Soichi Miwa

Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism

Kinetic refolding reactions of ferricytochrome c and β‐lactoglobulin have been studied by stopped‐flow circular dichroism by monitoring rapid ellipticity changes of peptide backbone and side‐chain chromophores. In both proteins, a transient intermediate accumulates within the dead time of stopped‐flow mixing (18 ms), and the intermediate has an appreciable amount of secondary structure but possesses an unfolded tertiary structure. It is suggested that the rapid formation of a secondary structure framework in protein folding is a common property observed in a variety of globular proteins.

TLR3 and TLR7 Are Targeted to the Same Intracellular Compartments by Distinct Regulatory Elements

Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu727 to Asp749) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection.

Reciprocal changes in endothelium-derived hyperpolarizing factor- and nitric oxide-system in the mesenteric artery of adult female rats following ovariectomy

1 To explore the effects of estrogen on arterial functions, we examined endothelium‐derived hyperpolarizing factor (EDHF)‐ and NO‐mediated responses in isolated mesenteric arteries of female rats, 4 weeks after sham‐operation (CON), ovariectomy (OVX) and OVX plus chronic estrogen treatment (OVX+E2). Tissue levels of connexins‐40, 43 (major components of gap junction), inducible NOS (iNOS), endothelial NOS (eNOS) and eNOS regulator proteins such as calmodulin, heat shock protein 90 (hsp90) and caveolin‐1 were also examined using Western blot. 2 In OVX, acetylcholine (ACh)‐induced EDHF‐mediated relaxation and membrane hyperpolarization of arterial smooth muscles were reduced, whereas ACh‐induced NO‐mediated relaxation was enhanced, leading to no change in ACh‐induced relaxation. 3 In OVX, connexin‐40 and 43 were decreased. Tissue levels of eNOS and its positive regulators (calmodulin and hsp90) were unchanged, but that of its negative regulator, caveolin‐1, was decreased. The levels of iNOS in mesenteric artery and aorta and plasma levels of NO metabolites and cholesterol were elevated. 4 In OVX, contraction of the artery by phenylephrine was reduced, but augmented by nonspecific inhibitor of NOS to the comparable level as that in CON group. The contraction in OVX group unlike that in CON group was augmented by specific iNOS inhibitor, and the difference between contractions in the presence of nonspecific and specific inhibitor as an index of eNOS activity was increased. 5 In OVX+E2, all these changes were recovered. 6 In all groups, EDHF‐mediated relaxation was suppressed by 18β‐glycyrrhetinic acid, an inhibitor of gap junction. 7 These results indicate that estrogen deficiency does not change the diameter of mesenteric artery: it reduces EDHF‐mediated relaxation by decreasing gap junction, whereas it augments NO‐mediated relaxation via an increase in NO release. Increased NO result from increased activity of eNOS subsequent to a decrease in caveolin‐1 and from induction of iNOS. However, excessive NO generation with elevated plasma cholesterol would raise a risk for atherosclerosis.

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.

MyD88 is a cytoplasmic adaptor protein that is critical for Toll‐like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non‐TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll‐interleukin I receptor‐resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non‐TIR regions in the subcellular localization and signaling properties of MyD88.

Age-Related Nuclear Cataract and Indoleamine 2,3-Dioxygenase-Initiated Tryptophan Metabolism in the Human Lens

Tryptophan-derived UV filters (kynurenine and 3-hydroxylkynurenine glucoside) have recently been shown to bind to human lens proteins. These UV filter adducts increase in amount with age and appear to be mainly responsible for the yellowing of the lens in man. On the basis of research performed in other tissues, it has been assumed that indoleamine 2,3-dioxygenase (IDO) may be the first and probably rate-limiting enzyme in UV filter biosynthesis. In this study, 25 human lenses were examined by a reliable and sensitive assay method with a monoclonal antibody specific for IDO. IDO activity was detected in all lenses ranging from 26 to 80 years, and there was no clear relationship of IDO activity with age. The mean activity was 0.85 + 0.49 nmol of kynurenine/h/lens. The level in the iris/ciliary body was negligible (<0.05 nmol of kynurenine/h). The lens IDO activity is consistent with UV filter turnover values obtained previously. These findings indicate that IDO is the first enzyme in the UV filter pathway and that UV filter biosynthesis is active even in aged lenses. Yellowing of the aged lens may therefore be preventable by drug-induced suppression of IDO activity.

Carbonyl Compounds in the Gas Phase of Cigarette Mainstream Smoke and Their Pharmacological Properties

Cigarette mainstream smoke is composed of gas and tar phases and contains >4000 chemical constituents, including nicotine and tar. The substances in the gas phase but not in the tar phase can pass through the airway epithelial barrier, enter the systemic circulation via the pulmonary circulation, and increase systemic oxidative damage, leading to the development of cigarette smoking-related diseases such as atherosclerosis. Recently, we identified some stable carbonyl compounds, including acrolein (ACR) and methyl vinyl ketone (MVK), as major cytotoxic factors in nicotine- and tar-free cigarette smoke extract (CSE) of the gas phase. CSE, ACR, and MVK induce protein kinase C (PKC)-dependent activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) via NOX, causing plasma membrane damage and cell apoptosis. CSE, ACR, and MVK also trigger carbonylation of PKC, which is an irreversible oxidative modification. Cell damage and PKC carbonylation in response to treatment with CSE, ACR, or MVK are abolished by thiol-containing antioxidants such as N-acetyl-L-cysteine and reduced glutathione. Thus pharmacological modulation of PKC and NOX activities and the trapping of ROS are potential strategies for the prevention of diseases related to cigarette smoking.

A Simple and Rapid Method for Standard Preparation of Gas Phase Extract of Cigarette Smoke

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice

Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

Background: Agonist stimulation induces different intracellular trafficking of endothelin receptors (ETA/BR) after internalization. The mechanism is unclear. Results: Stimulation induces ubiquitination, lysosomal targeting, and decreased cell surface ETBR levels. Non-ubiquitinated ETAR and ETBR mutant recycled to plasma membrane with smaller changes in the levels. Conclusion: Ubiquitination determines intracellular trafficking of endothelin receptors. Significance: ETBR ubiquitination fine tunes cellular responses to agonist by regulating cellular receptor levels. Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated “5KR mutant”) in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca2+ concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated “4KR mutant”), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

CCCP enhances catecholamine release from the perfused rat adrenal medulla

The present study was designed to investigate the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, on secretion of catecholamines from the isolated perfused model of the rat adrenal gland and to establish the mechanism of its adrenomedullary secretion. The perfusion of CCCP (3x10(-5) M) into an adrenal vein of for 90 min caused a great increase in catecholamine secretion. Tachyphylaxis to catecholamine-releasing effect of CCCP was not observed by repeated perfusion of it. The net catecholamine-releasing effects of CCCP were depressed by pretreament with pirenzepine (a selective muscarinic M(1)-receptor antagonist), chlorisondamine (a selective neuronal nicotinic receptor antagonist), nicardipine (an L-type Ca2+-channel antagonist), TMB-8 (an intracellular Ca2+-antagonist), and the perfusion of EGTA plus Ca2+-free medium, respectively. In the presence of CCCP (3x10(-5) M), catecholamine secretory responses induced by ACh (5.32x10(-3) M), high K+ (5.6x10(-2) M, a direct membrane depolarizer), DMPP (10(-4) M, (a selective neuronal nicotinic receptor agonist), and McN-A-343 (10(-4) M, (a selective muscarinic M1-receptor agonist) were significantly enhanced. CCCP also significantly enhanced the catecholamine secretory responses evoked by Bay-K-8644 (10(-5) M), L-type Ca2+ channel activator, and cyclopiazonic acid (10(-5) M), an inhibitor of Ca2+-ATPase. Furthermore, the perfusion of FCCP (3x10(-5) M), a similar mitochondrial uncoupler, into an adrenal vein of for 90 min also caused a great increase in catecholamine secretion in a similar pattern with CCCP. Taken together, the results demonstrate that CCCP causes the catecholamine secretion from the perfused rat adrenal medulla in a calcium-dependent fashion. It is thought that this catecholamine secretory enhancement of CCCP may be mediated by both cholinergic receptor stimulation and membrane depolarization, which are relevant to the cytoplasmic Ca2+ increase by stimulation of the Ca2+ influx as well as by the inhibition of Ca2+ uptake into the cytoplasmic Ca2+ stores (both endoplasmic reticulum and mitochondria in chromaffin cells). It also seems that protonophores, such as CCCP, suppress mitochondrial Ca2+ uptake and increase the stimulated secretion of catecholamine by the secretagogues. These results indicate that mitochondria modulate catecholamine secretion by regulating the Ca2+ mobilization for exocytosis.