Structure-Based Understanding of ABCA3 Variants

ABCA3 is a crucial protein of pulmonary surfactant biosynthesis, associated with recessive pulmonary disorders such as neonatal respiratory distress and interstitial lung disease. Mutations are mostly private, and accurate interpretation of variants is mandatory for genetic counseling and patient care. We used 3D structure information to complete the set of available bioinformatics tools dedicated to medical decision. Using the experimental structure of human ABCA4, we modeled at atomic resolution the human ABCA3 3D structure including transmembrane domains (TMDs), nucleotide-binding domains (NBDs), and regulatory domains (RDs) in an ATP-bound conformation. We focused and mapped known pathogenic missense variants on this model. We pinpointed amino-acids within the NBDs, the RDs and within the interfaces between the NBDs and TMDs intracellular helices (IHs), which are predicted to play key roles in the structure and/or the function of the ABCA3 transporter. This theoretical study also highlighted the possible impact of ABCA3 variants in the cytosolic part of the protein, such as the well-known p.Glu292Val and p.Arg288Lys variants.     

PSEN2 Thr421Met Mutation in a Patient with Early Onset Alzheimer’s Disease

Presenilin-2 (PSEN2) mutation Thr421Met was identified from a 57-years old patient with early onset Alzheimer’s disease (EOAD) for the first time in Korea. Previously, this mutation was discovered in an EOAD patient in Japan without a change on amyloid production from the cellular study. Both Korean and Japanese patients developed the disease in their 50s. Memory loss was prominent in both cases, but no additional clinical information was available on the Japanese patient. Magnetic resonance imaging (MRI) images of the Korean patient revealed asymmetric atrophies in both temporo-parietal lobes. In addition, amyloid positron emission tomography (PET) also revealed amyloid deposits in the gray matter of the temporo-parietal lobes asymmetrically. PSEN2 Thr421 was conserved among a majority of vertebrates (such as zebras, elephants, and giant pandas); hence, Thr421 could play an important role in its functions and any mutations could cause detrimental ramifications in its interactions. Interestingly, PSEN2 Thr421 could have homology with PSEN1 Thr440, as PSEN1 T440del mutations were reported from patients with AD or dementia with Lewy bodies. Hence, the changed amino acid from threonine to methionine of PSEN2 Thr421 could cause significant structural alterations in causing local protein dynamics, leading to its pathogenicity in EOAD. Lastly, PSEN2 Thr421Met may interact with other mutations in neurodegenerative disease related genes, which were found in the proband patient, such as ATP binding cassette subfamily A member 7 (ABCA7), Notch Receptor 3 (NOTCH3), or Leucine-rich repeat kinase 2 (LRRK2). These interactions of pathway networks among PSEN2 and other disease risk factors could be responsible for the disease phenotype through other pathways. For example, PSEN2 and ABCA7 may impact amyloid processing and reduce amyloid clearance. Interaction between PSEN2 and NOTCH3 variants may be associated with abnormal NOTCH signaling and a lower degree of neuroprotection. Along with LRRK2 variants, PSEN2 Thr421Met may impact neurodegeneration through Wnt related pathways. In the future, cellular studies of more than one mutation by CRISPR-Cas9 method along with biomarker profiles could be helpful to understand the complicated pathways.     

The structural basis of hyper IgM deficiency - CD40L mutations

X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency characterised by an inability to produce immunoglobulins of the IgG, IgA and IgE isotypes. It is caused by mutations of CD40 ligand (CD40L, CD154), expressed on T-lymphocytes. The interaction of CD40L on T-cells and its receptor CD40 on B-cells is essential for lymphocyte signalling leading to immunoglobulin class switching and B-cell maturation. To understand the structural basis for XHIGM, we utilised bioinformatics methods to analyse all the known CD40L missense mutations at both the sequence and structural level. Our results demonstrate that the 35 different missense mutations have diverse effects on CD40L structure and function, affecting structural disorder and aggregation tendencies, stability maintaining contacts and electrostatic properties. Several mutations also affect residues essential in receptor binding and trimerisation. Experimental study of effects of mutations is laborious and time-consuming and at the structural level often almost impossible. By contrast, precise and useful information about effects of mutations on protein structure and function can readily be obtained by theoretical methods. In this study, all the XHIGM causing missense mutations could be explained in terms of CD40L structure and function. Thus, the molecular basis of the syndrome could be elucidated.     

Role of ABCA7 in Human Health and in Alzheimer’s Disease

Several studies, including genome wide association studies (GWAS), have strongly suggested a central role for the ATP-binding cassette transporter subfamily A member 7 (ABCA7) in Alzheimer’s disease (AD). This ABC transporter is now considered as an important genetic determinant for late onset Alzheimer disease (LOAD) by regulating several molecular processes such as cholesterol metabolism and amyloid processing and clearance. In this review we shed light on these new functions and their cross-talk, explaining its implication in brain functioning, and therefore in AD onset and development.     

Rosmarinic Acid Increases Macrophage Cholesterol Efflux through Regulation of ABCA1 and ABCG1 in Different Mechanisms

Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.     

FTY720 Reduces Lipid Accumulation by Upregulating ABCA1 through Liver X Receptor and Sphingosine Kinase 2 Signaling in Macrophages

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis.     

Ethanolic Extract of Propolis Promotes Reverse Cholesterol Transport and the Expression of ATP-Binding Cassette Transporter A1 and G1 in Mice

The ethanolic extract of propolis (EEP) is beneficial in increasing high density lipoprotein (HDL) cholesterol (HDL-C) and diminishing risks of atherosclerosis. In this study, we examined the effects of EEP on reverse cholesterol transport in mice. ³H -cholesterol laden macrophage was injected intraperitoneally into mice fed by gastric gavage with EEP. Plasma lipid level was determined and ³H-cholesterol was traced in plasma, liver and feces. The effects of EEP on ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) and scavenger receptor BI (SR-BI) in mice liver and in cultured cells were also investigated. EEP administration led to a significant increase in HDL-C and peritoneal macrophage-original ³H-cholesterol in plasma, liver and feces. Liver protein expressions of ABCA1 and ABCG1 were increased but SR-B1 was not. In vitro experiments with HepG2 and Raw264.7 cell lines confirmed the above results. The finding of these studies shows that EEP-enhanced reverse cholesterol transport may have resulted from EEP stimulated plasma HDL level and hepatic ABCA1 and ABCG1 expression.     

Differential Regulation of ABCA1 and Macrophage Cholesterol Efflux by Elaidic and Oleic Acids

Trans fatty acid consumption is associated with an increased risk of coronary heart disease. This increased risk has been attributed to decreased levels of HDL cholesterol and increased levels of LDL cholesterol. However, the mechanism by which trans fatty acid modulates cholesterol transit remains poorly defined. ATP-binding cassette transporter A1 (ABCA1)-mediated macrophage cholesterol efflux is the rate-limiting step initiating apolipoprotein A-I lipidation. In this study, elaidic acid, the most abundant trans fatty acid in partially hydrogenated vegetable oil, was shown to stabilize macrophage ABCA1 protein levels in comparison to that of its cis fatty acid isomer, oleic acid. The mechanism responsible for the disparate effects of oleic and elaidic acid on ABCA1 levels was through accelerated ABCA1 protein degradation in cells treated with oleic acid. In contrast, no apparent differences were observed in ABCA1 mRNA levels, and only minor changes were observed in Liver X receptor/Retinoic X receptor promoter activity in cells treated with elaidic and oleic acid. Efflux of both tracers and cholesterol mass revealed that elaidic acid slightly increased ABCA1-mediated cholesterol efflux, while oleic acid led to decreased ABCA1-mediated efflux. In conclusion, these studies show that cis and trans structural differences in 18 carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation.     

Physicochemical feature-based classification of amino acid mutations

A huge quantity of gene and protein sequences have become available during the post-genomic era, and information about genetic variations, including amino acid substitutions and SNPs, is accumulating rapidly. To understand the effects of these changes, it is often essential to apply bioinformatics tools. Where there is a lack of homologous sequences or a three-dimensional structure, it becomes essential to predict the effects of mutations based solely on protein sequence information. Several computational methods utilizing machine learning techniques have been developed. These predictions generally use the 20-alphabet amino acid code to train the model. With limited available data, the 20-alphabet amino acid features may introduce so many parameters that the model becomes over-fitted. To decrease the number of parameters, we propose a physicochemical feature-based method to forecast the effects of amino acid substitutions on protein stability. Protein structure alterations caused by mutations can be classified as stabilizing or destabilizing. Based on experimental folding-unfolding free energy (DeltaDeltaG) values, we trained a support vector machine with a cleaned data set. The physicochemical properties of the mutated residues, the number of neighboring residues in the primary sequence and the temperature and pH were used as input attributes. Different kernel functions, attributes and window sizes were optimized. An average accuracy of 80% was obtained in cross-validation experiments.     

Cigarette Smoke Affects ABCAl Expression via Liver X Receptor Nuclear Translocation in Human Keratinocytes

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.     

The Role of the ATP-Binding Cassette A1 (ABCA1) in Human Disease

Cholesterol homeostasis is essential in normal physiology of all cells. One of several proteins involved in cholesterol homeostasis is the ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein widely expressed in many tissues. One of its main functions is the efflux of intracellular free cholesterol and phospholipids across the plasma membrane to combine with apolipoproteins, mainly apolipoprotein A-I (Apo A-I), forming nascent high-density lipoprotein-cholesterol (HDL-C) particles, the first step of reverse cholesterol transport (RCT). In addition, ABCA1 regulates cholesterol and phospholipid content in the plasma membrane affecting lipid rafts, microparticle (MP) formation and cell signaling. Thus, it is not surprising that impaired ABCA1 function and altered cholesterol homeostasis may affect many different organs and is involved in the pathophysiology of a broad array of diseases. This review describes evidence obtained from animal models, human studies and genetic variation explaining how ABCA1 is involved in dyslipidemia, coronary heart disease (CHD), type 2 diabetes (T2D), thrombosis, neurological disorders, age-related macular degeneration (AMD), glaucoma, viral infections and in cancer progression.     

Functional Characterization of ABCA4 Missense Variants Linked to Stargardt Macular Degeneration

ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate, N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype–phenotype correlations and evaluating therapeutic treatments for STGD1.     

Analysis of the 3D distribution of stacked self-assembled quantum dots by electron tomography

The 3D distribution of self-assembled stacked quantum dots (QDs) is a key parameter to obtain the highest performance in a variety of optoelectronic devices. In this work, we have measured this distribution in 3D using a combined procedure of needle-shaped specimen preparation and electron tomography. We show that conventional 2D measurements of the distribution of QDs are not reliable, and only 3D analysis allows an accurate correlation between the growth design and the structural characteristics.     

New Developments and Possibilities in Reanalysis and Reinterpretation of Whole Exome Sequencing Datasets for Unsolved Rare Diseases Using Machine Learning Approaches

Rare diseases impact the lives of 300 million people in the world. Rapid advances in bioinformatics and genomic technologies have enabled the discovery of causes of 20–30% of rare diseases. However, most rare diseases have remained as unsolved enigmas to date. Newer tools and availability of high throughput sequencing data have enabled the reanalysis of previously undiagnosed patients. In this review, we have systematically compiled the latest developments in the discovery of the genetic causes of rare diseases using machine learning methods. Importantly, we have detailed methods available to reanalyze existing whole exome sequencing data of unsolved rare diseases. We have identified different reanalysis methodologies to solve problems associated with sequence alterations/mutations, variation re-annotation, protein stability, splice isoform malfunctions and oligogenic analysis. In addition, we give an overview of new developments in the field of rare disease research using whole genome sequencing data and other omics.     

Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα

ATP binding cassette transporter A1 (ABCA1) plays a key role in atherogenesis. Hydrogen sulfide (H₂S), a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H₂S regulates ABCA1 expression. The effect of H₂S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE^−/− mice with a high-cholesterol diet. NaHS (an exogenous H₂S donor) treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H₂S generator cystathionine γ-lyase (CSE) by small RNA interference (siRNA) significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL), diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE^−/− mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα) nuclear translocation. H₂S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H₂S. H₂S may be a promising potential drug candidate for the treatment of atherosclerosis.     

Zinc as an Imaging Biomarker of Prostate Cancer

Zinc has long been the focus of many biological investigations because of its essential role in biology including a catalytic role in many enzymes, a structural role in the many zinc finger proteins, and a physiological role in many secretory cell processes. Divalent zinc is known to be highly abundant in healthy prostate tissues and lower in prostate cancer (PCa). Given the need for newer diagnostic methods for detection of prostate cancer, zinc-responsive probes of various types have been considered as imaging tools for detecting tissue levels of zinc. Among them, recent zinc-responsive MRI probes show great promise for non-invasive detection of zinc ion secretion from the prostate and other tissues in vivo. In this review, we summarize the need for new diagnostic tools and demonstrate how responsive zinc probes and MRI could satisfy this unmet need.     

The Multiverse of Plant Small RNAs: How Can We Explore It?

Plant small RNAs (sRNAs) are a heterogeneous group of noncoding RNAs with a length of 20–24 nucleotides that are widely studied due to their importance as major regulators in various biological processes. sRNAs are divided into two main classes—microRNAs (miRNAs) and small interfering RNAs (siRNAs)—which differ in their biogenesis and functional pathways. Their identification and enrichment with new structural variants would not be possible without the use of various high-throughput sequencing (NGS) techniques, allowing for the detection of the total population of sRNAs in plants. Classifying sRNAs and predicting their functional role based on such high-performance datasets is a nontrivial bioinformatics task, as plants can generate millions of sRNAs from a variety of biosynthetic pathways. Over the years, many computing tools have been developed to meet this challenge. Here, we review more than 35 tools developed specifically for plant sRNAs over the past few years and explore some of their basic algorithms for performing tasks related to predicting, identifying, categorizing, and quantifying individual sRNAs in plant samples, as well as visualizing the results of these analyzes. We believe that this review will be practical for biologists who want to analyze their plant sRNA datasets but are overwhelmed by the number of tools available, thus answering the basic question of how to choose the right one for a particular study.     

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.