Assessment in marine environment for a hypothetic nuclear accident based on the database of tidal harmonic constants

The eleven nuclear power plants in operation, under construction and a well-planned plant in the east coast of China generally use seawater for reactor cooling. In this study, an oceanic dispersion assessment system based on a database of tidal harmonic constants is developed. This system can calculate the tidal current without a large computational cost, and it is possible to calculate real-time predictions of pollu-tant dispersions in the ocean. Calculated amplitudes and phases have maximum errors of 10% and 20%with observations, respectively. A number of hypothetical simulations were performed according to vary-ing of the release starting time and duration of pollutant for the six nuclear sites in China. The developed system requires a computational time of one hour for one month of real-time forecasting in Linux OS. Thus, it can use to evaluate rapidly the dispersion characteristics of the pollutants released into the sea from a nuclear accident.European Union FP7 EURATOM project PREPARE 32328

The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasomemediated degradation of PPAR gamma

Peroxisome proliferator-activated receptor gamma (PPAR??) is a ligand-dependent transcription factor that regulates adipocyte differentiation and glucose homeostasis. The transcriptional activity of PPAR?? is regulated not only by ligands but also by post-translational modifications (PTMs). In this study, we demonstrate that a novel E3 ligase of PPAR??, tripartite motif-containing 25 (TRIM25), directly induced the ubiquitination of PPAR??, leading to its proteasome-dependent degradation. During adipocyte differentiation, both TRIM25 mRNA and protein expression significantly decreased and negatively correlated with the expression of PPAR??. The stable expression of TRIM25 reduced PPAR?? protein levels and suppressed adipocyte differentiation in 3T3-L1 cells. In contrast, the specific knockdown of TRIM25 increased PPAR?? protein levels and stimulated adipocyte differentiation. Furthermore, TRIM25-knockout mouse embryonic fibroblasts (MEFs) exhibited an increased adipocyte differentiation capability compared with wild-type MEFs. Taken together, these data indicate that TRIM25 is a novel E3 ubiquitin ligase of PPAR?? and that TRIM25 is a novel target for PPAR??-associated metabolic diseases

Modulation mechanisms of voltage-gated calcium channels

Voltage-gated calcium (CaV) channels mediate diverse essential physiological functions through membrane depolarization-induced Ca2+ influx. Malfunction of CaV channels causes various pathophysiological disorders. A wide range of cellular signals, including phosphoinositides, auxiliary CaV β subunits, Ca2+/calmodulin, and phosphorylation, are crucial for finely controlling the activity of CaV channels. Various interacting proteins, such as 14-3-3 protein, densin, and stac3, are also important in regulating the surface expression of CaV channels. Here, this review provides recent advances in the regulatory mechanisms of CaV channel gating and trafficking. © 2018 Elsevier LtdFALS

Molecular Mechanism of Voltage-Gated Ca2+ Channel Regulation by Membrane PIP2

Voltage-gated calcium (CaV) channels play essential roles in adjusting calcium influx upon membrane depolarization. CaV2 (N-, P/Q- and R-type) channels are concentrated in the presynaptic nerve terminals and important for the neurotransmitter release. Adjusting the presynaptic calcium channel gating exerts potent influence on synaptic plasticity. CaV channels need auxiliary subunits for proper trafficking to the plasma membrane and the channel gating. Especially CaVβ subunit plays crucial roles in the surface expression of CaV channels and fine-tuning of channel gating. It has been known that CaV channels are modulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2). The binding affinity between ion channel and PIP2 is important for the channel gating normally, but molecular mechanism of PIP2 regulation remains unclear. It was recently reported that subcellular localization of β subunit is a key factor for the control of PIP2 sensitivity of CaV channels. Here we found that the intracellular movement of I-II linker in a1 subunit is important for determining the PIP2 sensitivity of CaV channels. When the I-II linker was shifted to the plasma membrane, current inhibition by PIP2 depletion significantly decreased as like the responses triggered by membrane-tethered β subunit. Consistently we also found that inserting a flexible linker between membrane-tethered Lyn and GK domain of β subunit increased the PIP2 sensitivity of CaV channels. Polybasic motif at the C-terminal end of the I-II linker of CaV channels is a potential PIP2 interaction site. Neutralization of the polybasic motif of I-II linker abolished PIP2 sensitivity of CaV channels. Together, our results indicate that the conformational shift of I-II linker to the plasma membrane is the key mechanism for decreasing the PIP2 sensitivity of CaV channels and this shift is mainly regulated by auxiliary CaVβ subunit in physiological condition.1

The HOOK region of voltage-gated Ca2+ channel beta subunits senses and transmits PIP2 signals to the gate

The beta subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the alpha 1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV beta subunit is critical for this effect; N-terminal-dependent membrane targeting of the beta subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the beta subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a beta subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a beta subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The beta 2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the beta subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane

Molecular basis of the PIP2-dependent regulation of Ca(v)2.2 channel and its modulation by Ca-v beta subunits

High-voltage-activated Ca2+ (CaV) channels that adjust Ca2+ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP2) in an auxiliary CaV β subunit-dependent manner. However, the molecular mechanism by which the β subunits control the PIP2 sensitivity of CaV channels remains unclear. By engineering various α1B and β constructs in tsA-201 cells, we reported that at least two PIP2-binding sites, including the polybasic residues at the C-terminal end of I–II loop and the binding pocket in S4II domain, exist in the CaV2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled CaV β2 subunits. The membrane-anchored β subunit abolished the PIP2 interaction of the phospholipid-binding site in the I–II loop, leading to lower PIP2 sensitivity of CaV2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4II domain of CaV2.2 channels regardless of β2 isotype. Our data demonstrated that the anchoring properties of CaV β2 subunits to the plasma membrane determine the biophysical states of CaV2.2 channels by regulating PIP2 coupling to the nonspecific phospholipid-binding site in the I–II loop. © 2022, eLife Sciences Publications Ltd. All rights reserved.TRU

Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains

Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts. © 2021 National Academy of Sciences. All rights reserved.1

Translocatable voltage-gated Ca2+ channel beta subunits in alpha 1-beta complexes reveal competitive replacement yet no spontaneous dissociation

β subunits of high voltage-gated Ca2+ (CaV) channels promote cellsurface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: Plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells. © 2018 National Academy of Sciences. All Rights Reserved.1