Conditional control of target proteins using the auxin-inducible degron (AID) system provides a powerful tool for investigating protein function in eukaryotes. Here, we established an Affinity-linker ...based super-sensitive auxin-inducible degron (AlissAID) system in budding yeast by using a single domain antibody (a nanobody). In this system, target proteins fused with GFP or mCherry were degraded depending on a synthetic auxin, 5-Adamantyl-IAA (5-Ad-IAA). In AlissAID system, nanomolar concentration of 5-Ad-IAA induces target degradation, thus minimizing the side effects from chemical compounds. In addition, in AlissAID system, we observed few basal degradations which was observed in other AID systems including ssAID system. Furthermore, AlissAID based conditional knockdown cell lines are easily generated by using budding yeast GFP Clone Collection. Target protein, which has antigen recognition sites exposed in cytosol or nucleus, can be degraded by the AlissAID system. From these advantages, the AlissAID system would be an ideal protein-knockdown system in budding yeast cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these ...antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10−5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10−6 M) and ONO-8130 (a prostanoid EP1 receptor antagonist, 10−7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10−7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10−5 M) or L-798,106 (a prostanoid EP3 receptor antagonist, 10−8 M). NP-1815-PX (10−5–10−4 M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F2α (PGF2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10−5–10−4 M) strongly suppressed the U46619-induced increase in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10−4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.
Yeast cells adapt to alkaline conditions by activating the Rim101 alkali-responsive pathway. Rim21 acts as a sensor in the Rim101 pathway and detects extracellular alkalization. Interestingly, Rim21 ...is also known to be activated by alterations involving the lipid asymmetry of the plasma membrane. In this study, we briefly summarize the mechanism of activation and the signal transduction cascade of the Rim101 pathway and propose a hypothesis on how Rim21 is able to detect distinct signals, particularly external alkalization, and altered lipid asymmetry. We found that external alkalization can suppress transbilayer movements of phospholipids between the two leaflets of the plasma membrane, which may lead to the disturbance of the lipid asymmetry of the plasma membrane. Therefore, we propose that external alteration is at least partly sensed by Rim21 through alterations in lipid asymmetry. Understanding this activation mechanism could greatly contribute to drug development against fungal infections.
In yeast, external alkalization and alteration in plasma membrane lipid asymmetry are sensed by the Rim101 pathway. It is currently under debate whether the signal elicited by external alkalization ...is transduced to downstream molecules at the plasma membrane or via endocytosis of the Rim21 sensor protein at the late endosome. We found that the downstream molecules, including arrestin-related protein Rim8, calpain-like protein Rim13, and scaffold protein Rim20, accumulated at the plasma membrane upon external alkalization and that the accumulation was dependent on Rim21. Snf7, an
e
ndosomal
s
orting
c
omplex
r
equired for
t
ransport (ESCRT) III subunit also essential for the Rim101 pathway, localized to the plasma membrane, in addition to the late endosome, under alkaline conditions. Snf7 at the plasma membrane but not at the late endosome was shown to be involved in Rim101 signaling. In addition, the Rim101 pathway was normally activated, even when endocytosis was severely impaired. Considering this information as a whole, we propose that Rim101 signaling proceeds at the plasma membrane. We also found that activity of the Rsp5 ubiquitin ligase was required for recruiting the downstream molecules to the plasma membrane, suggesting that ubiquitination mediates Rim101 signaling at the plasma membrane.
In this study, we investigated the effects of platelet-activating factor (PAF) on the basal tone and spontaneous contractile activities of guinea pig (GP) and mouse urinary bladder (UB) smooth muscle ...(UBSM) tissues to determine whether PAF could induce UBSM tissue contraction. In addition, we examined the mRNA expression of the PAF receptor, PAF-synthesizing enzyme (lysophosphatidylcholine acyltransferase, LPCAT), and PAF-degrading enzyme (PAF acetylhydrolase, PAF-AH) in GP and mouse UB tissues using RT-qPCR. PAF (10
-10
M) strongly enhanced the basal tone and spontaneous contractile activities (amplitude and frequency) of GP and mouse UBSM tissues in a concentration-dependent manner. The enhancing effects of PAF (10
M) on both GP and mouse UBSM contractile activities were strongly suppressed by pretreatment with apafant (a PAF receptor antagonist, GP: 10
M; mouse: 3 × 10
M). The PAF receptor (Ptafr), LPCAT (Lpcat1, Lpcat2), and PAF-AH (Pafah1b3, Pafah2) mRNAs were detected in GP and mouse UB tissues. These findings reveal that PAF strongly enhances the contractile mechanical activities of UBSM tissues through its receptor and suggest that the PAF-synthesizing and -degrading system exists in UBSM tissues. PAF may serve as both an endogenous UBSM constrictor and an endogenous mediator leading to detrusor overactivity.
Atg18 is essential for both autophagy and the regulation of vacuolar morphology. The latter process is mediated by phosphatidylinositol 3,5-bisphosphate binding, which is dispensable for autophagy. ...Atg18 also binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro. Here, we investigate the relationship between PtdIns(3)P-binding of Atg18 and autophagy. Using an Atg18 variant, Atg18(FTTG), which is unable to bind phosphoinositides, we found that PtdIns(3)P binding of Atg18 is essential for full activity in both selective and nonselective autophagy. Atg18(FTTG) formed a complex with Atg2 in a normal manner, and Atg18-Atg2 complex formation occurred in cells in the absence of PtdIns(3)P, indicating that Atg18-Atg2 complex formation is independent of PtdIns(3)P-binding of Atg18. Atg18 localized to endosomes, the vacuolar membrane, and autophagic membranes, whereas Atg18(FTTG) did not localize to these structures. The localization of Atg2 to autophagic membranes was also lost in Atg18(FTTG) cells. These data indicate that PtdIns(3)P-binding of Atg18 is involved in directing the Atg18-Atg2 complex to autophagic membranes. Connection of a 2×FYVE domain, a specific PtdIns(3)P-binding domain, to the C terminus of Atg18(FTTG) restored the localization of Atg18-Atg2 to autophagic membranes and full autophagic activity, indicating that PtdIns(3)P-binding by Atg18 is dispensable for the function of the Atg18-Atg2 complex but is required for its localization. This also suggests that PtdIns(3)P does not act allosterically on Atg18. Taken together, Atg18 forms a complex with Atg2 irrespective of PtdIns(3)P binding, associates tightly to autophagic membranes by interacting with PtdIns(3)P, and plays an essential role.
Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including ...gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10−6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10−5 M), diltiazem (10−5 M), and nifedipine (10−7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10−5 M) was strongly suppressed by SKF-96365 (3 × 10−5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10−5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.
Yeast cells sense alterations in the plasma membrane (PM) lipid asymmetry and external alkalization by the sensor protein Rim21, which functions in the Rim101 pathway. Rim101 signaling is initiated ...at the PM by the recruitment of the Rim101 signaling complex. The PM physically associates with the cortical endoplasmic reticulum (ER) to form ER-PM contact sites, where several signaling events, lipid exchange, and ion transport take place. In the present study, we investigated the spatial relationship between ER-PM contact sites and the sites of Rim101 signaling. Rim101 signaling mostly proceeds outside ER-PM contact sites in the PM and did not require intact ER-PM contact for its activation. Rather, the Rim101 pathway was constitutively activated by ER-PM contact site disruption, which is known to cause ER stress. ER stress induced by tunicamycin treatment activated the Rim101 pathway. Furthermore, the sensitivity of cells to tunicamycin without ER-PM contact was considerably elevated by the deletion of RIM21. These results suggest that the Rim101 pathway is important for the adaptation to ER stress by compensating for alterations in PM lipid asymmetry induced by ER stress.
Alzheimer’s disease (AD) is accompanied by behavioral and psychological symptoms of dementia (BPSD), which is often alleviated by treatment with psychotropic drugs, such as antidepressants, ...hypnotics, and anxiolytics. If these drugs also inhibit acetylcholinesterase (AChE) activity, they may contribute to the suppression of AD progression by increasing brain acetylcholine concentrations. We tested the potential inhibitory effects of 31 antidepressants, 21 hypnotics, and 12 anxiolytics on recombinant human AChE (rhAChE) activity. At a concentration of 10−4 M, 22 antidepressants, 19 hypnotics, and 11 anxiolytics inhibited rhAChE activity by <20%, whereas nine antidepressants (clomipramine, amoxapine, setiptiline, nefazodone, paroxetine, sertraline, citalopram, escitalopram, and mirtazapine), two hypnotics (triazolam and brotizolam), and one anxiolytic (buspirone) inhibited rhAChE activity by ≥20%. Brotizolam (≥10−6 M) exhibited stronger inhibition of rhAChE activity than the other drugs, with its pIC50 value being 4.57 ± 0.02. The pIC50 values of the other drugs were <4, and they showed inhibitory activities toward rhAChE at the following concentrations: ≥3 × 10−6 M (sertraline and buspirone), ≥10−5 M (amoxapine, nefazodone, paroxetine, citalopram, escitalopram, mirtazapine, and triazolam), and ≥3 × 10−5 M (clomipramine and setiptiline). Among these drugs, only nefazodone inhibited rhAChE activity within the blood concentration range achievable at clinical doses. Therefore, nefazodone may not only improve the depressive symptoms of BPSD through its antidepressant actions but also slow the progression of cognitive symptoms of AD through its AChE inhibitory actions.
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