A simple search of TM segments in polytopic membrane protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Using both high performance liquid chromatography (HPLC) and amino acid sequencing (AAS), we previously analyzed band 3 TM peptide-segments that make up the transmembrane protein structure. However, the HPLC/AAS combination method was highly time-consuming. Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry is used to obtain accurate molecular weight information for proteins/peptides simply and sensitively. We applied the MALDI-TOF mass spectrometry technique to search for TM segments in membrane proteins. In combination with trypsin cleavages after alkali treatments (pH12 or 13) and sample preparation using organic solvents for MALDI-TOF mass spectrometry, we determined the TM segments of band 3 and glycophorin A in erythrocyte membrane. The method can be applied to other polytopic membrane proteins in erythrocyte membrane.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

m-Carborane bisphenol structure as a pharmacophore for selective estrogen receptor modulators

A series of m-carborane derivatives was prepared based upon the structures of antiestrogenic drugs and their activities were evaluated by estrogen receptor alpha (ERalpha) binding assay and transactivation assay using human breast cancer cell line, MCF-7 cells. The m-carborane bisphenol 5 exhibited about a thousand times more potent ER agonistic activity than the o-carborane bisphenol 11. The m-carborane bisphenol structure appears to be a favorable hydrophobic pharmacophore for the development of novel selective estrogen receptor modulators (SERMs).     

Lycopladines B-D and lyconadin B, new alkaloids from Lycopodium complanatum

Four new alkaloids, lycopladines B-D (1-3) and lyconadin B (4), have been isolated from the club moss Lycopodium complanatum and the structures including the stereochemistry were elucidated on the basis of spectral data and modified Mosher's method. Lyconadin B (4) elevated NGF mRNA expression in 1321N1 human astrocytoma cells.     

Superoxide production in human neutrophils is enhanced by treatment with transmembrane peptides derived from human formyl peptide receptor

Formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been performed on the ligand binding site of FPR, FPR dynamic behavior such as receptor dimerization on the cell surface remains unknown. Recently, peptides derived from the transmembrane (TM) domains of GPCRs were shown to disrupt dimer formation by receptors and to result in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from FPR were synthesized, and their biological activities were evaluated with human neutrophils. Synthetic peptides did not exhibit agonistic or antagonistic activity toward superoxide anion production. However, Neutrophils treated with hFPRTM4 produced 4-fold superoxide anion compared with untreated cells when stimulated with FPR agonist fMLP. Short peptide fragments derived from the fourth TM region of FPR did not enhance superoxide anion production, which suggests that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides were inserted into the membrane. The addition of hFPRTM4 increased the intracellular calcium concentration, which meant the peptide activated some membrane protein on the cell surface. The present study suggests that the fourth TM domain of FPR has a function related to a priming effect.     

Involvement of TNF receptor-associated factor 6 in IL-25 receptor signaling

IL-25 (IL-17E) induces IL-4, IL-5, and IL-13 production from an unidentified non-T/non-B cell population and subsequently induces Th2-type immune responses such as IgE production and eosinophilic airway inflammation. IL-25R is a single transmembrane protein with homology to IL-17R, but the IL-25R signaling pathways have not been fully understood. In this study, we investigated the signaling pathway under IL-25R, especially the possible involvement of TNFR-associated factor (TRAF)6 in this pathway. We found that IL-25R cross-linking induced NF-kappaB activation as well as ERK, JNK, and p38 activation. We also found that IL-25R-mediated NF-kappaB activation was inhibited by the expression of dominant negative TRAF6 but not of dominant negative TRAF2. Furthermore, IL-25R-mediated NF-kappaB activation, but not MAPK activation, was diminished in TRAF6-deficient murine embryonic fibroblast. In addition, coimmunoprecipitation assay revealed that TRAF6, but not TRAF2, associated with IL-25R even in the absence of ligand binding. Finally, we found that IL-25R-mediated gene expression of IL-6, TGF-beta, G-CSF, and thymus and activation-regulated chemokine was diminished in TRAF6-deficient murine embryonic fibroblast. Taken together, these results indicate that TRAF6 plays a critical role in IL-25R-mediated NF-kappaB activation and gene expression.     

Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.     

Lipophilicity of capsaicinoids and capsinoids influences the multiple activation process of rat TRPV1

Analogs of capsaicin, such as capsaicinoids and capsinoids, activate a cation channel, transient receptor potential cation channel vanilloid subfamily 1 (TRPV1), and then increase the intracellular calcium concentration ([Ca2+]i). These compounds would be expected to activate TRPV1 via different mechanism(s), depending on their properties. We synthesized several capsaicinoids and capsinoids that have variable lengths of acyl moiety. The activities of these compounds towards TRPV1 heterologously expressed in HEK293 cells were determined by measuring [Ca2+]i. When an extracellular or intracellular Ca2+ source was removed, some agonists such as capsaicin could increase [Ca2+]i. However, a highly lipophilic capsaicinoid containing C18:0 and capsinoids containing C14:0, C18:0, or C18:1 (the latter was named olvanilate) could not elicit a large increase in [Ca2+]i in the absence of an extracellular or intracellular Ca2+ source. These results suggest that highly lipophilic compounds cause only a slight Ca2+ influx, via TRPV1 in the plasma membrane, and are not able to activate TRPV1 in the endoplasmic reticulum.     

Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells

The mood-stabilizing agent valproic acid (VPA) potently promotes neuronal differentiation. As yet, however, little is known about the underlying molecular mechanism. Here, we show that VPA upregulates cytohesin-2 and mediates neurite outgrowth in N1E-115 neuroblastoma cells. Cytohesin-2 is the guanine-nucleotide exchange factor (GEF) for small GTPases of the Arf family; it regulates many aspects of cellular functions including morphological changes. Treatment with the specific cytohesin family inhibitor SecinH3 or knockdown of cytohesin-2 with its siRNA results in blunted induction of neurite outgrowth in N1E-115 cells. The outgrowth is specifically inhibited by siRNA knockdown of Arf6, but not by that of Arf1. Furthermore, VPA upregulates Arl4D, an Arf-like small GTPase that has recently been identified as the regulator that binds to cytohesin-2. Arl4D knockdown displays an inhibitory effect on neurite outgrowth resulting from VPA, while expression of constitutively active Arl4D induces outgrowth. We also demonstrate that the addition of cell-permeable peptide, coupling the cytohesin-2-binding region of Arl4D into cells, reduces the effect of VPA. Thus, Arl4D is a previously unknown regulator of neurite formation through cytohesin-2 and Arf6, providing another example that the functional interaction of two different small GTPases controls an important cellular function.     

Mitotic Regulation of the Stability of Microtubule Plus-end Tracking Protein EB3 by Ubiquitin Ligase SIAH-1 and Aurora Mitotic Kinases

Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G₁ phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.Microtubule dynamics are essential in many cellular processes, including cell motility, intracellular transport, accurate mitosis, and cytokinesis in all eukaryotes. The regulatory factors for microtubule dynamics can be classified into two main types as follows: microtubule-destabilizing proteins, such as stathmin/Op18 (1) and the Kinesin-13 family (also known as MCAK/KIF2 family) (2), and microtubule-stabilizing proteins, the classic superfamily of microtubule-associated proteins (3). Additionally, the plus-end tracking proteins (+TIPs)³ have recently been identified; this family specifically accumulates at the ends of growing microtubules and regulates the microtubule plus-end targeting to the cell cortex or mitotic kinetochores (4, 5).The EB1 family is a member of the +TIPs family and consists of three homologs in mammals: EB1, EB2/RP1 (henceforth, EB2), and EB3 (6). As EB1 was originally identified as a protein that interacts with the well characterized tumor suppressor adenomatous polyposis coli (APC) protein (7), the function of EB1 has been investigated extensively. EB1 interacts with other +TIPs, including APC, p150^glued, CLIPs, and CLASP1/2, and the interaction network controls microtubule orientation and microtubule-cortex interaction during cell migration (5, 8, 9). EB1 functions not only in the regulation of interphase microtubule dynamics but also in mitotic spindle regulation. For accurate chromosomal segregation, sister chromatids become aligned to the metaphase plate during metaphase, and the alignment requires spindle-kinetochore attachment. Two models have been proposed; in the first, termed the “search-and-capture” model, EB1 localized at the growing microtubule plus-ends searches for binding partners located on kinetochores (10, 11). In the second model proposed recently, EB1 makes kinetochore fibers and centrosomal microtubules connect, and it is essential for the formation of a functional bipolar spindle (12). Thus, EB1 is thought to be a master controller of microtubule plus-ends; however, little is known about other EB1 family members. Given that EB3 is localized on the microtubule network and binds to APC and CLIPs identically to EB1, it is possible that EB3 acts as an EB1 analog in cells (13–15).Cell division is precisely regulated by several post-translational modifications of proteins, mainly reversible phosphorylation and ubiquitination, which is followed by degradation. Accurate mitotic phase progression requires the appropriate phosphorylation of various proteins by mitotic kinases (16, 17). One of the key mitotic kinases is the Aurora family that has been highly conserved from yeast to humans. There are three homologs (Aurora-A, -B, and -C) in human and mouse (18). Although their homology at the protein level is more than 84%, their functions and subcellular localizations are distinct. Aurora-A is located in the centrosomes and spindle and is required for mitotic entry, centrosome maturation/separation, and spindle assembly (19). Aurora-B is a chromosomal passenger protein that localizes on the inner centromere of the chromosomes until metaphase to regulate the spindle-kinetochore attachment, and from anaphase, it translocates to the central spindle and then accumulates in the midbody for cytokinesis (20, 21). The numerous substrates of the Aurora family include regulatory factors for microtubule dynamics, such as the microtubule-destabilizing proteins MCAK and stathmin, which help to establish the bipolar attachment and spindle assembly, respectively (22–24). It is possible that the Aurora family regulates the EB1 family by phosphorylation.In this study, we performed yeast two-hybrid screening and obtained the EB1 yeast homolog Bim1 as a protein that interacts with Ipl1, a yeast counterpart of Aurora. Here we demonstrate the novel regulatory mechanisms of EB3 by two cell cycle-dependent post-translational modifications, phosphorylation and ubiquitin-proteasome-mediated degradation.