Endoplasmic reticulum protein 29 (ERp29) belongs to the redox-inactive PDI-Dβ-subfamily of PDI-proteins. ERp29 is expressed in all mammalian tissues examined. Especially high levels of expression ...were observed in secretory tissues and in some tumors. However, the biological role of ERp29 remains unclear. In the present study we show, by using thyrocytes and primary dermal fibroblasts from adult ERp29
−/−
mice, that ERp29 deficiency affects the activation of the ATF6–CHOP-branch of unfolded protein response (UPR) without influencing the function of other UPR branches, like the ATF4-eIF2α-XBP1 signaling pathway. As a result of impaired ATF6 activation, dermal fibroblasts and adult thyrocytes from ERp29
−/−
mice display significantly lower apoptosis sensitivities when treated with tunicamycin and hydrogen peroxide. However, in contrast to previous reports, we could demonstrate that ERp29 deficiency does not alter thyroglobulin expression levels. Therefore, our study suggests that ERp29 acts as an escort factor for ATF6 and promotes its transport from ER to Golgi apparatus under ER stress conditions.
The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel ...functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N-end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time.
We gained mechanistic insights into PRT1 substrate preference and activation by monitoring live ubiquitination using a fluorescent chemical probe coupled to artificial substrate reporters. Ubiquitination was measured by rapid in-gel fluorescence scanning as well as in real time by fluorescence polarization.
The enzymatic activity, substrate specificity, mechanisms and reaction optimization of PRT1-mediated ubiquitination were investigated ad hoc instantaneously and with significantly reduced reagent consumption.
We demonstrated that PRT1 is indeed an E3 ligase, which has been hypothesized for over two decades. These results demonstrate that PRT1 has the potential to be involved in polyubiquitination of various substrates and therefore pave the way to understanding recently discovered phenotypes of prt1 mutants.
•Cyclophilin A (CypA) is a host factor for human coronavirus NL63 replication.•CypA is a target for anti-coronaviral therapy.•Non-immunosuppressive CsA derivatives (Alisporivir, NIM811) inhibit CoV ...replication.•New classes of non-immunosuppressive CsA/FK506 derivatives inhibit CoV replication.
Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Müller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schöpf, J., Kögl, M., Friedel, C., Müller, M.A., Stellberger, T., von Dall’Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Züst, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Weßels, C., Pöhlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivatives of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concentrations in cell culture. We show by qPCR analysis that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.
The synthesis of a monofluorinated α-glycosidase inhibitor from miglitol is reported.
Selective monofluorination of the α-glycosidase inhibitor and antidiabetic agent Miglitol at position C(2) ...creates an competitive inhibitor of five times higher activity than the parent compound. Its screening against a panel of human cell lines showed a low cytotoxicity therefore making this compound an interesting candidate for further clinical investigations.
Inflammatory processes in the asthmatic lung involve the large and small airway and alveolar sites. Leukotriene B4 (LTB₄) is an important disease marker, but its role in inflammation of the small ...airways in asthma has not been established yet.
To distinguish between large and small airway or alveolar LTB₄ concentrations in children with asthma using the new technique of fractionated exhaled breath condensate sampling.
Sixty-eight children (9-17 years old, 33 children with asthma and 35 controls) underwent fractional exhaled nitric oxide (FeNO) measurements, lung function testing, and collection of fractionated exhaled breath condensate using a capnograph-based approach. The LTB₄ concentrations in the small airway or alveolar and large airway fractions were correlated to disease status, lung function impairment, and clinical parameters.
Children with asthma had significantly higher LTB₄ concentrations in the small airway or alveolar fraction than controls (5.58 pg/mL; 95% interquartile range IQR, 2.0-11.77 pg/mL; vs 2.0 pg/mL; 95% IQR, 2.0-6.2 pg/mL; P = .003). No difference was found between the groups in the large airway fraction. Children with obstructive lung function impairment (forced expiratory volume in 1 second z score <-1.65) had increased small airway or alveolar LTB₄ concentrations compared with children without impairment (2.0 pg/mL; 95% IQR, 2.0-9.21 pg/mL; vs 18.32 pg/mL; 95% IQR, 3.7-23.02 pg/mL; P = .04). Children with asthma but without pathologic obstructive lung function still had higher LTB₄ concentrations than controls (5.57 pg/mL; 95% IQR, 2.00-10.60 pg/mL; vs 2.00 pg/mL; 95% IQR, 2.00-6.20 pg/mL; P = .01).
LTB₄ is detectable and elevated in the small airway or alveolar fraction of exhaled breath condensate in pediatric asthma. Because of the possibility of detecting elevated levels in patients without lung function impairment in controlled disease, it may be used as a noninvasive marker of small airways disease; however, future long-term studies are needed.
Selective monofluorination of the α‐glycosidase inhibitor and antidiabetic agent miglitol at positions C(2′) or C(6) creates competitive inhibitors of glycosidases. Introducing a fluorine substituent ...at position C(6) results in a reduced binding to the enzyme whereas fluorination at C(2′) produces an inhibitor with an activity four times higher than the parent compound. This compound is selective for the α‐galactosidase from green coffee beans. Its screening against a panel of human cell lines showed a low cytotoxicity, therefore, making this compound an interesting candidate for further clinical investigations.
Diabetes affects about 285 million people worldwide and the number of patients is still increasing. To create a competitive inhibitor for α‐galactosidase, the α‐glycosidase inhibitor and antidiabetic agent miglitol was selectively monofluorinated at position C(2′) and then biologically evaluated.
Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and ...treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis.
Purpose
Determining the glomerular filtration rate (GFR) is essential for clinical medicine but also for pre-clinical animal studies. Functional imaging using positron emission tomography (PET) ...allows repetitive almost non-invasive measurements. The aim of the study was the development and evaluation of easily synthesizable PET tracers for GFR measurements in small animals.
Procedures
Diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) were labeled with Ga-68. The binding to blood cells and plasma proteins was tested
in vitro
. The distribution of the tracers in rats was analyzed by PET imaging and
ex vivo
measurements. From the time-activity-curve of the blood compartment (heart) and the total tracer mass excreted by the kidney, the GFR was calculated. These values were compared directly with the inulin clearance in the same animals.
Results
Both tracers did not bind to blood cells.
68
GaDPTA but not
68
GaEDTA showed strong binding to plasma proteins. For this reason,
68
GaDPTA stayed much longer in the blood and only 30 % of the injected dose was eliminated by the kidney within 60 min whereas the excretion of
68
GaEDTA was 89 ± 1 %. The calculated GFR using
68
GaEDTA was comparable to the measured inulin clearance in the same animal. Using
68
Ga-DPTA, the measurements led to values which were 80 % below the normal GFR. The results also revealed that definition of the volume of interest for the blood compartment affects the calculation and may lead to a slight overestimation of the GFR.
Conclusions
68
GaEDTA is a suitable tracer for GFR calculation from PET imaging in small animals. It is easy to be labeled, and the results are in good accordance with the inulin clearance.
68
GaDTPA led to a marked underestimation of GFR due to its strong binding to plasma proteins and is therefore not an appropriate tracer for GFR measurements.
Leukocyte trafficking and recruitment is a critical process in host immune surveillance and in inflammatory diseases. Extracellular cyclophilins (eCyps) have been identified as a novel class of ...chemotactic mediators. The impact of eCyp/CD147 interactions for the recruitment of leukocytes during inflammation was analyzed using a structurally simplified cell-impermeable eCyp inhibitor. This compound was highly effective at inhibiting leukocyte migration toward CypA in vitro as well as in the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction. By using CD147–/– mice in combination with the cell-impermeable eCyp inhibitor, we were able to show that the action of eCyps in inflammation is exclusively mediated by interaction with CD147. Our findings suggest that blocking eCyps may be an effective therapeutic target for reducing inflammatory diseases associated with leukocyte recruitment.
The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the ...protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective
N
-hydroxyalkylation of the residues Val5 and
d
-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5
N
-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the
N
-alkylation of
d
-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.
N
-Hydroxyalkylation of cyclosporine A residues Val5 and
d
-Ala8 significantly influenced their conformation behavior and pharmacological properties.
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