In Silico Study of Rett Syndrome Treatment-Related Genes, MECP2, CDKL5, and FOXG1, by Evolutionary Classification and Disordered Region Assessment

Rett syndrome (RTT), a neurodevelopmental disorder, is mainly caused by mutations in methyl CpG-binding protein 2 (MECP2), which has multiple functions such as binding to methylated DNA or interacting with a transcriptional co-repressor complex. It has been established that alterations in cyclin-dependent kinase-like 5 (CDKL5) or forkhead box protein G1 (FOXG1) correspond to distinct neurodevelopmental disorders, given that a series of studies have indicated that RTT is also caused by alterations in either one of these genes. We investigated the evolution and molecular features of MeCP2, CDKL5, and FOXG1 and their binding partners using phylogenetic profiling to gain a better understanding of their similarities. We also predicted the structural order–disorder propensity and assessed the evolutionary rates per site of MeCP2, CDKL5, and FOXG1 to investigate the relationships between disordered structure and other related properties with RTT. Here, we provide insight to the structural characteristics, evolution and interaction landscapes of those three proteins. We also uncovered the disordered structure properties and evolution of those proteins which may provide valuable information for the development of therapeutic strategies of RTT

Identification of endogenous small peptides involved in rice immunity through transcriptomics- and proteomics-based screening

Small signalling peptides, generated from larger protein precursors, are important components to orchestrate various plant processes such as development and immune responses. However, small signalling peptides involved in plant immunity remain largely unknown. Here, we developed a pipeline using transcriptomics- and proteomics-based screening to identify putative precursors of small signalling peptides: small secreted proteins (SSPs) in rice, induced by rice blast fungus Magnaporthe oryzae and its elicitor, chitin. We identified 236 SSPs including members of two known small signalling peptide families, namely rapid alkalinization factors and phytosulfokines, as well as many other protein families that are known to be involved in immunity, such as proteinase inhibitors and pathogenesis-related protein families. We also isolated 52 unannotated SSPs and among them, we found one gene which we named immune response peptide (IRP) that appeared to encode the precursor of a small signalling peptide regulating rice immunity. In rice suspension cells, the expression of IRP was induced by bacterial peptidoglycan and fungal chitin. Overexpression of IRP enhanced the expression of a defence gene, PAL1 and induced the activation of the MAPKs in rice suspension cells. Moreover, the IRP protein level increased in suspension cell medium after chitin treatment. Collectively, we established a simple and efficient pipeline to discover SSP candidates that probably play important roles in rice immunity and identified 52 unannotated SSPs that may be useful for further elucidation of rice immunity. Our method can be applied to identify SSPs that are involved not only in immunity but also in other plant functions

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication

Dynamic Regulation of Myosin Light Chain Phosphorylation by Rho-kinase

Myosin light chain (MLC) phosphorylation plays important roles in various cellular functions such as cellular morphogenesis, motility, and smooth muscle contraction. MLC phosphorylation is determined by the balance between activities of Rho-associated kinase (Rho-kinase) and myosin phosphatase. An impaired balance between Rho-kinase and myosin phosphatase activities induces the abnormal sustained phosphorylation of MLC, which contributes to the pathogenesis of certain vascular diseases, such as vasospasm and hypertension. However, the dynamic principle of the system underlying the regulation of MLC phosphorylation remains to be clarified. Here, to elucidate this dynamic principle whereby Rho-kinase regulates MLC phosphorylation, we developed a mathematical model based on the behavior of thrombin-dependent MLC phosphorylation, which is regulated by the Rho-kinase signaling network. Through analyzing our mathematical model, we predict that MLC phosphorylation and myosin phosphatase activity exhibit bistability, and that a novel signaling pathway leading to the auto-activation of myosin phosphatase is required for the regulatory system of MLC phosphorylation. In addition, on the basis of experimental data, we propose that the auto-activation pathway of myosin phosphatase occurs in vivo. These results indicate that bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to this feature of bistability

Molecular Mechanisms Regulating Vascular Endothelial Permeability

Vascular endothelial cells form a monolayer in the vascular lumen and act as a selective barrier to control the permeability between blood and tissues. To maintain homeostasis, the endothelial barrier function must be strictly integrated. During acute inflammation, vascular permeability temporarily increases, allowing intravascular fluid, cells, and other components to permeate tissues. Moreover, it has been suggested that the dysregulation of endothelial cell permeability may cause several diseases, including edema, cancer, and atherosclerosis. Here, we reviewed the molecular mechanisms by which endothelial cells regulate the barrier function and physiological permeability

CRMP-2 Is Involved in Kinesin-1-Dependent Transport of the Sra-1/WAVE1 Complex and Axon Formation

A neuron has two types of highly polarized cell processes, the single axon and multiple dendrites. One of the fundamental questions of neurobiology is how neurons acquire such specific and polarized morphologies. During neuronal development, various actin-binding proteins regulate dynamics of actin cytoskeleton in the growth cones of developing axons. The regulation of actin cytoskeleton in the growth cones is thought to be involved in axon outgrowth and axon-dendrite specification. However, it is largely unknown which actin-binding proteins are involved in axon-dendrite specification and how they are transported into the developing axons. We have previously reported that collapsin response mediator protein 2 (CRMP-2) plays a critical role in axon outgrowth and axon-dendrite specification (N. Inagaki, K. Chihara, N. Arimura, C. Menager, Y. Kawano, N. Matsuo, T. Nishimura, M. Amano, and K. Kaibuchi, Nat. Neurosci. 4:781-782, 2001). Here, we found that CRMP-2 interacted with the specifically Rac1-associated protein 1 (Sra-1)/WASP family verprolin-homologous protein 1 (WAVE1) complex, which is a regulator of actin cytoskeleton. The knockdown of Sra-1 and WAVE1 by RNA interference canceled CRMP-2-induced axon outgrowth and multiple-axon formation in cultured hippocampal neurons. We also found that CRMP-2 interacted with the light chain of kinesin-1 and linked kinesin-1 to the Sra-1/WAVE1 complex. The knockdown of CRMP-2 and kinesin-1 delocalized Sra-1 and WAVE1 from the growth cones of axons. These results suggest that CRMP-2 transports the Sra-1/WAVE1 complex to axons in a kinesin-1-dependent manner and thereby regulates axon outgrowth and formation

Comparison of the Diagnostic Power of Transthoracic and Transesophageal Echocardiography to Detect Ruptured Chordae Tendineae

Preoperative information concerning the severity and etiology of MR is very important for selecting the most appropriate surgical strategy. Ruptured chordae tendineae (RCT) are one of the most important preoperative findings. We compared the diagnostic power of transesophageal echocardiography (TEE) and transthoracic echocardiography (TTE) to detect RCT in patients with MR. We studied 61 patients with MR (30 men, 31 women; mean age, 61 ± 12 years) who underwent mitral valve repair or replacement. Both TTE and TEE were performed before the operations, and the sensitivity and specificity of TTE and TEE to detect RCT were determined. In addition, other factors that influenced the detection of RCT by these two methods were investigated. At the time of an operation, RCT was confirmed in 39 of 61 cases. Transesophageal echocardiography had a higher sensitivity than TTE (74% versus 44%; P = 0.006) to detect RCT, although the specificity was not significantly different. In patients with a body mass index (BMI) > 22 (P = 0.023) or MR grade 4 (P = 0.026), TEE had a significantly higher diagnostic sensitivity than TTE, although there was no significant difference in patients with BMI < 22 or MR grade ≤ 3. In the lateral and medial segments of the mitral valve, TEE had a significantly higher diagnostic sensitivity to detect RCT than TTE (P = 0.0012), although there was no significant difference in the middle segments. There was no significant difference between TTE and TEE with respect to the sensitivity to detect RCT in myxomatous mitral valves. Although the sensitivity of TEE was higher than that of TTE to detect RCT, it was affected by BMI, MR grade, the RCT-presenting segments, and the etiology of MR

α-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

<div><p>Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.</p></div

Knockdown of α-taxilin induces the degradation of TfnR.

<p>(A) Top: HeLaS3 cells transfected with control (Con) or α-taxilin siRNA (#2, #3 and #4) were lysed, and the cell lysates were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.0005; **, P<0.005; ***, P<0.005, by Student's <i>t</i>-test. (B) Top: HeLaS3 cells stably expressing control (Con) or α-taxilin shRNA (#2, #7) were lysed, and the cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.005; **, P<0.005, by Student's <i>t</i>-test. (C) Total RNA was extracted from HeLaS3 cells transfected with control (Con) or α-taxilin siRNA (#2, #3 and #4) for 48 h, and <i>TFNR</i> and <i>GAPDH</i> mRNA were analyzed by RT-PCR. The ratio of the <i>TFNR</i> mRNA level relative to the <i>GAPDH</i> mRNA level was expressed as arbitrary units. <i>TFNR</i> mRNA level relative to <i>GAPDH</i> mRNA level in control HeLaS3 cells was set to 1.0. The results shown are means ± s.e.m. from three independent experiments. Ns, not significant, by Student's <i>t</i>-test. (D) Top: HeLaS3 cells transfected with control (Con) or α-taxilin (αTax) siRNA (#3) were treated with 0.1% DMSO, 10 μM lactacystin or 100 nM bafilomycin A1 (BafA1) for 24 h. The cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. Bottom: the amount of TfnR was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of TfnR in α-taxilin knockdown cells to TfnR in control cells from three independent experiments. *, P<0.005; **, P<0.01; ns, not significant, by Student's <i>t</i>-test. (E) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with 0.1% DMSO or 100 nM bafilomycin A1 (BafA1) for 24 h, and then the cells were immunostained with anti-TfnR and anti-α-taxilin antibodies. The results shown are representative of three independent experiments. Scale bars, 10 μm. (F) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were stimulated with EGF (100 ng/ml) for the indicated time periods. Cell lysates were probed with the indicated antibodies. The results shown are representative of three independent experiments. (G) The amount of EGFR in (F) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of EGFR at each time point to EGFR at time zero from three independent experiments. Values at time zero are set to 100%. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 15, 30, 60 min) determined by Student's <i>t</i>-test was not significant.</p

Knockdown of α-taxilin impedes the recycling of Tfn.

<p>(A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's <i>t</i>-test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. <i>P</i>-values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's <i>t</i>-test. *, P<0.005; **, P<0.001. <i>P</i>-values at 10 min was not significant. (E) The intensity of Tfn-488 signal of HeLaS3 cells treated or untreated with 200 μg/ml leupeptin in (C) was calculated as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at 20 and 40 min to Tfn-488 at time zero. Values at time zero are set to 1.0. <i>P</i>-values (the cells untreated with leupeptin vs. the cells treated with leupeptin at 20 and 40 min) are determined by Student's <i>t</i>-test. *, P<0.05; **, P<0.05; ns, not significant. Con, control; α-Tax, α-taxilin.</p