Ca2+/calmodulin regulates Kvβ1.1-mediated inactivation of voltage-gated K+ channels

A-type K+ channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K+ channel’s α or β subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvβ1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca2+ concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca2+ dependence is gained by binding of calmodulin to the “chain” segment of Kvβ1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca2+/calmodulin does not interfere with the β subunit’s enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvβ1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

© FASEB. Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-ciseicosatetraenoic acid] and its subsequent transformation to LTA4 . 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site

Cell-free DNA in patients with sepsis: long term trajectory and association with 28-day mortality and sepsis-associated acute kidney injury

IntroductionOutcome-prediction in patients with sepsis is challenging and currently relies on the serial measurement of many parameters. Standard diagnostic tools, such as serum creatinine (SCr), lack sensitivity and specificity for acute kidney injury (AKI). Circulating cell-free DNA (cfDNA), which can be obtained from liquid biopsies, can potentially contribute to the quantification of tissue damage and the prediction of sepsis mortality and sepsis-associated AKI (SA-AKI).MethodsWe investigated the clinical significance of cfDNA levels as a predictor of 28-day mortality, the occurrence of SA-AKI and the initiation of renal replacement therapy (RRT) in patients with sepsis. Furthermore, we investigated the long-term course of cfDNA levels in sepsis survivors at 6 and 12 months after sepsis onset. Specifically, we measured mitochondrial DNA (mitochondrially encoded NADH-ubiquinone oxidoreductase chain 1, mt-ND1, and mitochondrially encoded cytochrome C oxidase subunit III, mt-CO3) and nuclear DNA (nuclear ribosomal protein S18, n-Rps18) in 81 healthy controls and all available samples of 150 intensive care unit patients with sepsis obtained at 3 ± 1 days, 7 ± 1 days, 6 ± 2 months and 12 ± 2 months after sepsis onset.ResultsOur analysis revealed that, at day 3, patients with sepsis had elevated levels of cfDNA (mt-ND1, and n-Rps18, all p<0.001) which decreased after the acute phase of sepsis. 28-day non-survivors of sepsis (16%) had higher levels of cfDNA (all p<0.05) compared with 28-day survivors (84%). Patients with SA-AKI had higher levels of cfDNA compared to patients without AKI (all p<0.05). Cell-free DNA was also significantly increased in patients requiring RRT (all p<0.05). All parameters improved the AUC for SCr in predicting RRT (AUC=0.88) as well as APACHE II in predicting mortality (AUC=0.86).ConclusionIn summary, cfDNA could potentially improve risk prediction models for mortality, SA-AKI and RRT in patients with sepsis. The predictive value of cfDNA, even with a single measurement at the onset of sepsis, could offer a significant advantage over conventional diagnostic methods that require repeated measurements or a baseline value for risk assessment. Considering that our data show that cfDNA levels decrease after the first insult, future studies could investigate cfDNA as a “memoryless” marker and thus bring further innovation to the complex field of SA-AKI diagnostics

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials

Sphingosine-1-phosphate promotes barrier-stabilizing effects in human microvascular endothelial cells via AMPK-dependent mechanisms.

Breakdown of the endothelial barrier is a critical step in the development of organ failure in severe inflammatory conditions such as sepsis. Endothelial cells from different tissues show phenotypic variations which are often neglected in endothelial research. Sphingosine-1-phosphate (S1P) and AMP-dependent kinase (AMPK) have been shown to protect the endothelium and phosphorylation of AMPK by S1P was shown in several cell types. However, the role of the S1P-AMPK interrelationship for endothelial barrier stabilization has not been investigated. To assess the role of the S1P-AMPK signalling axis in this context, we established an in vitro model allowing real-time monitoring of endothelial barrier function in human microvascular endothelial cells (HMEC-1) and murine glomerular endothelial cells (GENCs) with the electric cell-substrate impedance sensing (ECIS™) system. Following the disruption of the cell barrier by co-administration of LPS, TNF-α, IL-1ß, IFN-γ, and IL-6, we demonstrated self-recovery of the disrupted barrier in HMEC-1, while the barrier remained compromised in GENCs. Under physiological conditions we observed a rapid phosphorylation of AMPK in HMEC-1 stimulated with S1P, but not in GENCs. Consistently, S1P enhanced the basal endothelial barrier in HMEC-1 exclusively. siRNA-mediated knockdown of AMPK in HMEC-1 led to a less pronounced barrier enhancement. Thus we present evidence for a functional role of AMPK in S1P-mediated barrier stabilization in HMEC-1 and we provide insight into cell-type specific differences of the S1P-AMPK-interrelationship, which might influence the development of interventional strategies targeting endothelial barrier dysfunction

Deletion of Sphingosine Kinase 2 Attenuates Acute Kidney Injury in Mice with Hemolytic-Uremic Syndrome

Typical hemolytic uremic syndrome (HUS) can occur as a severe systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli. Its pathology can be induced by Stx types, resulting in toxin-mediated damage to renal barriers, inflammation, and the development of acute kidney injury (AKI). Two sphingosine kinase (SphK) isozymes, SphK1 and SphK2, have been shown to be involved in barrier maintenance and renal inflammatory diseases. Therefore, we sought to determine their role in the pathogenesis of HUS. Experimental HUS was induced by the repeated administration of Stx2 in wild-type (WT) and SphK1 (SphK1−/−) or SphK2 (SphK2−/−) null mutant mice. Disease severity was evaluated by assessing clinical symptoms, renal injury and dysfunction, inflammatory status and sphingolipid levels on day 5 of HUS development. Renal inflammation and injury were found to be attenuated in the SphK2−/− mice, but exacerbated in the SphK1−/− mice compared to the WT mice. The divergent outcome appeared to be associated with oppositely altered sphingolipid levels. This study represents the first description of the distinct roles of SphK1−/− and SphK2−/− in the pathogenesis of HUS. The identification of sphingolipid metabolism as a potential target for HUS therapy represents a significant advance in the field of HUS research

5‐Lipoxygenase‐activating protein rescues activity of 5‐lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA(4). 5-LOX is thought to receive arachidonic acid from the nuclear membrane–embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX–wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60–70% 5-LOX-wt levels) but not of LTA(4) hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA(4) hydrolysis products. Coexpression of FLAP partially restored LTA(4) hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.—Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors

In this work the synthesis, structure activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 mu M in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization. (C) 2015 Published by Elsevier Masson SAS