HSP40^K32 were identified that with important roles in F. oxysporum conidiation. Specifically, K70 acetylation (K70ac) was found to be crucial for maintaining stability and activity of protein disulphide isomerase and the K604ac of HSP70 and K32ac of HSP40 suppressed the detoxification ability of these heat shock proteins, resulting in higher levels of protein aggregation. During conidial formation, an increased level of PDI^K70ac and decreased levels of HSP70^K604ac and HSP40^K32ac contributed to the proper processing of unfolded proteins and eliminated protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation in F. oxysporum.     

Differential gene expression associated with flower development of mango (Mangifera indica L.) varieties with different shelf-life

Mango (Mangifera indica L.) is one of the most important commercial fruit crop grown in many parts of the world. Major challenges affecting mango trade are short shelf-life, high susceptibility to chilling injury, post-harvest diseases and consumer demand for improved fruit quality. The objective of the present study was to reveal the key regulators present in bud and flower tissues during flower development stage, associated with fruit development and affect the shelf-life of the mango fruit. RNA-sequencing of contrasting genotypes having short and long shelf-life, was carried out. Comparative differential expression pathway studies of long shelf-life (Totapuri) and short shelf-life (Bombay Green) mango genotypes revealed a total of 177 highly differentially expressed genes. Out of 177 total genes, 101 genes from endoplasmic reticulum pathway and very few from gibberellins (3) and jasmonic acid (1) pathway were identified. Genes from endoplasmic reticulum pathway like hsp 90, SRC2, DFRA, CHS, BG3 and ASPG1 mainly up regulated in Bombay Green. Uniprotein B9R8D3 also shows up regulation in Bombay Green. Ethylene insensitive pathway gene EIL1 up regulated in Bombay Green. Gene CAD1 from phenylpropanoid pathway mainly up regulated in Bombay Green. A total of 4 SSRs and 227 SNPs were mined from these pathways specific to the shelf-life. Molecular studies of endoplasmic reticulum, phenylpropanoid, ethylene, polygalacturonase and hormone pathways at the time of bud and flower formation revealed key regulators that determine the shelf-life of mango fruit.     

Structure of the Human Cholesterol Transporter ABCG1

ABCG1 is an ATP binding cassette (ABC) transporter that removes excess cholesterol from peripheral tissues. Despite its role in preventing lipid accumulation and the development of cardiovascular and metabolic disease, the mechanism underpinning ABCG1-mediated cholesterol transport is unknown. Here we report a cryo-EM structure of human ABCG1 at 4 Å resolution in an inward-open state, featuring sterol-like density in the binding cavity. Structural comparison with the multidrug transporter ABCG2 and the sterol transporter ABCG5/G8 reveals the basis of mechanistic differences and distinct substrate specificity. Benzamil and taurocholate inhibited the ATPase activity of liposome-reconstituted ABCG1, whereas the ABCG2 inhibitor Ko143 did not. Based on the structural insights into ABCG1, we propose a mechanism for ABCG1-mediated cholesterol transport.     

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.     

Molecular dynamics and simulation analysis against superoxide dismutase (SOD) target of Micrococcus luteus with secondary metabolites from Bacillus licheniformis recognized by genome mining approach

Micrococcus luteus, also known as M. luteus, is a bacterium that inhabits mucous membranes, human skin, and various environmental sources. It is commonly linked to infections, especially among individuals who have compromised immune systems. M. luteus is capable of synthesizing the enzyme superoxide dismutase (SOD) as a component of its protective response to reactive oxygen species (ROS). This enzyme serves as a promising target for drug development in various diseases. The current study utilized a subtractive genomics approach to identify potential therapeutic targets from M. luteus. Additionally, genome mining was employed to identify and characterize the biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites in Bacillus licheniformis (B. licheniformis), a bacterium known for its production of therapeutically relevant secondary metabolites. Subtractive genomics resulted in identification of important extracellular protein SOD as a drug target that plays a crucial role in shielding cells from damage caused by ROS. Genome mining resulted in identification of five potential ligands (secondary metabolites) from B. licheniformis such as, Bacillibactin (BAC), Paenibactin (PAE), Fengycin (FEN), Surfactin (SUR) and Lichenysin (LIC). Molecular docking was used to predict and analyze the binding interactions between these five ligands and target protein SOD. The resulting protein–ligand complexes were further analyzed for their motions and interactions of atoms and molecules over 250 ns using molecular dynamics (MD) simulation analysis. The analysis of MD simulations suggests, Bacillibactin as the probable candidate to arrest the activities of SOD. All the five compounds reported in this study were found to act by directly/indirectly interacting with ROS molecules, such as superoxide radicals (O₂–) and hydrogen peroxide (H₂O₂), and transforming them into less reactive species. This antioxidant activity contributes to its protective effects against oxidative stress-induced damage in cells making them likely candidate for various applications, including in the development of antioxidant-based therapies, nutraceuticals, and functional foods.     

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.     

2-Hydroxypropyl-β-cyclodextrin mitigates pathological changes in a mouse model of retinal cholesterol dyshomeostasis

CYP46A1 is a CNS-specific enzyme, which eliminates cholesterol from the brain and retina by metabolism to 24-hydroxycholesterol, thus contributing to cholesterol homeostasis in both organs. 2-Hydroxypropyl-β-cyclodextrin (HPCD), a Food and Drug Administration-approved formulation vehicle, is currently being investigated off-label for treatment of various diseases, including retinal diseases. HPCD was shown to lower retinal cholesterol content in mice but had not yet been evaluated for its therapeutic benefits. Herein, we put Cyp46a1^?/? mice on high fat cholesterol-enriched diet from 1 to 14 months of age (control group) and at 12 months of age, started to treat a group of these animals with HPCD until the age of 14 months. We found that as compared with mature and regular chow-fed Cyp46a1^?/? mice, control group had about 6-fold increase in the retinal total cholesterol content, focal cholesterol and lipid deposition in the photoreceptor-Bruch’s membrane region, and retinal macrophage activation. In addition, aged animals had cholesterol crystals at the photoreceptor-retinal pigment epithelium interface and changes in the Bruch’s membrane ultrastructure. HPCD treatment mitigated all these manifestations of retinal cholesterol dyshomeostasis and altered the abundance of six groups of proteins (genetic information transfer, vesicular transport, and cytoskeletal organization, endocytosis and lysosomal processing, unfolded protein removal, lipid homeostasis, and Wnt signaling). Thus, aged Cyp46a1^?/? mice on high fat cholesterol-enriched diet revealed pathological changes secondary to retinal cholesterol overload and supported further studies of HPCD as a potential therapeutic for age-related macular degeneration and diabetic retinopathy associated with retinal cholesterol dyshomeostasis.