LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes.

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL's chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation.

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys34 of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys34 of albumin and homocysteine attached to lysine residues.

Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-ß signaling.

Cardiac hypertrophy and associated heart fibrosis remain a major cause of death worldwide. Phytochemicals have gained attention as alternative therapeutics for managing cardiovascular diseases. These include the extract from the plant Terminalia arjuna, which is a popular cardioprotectant and may prevent or slow progression of pathological hypertrophy to heart failure. Here, we investigated the mode of action of a principal bioactive T. arjuna compound, arjunolic acid (AA), in ameliorating hemodynamic load-induced cardiac fibrosis and identified its intracellular target. Our data revealed that AA significantly represses collagen expression and improves cardiac function during hypertrophy. We found that AA binds to and stabilizes the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) and increases its expression during cardiac hypertrophy. PPARα knockdown during AA treatment in hypertrophy samples, including angiotensin II-treated adult cardiac fibroblasts and renal artery-ligated rat heart, suggests that AA-driven cardioprotection primarily arises from PPARα agonism. Moreover, AA-induced PPARα up-regulation leads to repression of TGF-ß signaling, specifically by inhibiting TGF-ß-activated kinase1 (TAK1) phosphorylation. We observed that PPARα directly interacts with TAK1, predominantly via PPARα N-terminal transactivation domain (AF-1) thereby masking the TAK1 kinase domain. The AA-induced PPARα-bound TAK1 level thereby shows inverse correlation with the phosphorylation level of TAK1 and subsequent reduction in p38 MAPK and NF-κBp65 activation, ultimately culminating in amelioration of excess collagen synthesis in cardiac hypertrophy. In conclusion, our findings unravel the mechanism of AA action in regressing hypertrophy-associated cardiac fibrosis by assigning a role of AA as a PPARα agonist that inactivates non-canonical TGF-ß signaling.

Probing molecular forces in multi-component physiological membranes.

Biological membranes are remarkably heterogeneous, composed of diverse lipid mixtures with distinct chemical structure and composition. By combining molecular dynamics simulations and the newly developed Lipid-Force Distribution Analysis (L-FDA), we explore force transmission in complex multi-component membrane models mimicking eukaryotic organelles. We found that the chemical-moiety based segmentation at membrane interfaces revealed a distinctive distribution of bonded and non-bonded forces in diverse membrane environment. Our molecular stress analysis could have far-reaching implications in describing the relationship between membrane mechanical properties and functional states of chemically distinct lipids.

Selective inhibition of miR-21 by phage display screened peptide.

miRNAs are nodal regulators of gene expression and deregulation of miRNAs is causally associated with different diseases, including cancer. Modulation of miRNA expression is thus of therapeutic importance. Small molecules are currently being explored for their potential to downregulate miRNAs. Peptides have shown to have better potency and selectivity toward their targets but their potential in targeting and modulating miRNAs remain unexplored. Herein, using phage display we found a very selective peptide against pre-miR-21. Interestingly, the peptide has the potential to downregulate miR-21, by binding to pre-miR-21 and hindering Dicer processing. It is selective towards miR-21 inside the cell. By antagonising miR-21 function, the peptide is able to increase the expression of its target proteins and thereby increase apoptosis and suppress cell proliferation, invasion and migration. This peptide can further be explored for its anti-cancer activity in vivo and may be even extended to clinical studies.

Genetic and functional studies implicate synaptic overgrowth and ring gland cAMP/PKA signaling defects in the Drosophila melanogaster neurofibromatosis-1 growth deficiency.

Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3'-5' cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1(st) and 2(nd) chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise to NF1 tumors and that NF1 regulated ALK/RAS/ERK signaling appears conserved in man.

Deciphering structural aspects of reverse cholesterol transport: mapping the knowns and unknowns.

Atherosclerosis is a major contributor to the onset and progression of cardiovascular disease (CVD). Cholesterol-loaded foam cells play a pivotal role in forming atherosclerotic plaques. Induction of cholesterol efflux from these cells may be a promising approach in treating CVD. The reverse cholesterol transport (RCT) pathway delivers cholesteryl ester (CE) packaged in high-density lipoproteins (HDL) from non-hepatic cells to the liver, thereby minimising cholesterol load of peripheral cells. RCT takes place via a well-organised interplay amongst apolipoprotein A1 (ApoA1), lecithin cholesterol acyltransferase (LCAT), ATP binding cassette transporter A1 (ABCA1), scavenger receptor-B1 (SR-B1), and the amount of free cholesterol. Unfortunately, modulation of RCT for treating atherosclerosis has failed in clinical trials owing to our lack of understanding of the relationship between HDL function and RCT. The fate of non-hepatic CEs in HDL is dependent on their access to proteins involved in remodelling and can be regulated at the structural level. An inadequate understanding of this inhibits the design of rational strategies for therapeutic interventions. Herein we extensively review the structure-function relationships that are essential for RCT. We also focus on genetic mutations that disturb the structural stability of proteins involved in RCT, rendering them partially or completely non-functional. Further studies are necessary for understanding the structural aspects of RCT pathway completely, and this review highlights alternative theories and unanswered questions.

Identification of pseudobaptigenin as a novel polyphenol-based multi-target antagonist of different hormone receptors for breast cancer therapeutics.

Breast cancer (BC) is one of the most prevalent cancers in the world and is one of the major reasons for the death of women worldwide. BC is majorly categorized based on the presence or absence of three cell receptors ER, PR and HER2. The latest treatment for BC involves interfering with the production and action of hormones such as estrogen and progesterone. These hormones bind with receptors such as ER and PR and enhance the growth and proliferation of the BC cells. Although the available are effective, the increasing resistance and side effects related to hormonal imbalance are significant and hence there is a need for designing. On the other hand, plant-derivative products have gained a lot of popularity for their promising anti-cancerous activities. Polyphenols are one such group of plant derivatives that have proven to be useful against cancer. In the present study, an in-silico approach was used to search for a polyphenol that can inhibit ER. In this work, a total of 750 polyphenols were taken into consideration. This number was narrowed down to 55, based on their ADMET properties. These 55 polyphenols were then docked to the receptors, ER, PR and HER2. The molecular docking was followed by Molecular Dynamics (MD) simulations. Based on molecular docking and MD simulation results it was concluded that Pseudobaptigenin has the potential to be an inhibitor of ER, PR and HER2.Communicated by Ramaswamy H. Sarma.

Improved model quality assessment using ProQ2.

BACKGROUND: Employing methods to assess the quality of modeled protein structures is now standard practice in bioinformatics. In a broad sense, the techniques can be divided into methods relying on consensus prediction on the one hand, and single-model methods on the other. Consensus methods frequently perform very well when there is a clear consensus, but this is not always the case. In particular, they frequently fail in selecting the best possible model in the hard cases (lacking consensus) or in the easy cases where models are very similar. In contrast, single-model methods do not suffer from these drawbacks and could potentially be applied on any protein of interest to assess quality or as a scoring function for sampling-based refinement. RESULTS: Here, we present a new single-model method, ProQ2, based on ideas from its predecessor, ProQ. ProQ2 is a model quality assessment algorithm that uses support vector machines to predict local as well as global quality of protein models. Improved performance is obtained by combining previously used features with updated structural and predicted features. The most important contribution can be attributed to the use of profile weighting of the residue specific features and the use features averaged over the whole model even though the prediction is still local. CONCLUSIONS: ProQ2 is significantly better than its predecessors at detecting high quality models, improving the sum of Z-scores for the selected first-ranked models by 20% and 32% compared to the second-best single-model method in CASP8 and CASP9, respectively. The absolute quality assessment of the models at both local and global level is also improved. The Pearson's correlation between the correct and local predicted score is improved from 0.59 to 0.70 on CASP8 and from 0.62 to 0.68 on CASP9; for global score to the correct GDT_TS from 0.75 to 0.80 and from 0.77 to 0.80 again compared to the second-best single methods in CASP8 and CASP9, respectively. ProQ2 is available at http://proq2.wallnerlab.org.

Comparative structural and dynamics study of free and gRNA-bound FnCas9 and SpCas9 proteins.

The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulation of free and gRNA bound forms of both the Cas9 proteins was performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches.

Decrypting the mechanistic basis of CRISPR/Cas9 protein.

CRISPR/Cas system, a newly but extensively investigated genome-editing method, harbors practical solutions for various genetic problems. It relies on short guide RNAs (gRNAs) to recruit the Cas9 protein, a DNA cleaving enzyme, to its genomic target DNAs. The Cas9 enzyme exhibits some unique properties, like the ability to differentiate self vs. non-self - DNA strands using the base-pairing potential of crRNA, i.e., only CRISPR DNA is entirely complementary to the CRISPR repeat sequences at the crRNA whereas the presence of mismatches in the upstream region of the spacer permit CRISPR interference which is inhibited in case of CRISPR-DNA, allosteric regulation in its domains, and domain reorientation on sgRNA binding. Several groups have contributed their efforts in understanding the functioning of the CRISPR/Cas system, but even then, there is a lot more to explore in this area. The structural and sequence-based understanding of the whole CRISPR-associated bacterial ortholog family landscape is still ambiguous. A better understanding of the underlying energetics of the CRISPR/Cas9 system should reveal critical parameters to design better CRISPR/Cas9s.

Small molecule quercetin binds MALAT1 triplex and modulates its cellular function.

The triple-helix structure at the 3' end of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA, has been considered to be a target for modulating the oncogenic functions of MALAT1. This study examines the binding of quercetin-a known triplex binding molecule-to the MALAT1 triplex. By employing UV-visible spectroscopy, circular dichroism spectroscopy, and isothermal titration calorimetry, we observed that quercetin binds to the MALAT1 triplex with a stoichiometry of 1:1 and K d of 495 ± 61 nM, along with a negative change in free energy, indicating a spontaneous interaction. Employing real-time PCR measurements, we observed around 50% downregulation of MALAT1 transcript levels in MCF7 cells, and fluorescence in situ hybridization (FISH) experiments showed concomitantly reduced levels of MALAT1 in nuclear speckles. This interaction is likely a result of a direct interaction between the molecule and the RNA, as indicated by a transcription-stop experiment. Further, transcriptome-wide analysis of alternative splicing changes induced by quercetin revealed modulation of MALAT1 downstream genes. Collectively, our study shows that quercetin strongly binds to the MALAT1 triplex and modulates its functions. It can thus be used as a scaffold for further development of therapeutics or as a chemical tool to understand MALAT1 functions.

Comprehensive Assessment of Indian Variations in the Druggable Kinome Landscape Highlights Distinct Insights at the Sequence, Structure and Pharmacogenomic Stratum.

India confines more than 17% of the world's population and has a diverse genetic makeup with several clinically relevant rare mutations belonging to many sub-group which are undervalued in global sequencing datasets like the 1000 Genome data (1KG) containing limited samples for Indian ethnicity. Such databases are critical for the pharmaceutical and drug development industry where diversity plays a crucial role in identifying genetic disposition towards adverse drug reactions. A qualitative and comparative sequence and structural study utilizing variant information present in the recently published, largest curated Indian genome database (IndiGen) and the 1000 Genome data was performed for variants belonging to the kinase coding genes, the second most targeted group of drug targets. The sequence-level analysis identified similarities and differences among different populations based on the nsSNVs and amino acid exchange frequencies whereas a comparative structural analysis of IndiGen variants was performed with pathogenic variants reported in UniProtKB Humsavar data. The influence of these variations on structural features of the protein, such as structural stability, solvent accessibility, hydrophobicity, and the hydrogen-bond network was investigated. In-silico screening of the known drugs to these Indian variation-containing proteins reveals critical differences imparted in the strength of binding due to the variations present in the Indian population. In conclusion, this study constitutes a comprehensive investigation into the understanding of common variations present in the second largest population in the world and investigating its implications in the sequence, structural and pharmacogenomic landscape. The preliminary investigation reported in this paper, supporting the screening and detection of ADRs specific to the Indian population could aid in the development of techniques for pre-clinical and post-market screening of drug-related adverse events in the Indian population.

A High Throughput Lipidomics Method Using Scheduled Multiple Reaction Monitoring.

Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance <30%. We used this method to identify plasma lipids altered due to vitamin B12 deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B12 deficient samples. This method enables rapid screening of a large number of lipid species in a single experiment and would substantially advance our understanding of the role of lipids in biological processes.

Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by connectivity map-based analysis and in vitro studies.

BACKGROUND: Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Herbal medicines have been a source of various antiviral compounds such as Oseltamivir, extracted using shikimic acid from star anise (Illicium verum) and Acyclovir from Carissa edulis are FDA (Food and Drug Administration) approved antiviral drugs. In this study, we dissect the anti-coronavirus infection activity of Cissampelos pareira L (Cipa) extract using an integrative approach. METHODS: We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map ( https://clue.io/ ). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. Molecular docking analyses of constituents of with key targets of SARS-CoV2 protein viz. spike protein, RNA­dependent RNA­polymerase (RdRp) and 3C­like proteinase. was also performed. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. RESULTS: Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g. pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa. Amongst all the tested compounds, Magnoflorine and Salutaridine exhibited the most potent and consistent strong in silico binding profiles with SARS-CoV2 therapeutic targets.

Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

Model quality assessment for membrane proteins.

MOTIVATION: Learning-based model quality assessment programs have been quite successful at discriminating between high- and low-quality protein structures. Here, we show that it is possible to improve this performance significantly by restricting the learning space to a specific context, in this case membrane proteins. Since these are among the most important structures from a pharmaceutical point-of-view, it is particularly interesting to resolve local model quality for regions corresponding, e.g. to binding sites. RESULTS: Our new ProQM method uses a support vector machine with a combination of general and membrane protein-specific features. For the transmembrane region, ProQM clearly outperforms all methods developed for generic proteins, and it does so while maintaining performance for extra-membrane domains; in this region it is only matched by ProQres. The predictor is shown to accurately predict quality both on the global and local level when applied to GPCR models, and clearly outperforms consensus-based scoring. Finally, the combination of ProQM and the Rosetta low-resolution energy function achieve a 7-fold enrichment in selection of near-native structural models, at very limited computational cost. AVAILABILITY: ProQM is available as a server at +proqm.cbr.su.se+.

Elucidating the Structural Features of ABCA1 in its Heterogeneous Membrane Environment.

ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The other theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular "reservoir". This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within the extracellular domains (ECDs) but not in the TMDs, giving it an outward-facing conformation. This structure was hypothesized to substantiate the lateral access theory. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a large heterogeneous lipid environment and found that the protein undergoes several large conformational changes in its extremities. We observed that the cavity enclosed within ATP unbound form of ABCA1 is narrow at the distal ends of TMD as well as the ECD region substantiating the "lateral access" theory. We have also characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.