SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/sigma(E) stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle-targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.     

Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast

Recent data suggest that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subtype plays a pivotal role in the pathogenesis of effective disorders and in the action of antidepressant drugs. After chronic treatment with the antidepressants desipramine or paroxetine, we measured by immunoprecipitation and Western blotting, the changes in the interaction of AMPA receptor subunits with proteins involved in trafficking and/or stabilization of the subunits into synaptic membranes of the hippocampus. Both antidepressants increased the interaction of GluR1 subunit with stargazin and of GluR2/3 with NSF. Paroxetine increased the interaction of GluR1 with Rab4A, and desipramine markedly increased the interaction of GluR1 with SAP97. Paroxetine, but not desipramine, also increased membrane levels of CaMKII, autophosphorylated CaMKII and GluR1 phosphorylated at the CaMKII site. Interactions of GluR1 and GluR2/3 with proteins implicated in AMPA receptor trafficking and with scaffolding proteins appear to account for the enhanced membrane expression of AMPA receptors in the hippocampus after antidepressant treatment.     

Concerning the dynamic instability of actin homolog ParM

Apoptosis is a highly conserved procedure of cell death and occurs under various stimuli, including oxidative stress. A small heat shock protein, alphaB-crystallin, is found to process resistance to apoptosis in some cells and tissues. But the mechanisms under this protective role are not fully understood. In the present study, we reported the early protective role of alphaB-crystallin against hydrogen peroxide-induced apoptosis in mice myogenic C(2)C(12) cells. alphaB-Crystallin interacted with p53, a proapoptotic protein, during cell apoptosis and such protein interaction mainly occurred in the cytoplasm of the cells, suggesting that the interaction of alphaB-crystallin with p53 might prevent the translocation of p53 from cytoplasm to mitochondria. Hence, this study provides a hint that alphaB-crystallin affects on p53 mitochondrial translocation during oxidative stress-induced apoptosis.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.     

Colloidal Au replacement assay for highly sensitive quantification of low molecular weight analytes by surface plasmon resonance

A novel sensing method based on surface plasmon resonance (SPR) was developed for the highly sensitive quantification of low molecular weight (LMW) analytes (colloidal Au replacement assay). Gold nanoparticles (diameter = 20 nm) functionalized with lactosyl-poly(ethylene glycol) (PEG) were prepared and were specifically adsorbed onto a Ricinus communis agglutinin (RCA120)-immobilized SPR sensor chip surface. Subsequent injection of free d-galactose elicited the elution of the preadsorbed lactosyl-PEGylated gold nanoparticles in a manner proportional to the galactose concentration, achieving a substantial and quantitative analysis over a wide range of galactose concentrations (0.1-50 ppm). This method of d-galactose sensing through the substituted elution of preadsorbed nanoparticles from the sensor chip surface would be applicable for the highly sensitive SPR quantification of various LMW analytes, which are known to be difficult to detect by the conventional SPR sensing regime.     

Systematic comparison of catalytic mechanisms of hydrolysis and transfer reactions classified in the EzCatDB database

Catalytic mechanisms of 270 enzymes from 131 superfamilies, mainly hydrolases and transferases, were analyzed based on their enzyme structures. A method of systematic comparison and classification of the catalytic reactions was developed. Hydrolysis and transfer reactions closely resemble one another, displaying common mechanisms, single displacement, and double displacement. These displacement mechanisms might be further subclassified according to the type of catalytic factors and nucleophilic substitution involved. Several types of catalytic factors exist: nucleophile, acid, base, stabilizer, modulator, cofactors. Nucleophilic substitution might be categorized as S(N)1/S(N)2 (or dissociative/associative) reactions. The classification indicates that some mechanisms favor particular types of catalytic factors. In hydrolyses of amide bonds and phosphoric ester bonds, mechanisms with single displacement tend to use inorganic cofactors such as zinc and magnesium ions as important catalysts, whereas those with double displacement frequently do not use such cofactors. In contrast, hydrolyses of O-glycoside bond rarely use such cofactors, with one exception. The trypsin-like hydrolytic reaction, which is catalyzed by the classic catalytic triad comprising serine/histidine/aspartate, can be considered as a "super-reaction" because it is observed in at least three nonhomologous enzymes, whereas most reactions are singlets without any nonhomologous enzymes. By dividing complex reactions into several reactions, correlations between active site structures and catalytic functions can be suggested. This classification method is applicable to other reactions such as elimination and isomerization. Furthermore, it will facilitate annotation of enzyme functions from 3D patterns of enzyme active sites. The classification is available at http://mbs.cbrc.jp/EzCatDB/RLCP/index.html.     

Antibacterial activity of a virgatusin-related compound

The relationship between antibacterial activity of tetra-substituted tetrahydrofuran lignans (1-4) and their absolute configurations was tested. Only compound 4 among two virgatusins and two related compounds exhibited growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Listeria denitrificans. Compound 4 affected the growth of B. subtilis in a bactericidic manner, and its ability to dissipate the cytoplasmic membrane potential was investigated using the fluorescence probe 3,3'-dipropylthiadicarbocyanine iodide. These results suggested that compound 4 damages cells by causing the loss of the proton motive force and disruption of the cellular integrity of the membrane, leading to cell death. In addition, it was shown that the antibacterial activity of a lignan was closely related to its absolute configuration and functional groups.     

The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates

Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.     

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads

Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.     

Neuropeptide Y regulates the hematopoietic stem cell microenvironment and prevents nerve injury in the bone marrow

Many reports have revealed the importance of the sympathetic nervous system (SNS) in the control of the bone marrow environment. However, the specific role of neuropeptide Y (NPY) in this process has not been systematically studied. Here we show that NPY‐deficient mice have significantly reduced hematopoietic stem cell (HSC) numbers and impaired regeneration in bone marrow due to apoptotic destruction of SNS fibers and/or endothelial cells. Furthermore, pharmacological elevation of NPY prevented bone marrow impairments in a mouse model of chemotherapy‐induced SNS injury, while NPY injection into conditional knockout mice lacking the Y1 receptor in macrophages did not relieve bone marrow dysfunction. These results indicate that NPY promotes neuroprotection and restores bone marrow dysfunction from chemotherapy‐induced SNS injury through the Y1 receptor in macrophages. They also reveal a new role of NPY as a regulator of the bone marrow microenvironment and highlight the potential therapeutic value of this neuropeptide.