The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity

In-situ estimation of ice crystal properties at the South Pole using LED calibration data from the IceCube Neutrino Observatory

The IceCube Neutrino Observatory instruments about 1 km3 of deep, glacial ice at the geographic South Pole using 5160 photomultipliers to detect Cherenkov light emitted by charged relativistic particles. A unexpected light propagation effect observed by the experiment is an anisotropic attenuation, which is aligned with the local flow direction of the ice. Birefringent light propagation has been examined as a possible explanation for this effect. The predictions of a first-principles birefringence model developed for this purpose, in particular curved light trajectories resulting from asymmetric diffusion, provide a qualitatively good match to the main features of the data. This in turn allows us to deduce ice crystal properties. Since the wavelength of the detected light is short compared to the crystal size, these crystal properties do not only include the crystal orientation fabric, but also the average crystal size and shape, as a function of depth. By adding small empirical corrections to this first-principles model, a quantitatively accurate description of the optical properties of the IceCube glacial ice is obtained. In this paper, we present the experimental signature of ice optical anisotropy observed in IceCube LED calibration data, the theory and parametrization of the birefringence effect, the fitting procedures of these parameterizations to experimental data as well as the inferred crystal properties.</p

Understanding the structure and interactions of polycystin-2 through structural and simulation studies

Polycystin-2 (PC2) is a member of the transient receptor potential (TRP) superfamily of non-selective cation channels. It has a systemic distribution with relatively high expression in kidney tubules. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a extracellular/luminal domain (TOP domain) unique to TRPP and TRPML channels. Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD), which is one of the most prevalent genetic disorders in human. Despite that the genetic link between PC2 and ADPKD is well established, the molecular basis of the disease is still elusive. It has been proposed that dysfunction of ciliary PC2 is the main mechanism of pathology of PC2-associated ADPKD. Given the complex organisation of ciliary membrane, it is important to establish how PC2 interacts with specific lipids in its membrane environment. Lipid binding assay results suggest that detergent-solubilised PC2 can bind a range of phosphatidylinositol phosphates but not other simple anionic (phosphatidylserine, phosphatidic acid) or zwitterionic (phosphatidylethanolamine, phosphatidylcholine) lipids. Combining cryo-electron microscopy (cyro-EM) and multiscale molecular dynamics (MD) simulations, we identified a hydrophobic pocket in PC2 formed between S3, S4 transmembrane helices and S4-S5 linker, which showed a preference for phosphatidylinositol bisphosphate (PIP2) binding. Simulations of other members of the TRP channel family suggest this lipid-binding site may be shared amongst a number of TRP channels. In addition, our cryo-EM maps revealed a binding site for cholesterol on PC2, which is further characterised with MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organisation of ciliary membranes. Moreover, we used MD simulations to probe the ion selectivity of PC2. Our results suggest that PC2 is selective for Na+ over Ca2+, and not only the selectivity filter residues but also the TOP domain contribute to the selectivity of the channel.</p

Understanding the structure and interactions of polycystin-2 through structural and simulation studies

Polycystin-2 (PC2) is a member of the transient receptor potential (TRP) superfamily of non-selective cation channels. It has a systemic distribution with relatively high expression in kidney tubules. PC2 shares a common transmembrane fold with other TRP channels, in addition to having a extracellular/luminal domain (TOP domain) unique to TRPP and TRPML channels. Mutations in PC2 are associated with autosomal dominant polycystic kidney disease (ADPKD), which is one of the most prevalent genetic disorders in human. Despite that the genetic link between PC2 and ADPKD is well established, the molecular basis of the disease is still elusive. It has been proposed that dysfunction of ciliary PC2 is the main mechanism of pathology of PC2-associated ADPKD. Given the complex organisation of ciliary membrane, it is important to establish how PC2 interacts with specific lipids in its membrane environment. Lipid binding assay results suggest that detergent-solubilised PC2 can bind a range of phosphatidylinositol phosphates but not other simple anionic (phosphatidylserine, phosphatidic acid) or zwitterionic (phosphatidylethanolamine, phosphatidylcholine) lipids. Combining cryo-electron microscopy (cyro-EM) and multiscale molecular dynamics (MD) simulations, we identified a hydrophobic pocket in PC2 formed between S3, S4 transmembrane helices and S4-S5 linker, which showed a preference for phosphatidylinositol bisphosphate (PIP2) binding. Simulations of other members of the TRP channel family suggest this lipid-binding site may be shared amongst a number of TRP channels. In addition, our cryo-EM maps revealed a binding site for cholesterol on PC2, which is further characterised with MD simulations. These results help to position PC2 within an emerging model of the complex roles of lipids in the regulation and organisation of ciliary membranes. Moreover, we used MD simulations to probe the ion selectivity of PC2. Our results suggest that PC2 is selective for Na+ over Ca2+, and not only the selectivity filter residues but also the TOP domain contribute to the selectivity of the channel.</p

Modeling Flexible Molecules in Solution: A pKa Case Study

Continuum solvation models have been incredibly successful for the computationally efficient study of chemical reactions in solution. However, their development and application has generally been on focused on investigations of small, rigid molecules. Additional factors must be considered when studying large, flexible and multiply ionizable species. These include whether the use of thermocycle or entirely solution-phase approaches are more appropriate for the calculation of solution-phase free energies, which metrics can be used to reliably identify the conformation(s) adopted by flexible molecules in solution, and how errors due to inaccuracies in the prediction of low energy vibrational frequencies can be avoided. Here we explore these issues using the calculation of pKas for a diverse set of amine-containing species as a case study. We show that thermocycle-based approaches should only be applied where there are relatively small structural changes between the gas- and solution-phase molecular geometries, and that these methods are generally not appropriate for conformational searching. Using gas- or solution-phase energies or gas-phase free energies can also lead to errors in the identification of the most stable molecular conformation(s). Scaling of low energy vibrational modes (i.e., use of the quasi-harmonic oscillator approximation) is helpful, however care must be taken to ensure modes that change as part of the reaction are not disregarded. Entirely solution-phase approaches to the Gibbs free energy and hence pKa calculations were found to yield accurate pKa values for the amine test set studied when each charged site is complexed with an explicit water molecule and a proton exchange scheme is applied with an appropriately chosen reference acid.This work was funded via the Australian Research Council Centre of Excellence for Electromaterials Science (CE140100012)

Effects of Apelin on the fibrosis of retinal tissues and Müller cells in diabetes retinopathy through the JAK2/STAT3 signalling pathway

Retinal fibrosis was a key characteristic of diabetes retinopathy (DR). Apelin was found to be a candidate for tissue fibrosis. Nevertheless, the role of Apelin in the Müller cells in DR remains unclear. This study identified the function and mechanism of Apelin in Müller cells and the fibrosis of retinal tissue. Western blot was carried out to detect the Apelin, GFAP, Collagen I, α-SMA, JAK2 and STAT3 protein levels. Masson staining was performed to display the histopathological changes in retinal tissue of diabetic mellitus (DM) rats. The immunofluorescence staining was conducted to evaluate the Apelin levels in the retinal tissue. The levels of GFAP, Collagen I and α-SMA in the retinal tissue of DM rats was visualised by the immunohistochemistry staining. The results showed that Apelin, GFAP, Collagen I andα-SMA expression was prominently elevated in the retinal tissue of DM rats and high glucose (HG)-exposed Müller cells. The results of Masson staining showed that the epiretinal fibrotic membrane was observed in DM rats. Apelin knockdown declined the GFAP, Collagen I andα-SMA levels. Besides, the protein levels of p-JAK2 and p-STAT3 were elevated in the HG-treated Müller cells, while Apelin knockdown declined them. FLLL32 treatment neutralised the role of Apelin. In conclusion, Apelin facilitated the fibrogenic activity of Müller cells through activating the JAK2/STAT3 signalling pathway, and thus inducing the retinal fibrosis in DR

<i>Per1/Per2</i> Disruption Reduces Testosterone Synthesis and Impairs Fertility in Elderly Male Mice

Circadian rhythm disorders caused by genetic or environmental factors lead to decreased male fertility but the mechanisms are poorly understood. The current study reports that the mechanism of Per1/Per2 Double knockout (DKO) reduced the reproductive capacity of elderly male mice. The sperm motility and spermatogenic capacity of male DKO mice were weak. Hormone-targeted metabolomics showed reduced plasma levels of free testosterone in DKO male mice compared with WT male mice. Transcriptomic analysis of testicular tissue showed the down-regulation of testosterone synthesis-related enzymes (Cyp11a1, Cyp17a1, Hsd17b3, Hsd3b1, and Star) in the steroid hormone synthesis pathway. Spermatogenesis genes, Tubd1 and Pafah1b were down-regulated, influencing tubulin dynamics and leading to impaired motility. Seleno-compound metabolic loci, Scly and Sephs2, were up-regulated and Slc7a11 and Selenop were down-regulated. Western-blotting showed that steroid acute regulatory protein (StAR) and p-CREB, PKA and AC1 were reduced in testicular tissue of DKO mice compared to WT. Therefore, Per1/Per2 disruption reduced testosterone synthesis and sperm motility by affecting the PKA-StAR pathway, leading to decreased fertility

Simultaneous efficient removal of oxyfluorfen with electricity generation in a microbial fuel cell and its microbial community analysis

The performance of a microbial fuel cell (MFC) to degrade oxyfluorfen was investigated. Approximately 77% of 50 mg/L oxyfluorfen was degraded within 24 h by anodic biofilm. The temperature, pH, and initial oxyfluorfen concentration had a significant effect on oxyfluorfen degrading, and a maximum degradation rate of 94.95% could theoretically be achieved at 31.96 degrees C, a pH of 7.65, and an initial oxyfluorfen concentration of 120.05 mg/L. Oxyfluorfen was further catabolized through various microbial metabolism pathways. Moreover, the anodic biofilm exhibited multiple catabolic capacities to 4-nitrophenol, chloramphenicol, pyraclostrobin, and sulfamethoxazole. Microbial community analysis indicated that functional bacteria Arcobacter, Acinetobacter, Azospirillum, Azonexus, and Comamonas were the predominant genera in the anodic biofilm. In terms of the efficient removal of various organic compounds and energy recovery, the MFC seemed to be a promising approach for the treatment of environmental contaminants

Modeling Flexible Molecules in Solution: A p<i>K</i>_<i>a</i> Case Study

Continuum solvation models have been incredibly successful for the computationally efficient study of chemical reactions in solution. However, their development and application has generally been on focused on investigations of small, rigid molecules. Additional factors must be considered when studying large, flexible and multiply ionizable species. These include whether the use of thermocycle or entirely solution-phase approaches are more appropriate for the calculation of solution-phase free energies, which metrics can be used to reliably identify the conformation(s) adopted by flexible molecules in solution, and how errors due to inaccuracies in the prediction of low energy vibrational frequencies can be avoided. Here we explore these issues using the calculation of p<i>K</i>_<i>a</i>s for a diverse set of amine-containing species as a case study. We show that thermocycle-based approaches should only be applied where there are relatively small structural changes between the gas- and solution-phase molecular geometries, and that these methods are generally not appropriate for conformational searching. Using gas- or solution-phase energies or gas-phase free energies can also lead to errors in the identification of the most stable molecular conformation(s). Scaling of low energy vibrational modes (i.e., use of the quasi-harmonic oscillator approximation) is helpful, however care must be taken to ensure modes that change as part of the reaction are not disregarded. Entirely solution-phase approaches to the Gibbs free energy and hence p<i>K</i>_<i>a</i> calculations were found to yield accurate p<i>K</i>_<i>a</i> values for the amine test set studied when each charged site is complexed with an explicit water molecule and a proton exchange scheme is applied with an appropriately chosen reference acid