The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death.

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL-R-associated complex I as well as of the cytoplasmic TRAIL-induced complex II In both of these complexes, HOIP limits caspase-8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase-8-dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL-containing complex, LUBAC also restricts TRAIL-induced necroptosis. We identify RIPK1 and caspase-8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL-induced RIPK1-independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL-induced activation of NF-κB and, consequently, the production of cytokines, downstream of FADD, caspase-8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation

LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone

LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L

The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone

Correction for missed events based on a realistic model of a detector.

Quantitative patch-clamp analysis based on dwell-time histograms has to deal with the problem of missed events. The correction of the evaluated time constants has to take into account the characteristics of the detector used for the reconstruction of the time series. In previous approaches a simple model of the detector has been used, which is based on the assumption that all events shorter than the temporal resolution tres were missed, irrespective of the preceding events. Rather than the standard assumption of a fixed dead time, we introduce a more realistic model of a detector by a continuous-time version of the Hinkley detector. The combined state of the channel and the detector obeys a Markov model, which is governed by a Fokker-Planck-Kolmogorov partial differential equation. The steady-state solution leads to the determination of the apparent time constants tau o and tau c depending on the true rate constants koc and kco and the temporal resolution tres of the detector. Simulations with different kinds of detectors, including the Bessel filter with half-amplitude threshold detection, are performed. They show that our new equation predicts the dependence of tau c and tau o on koc, kco, and tres better than the standard equation used until now

Detection of jumps in single-channel data containing subconductance levels.

Detection algorithms are widely used for the analysis of single-channel data because they remove the background noise from the measured current signal and reconstruct the noise-free time series. Standard detection algorithms assume channels switching only between zero and full conductance. Many types of channels, however, show subconductance levels. A new detection algorithm for data containing sublevels, the so-called sublevel Hinkley-detector (SHD), calculates several test values in parallel, one for each possible jump. The velocity of increase has a maximum for the correct jump. This feature is used to detect the jump and to diagnose the new level of current. Because patch-clamp data are always filtered by an antialiasing low-pass filter before sampling, the algorithm is supplemented by a special diagnosis phase accounting for the distortion of the originally rectangular jumps. Along with the reconstructed (noise-free) time series the SHD also gives a matrix of the transition counts between the levels. This matrix is a useful statistical tool for the decision whether the observed channel(s) have in fact a subconductance conformation or if there are simply several channels of different conductivity contained within the patch

Fast single-channel measurements resolve the blocking effect of Cs+ on the K+ channel.

The Cs+ block of K+ channels has often been investigated by methods that allow only indirect estimation of the rate constants of blocking and re-opening. This paper presents single-channel records with high temporal resolution which make the direct observation of the fast transitions between the blocked and the unblocked state possible. The rate constants kOGb, kGbO of Cs(+)-dependent blocking and of re-opening are evaluated from the time constants found in the open-time and closed-time histograms. The blocking rate constant kOGb between 1000 and 50000 s-1 depends linearly on the Cs+ concentration and strongly on voltage, increasing by a factor of 1.44 per 10 mV hyperpolarization. The re-opening rate constant kGbO approximately 30000 s-1 is independent of Cs+ concentration and only slightly voltage-dependent. Formally, the results can be described by a Woodhull-model. The strong voltage dependence with d > 1, however, weakens its plausibility. The results are interpreted in terms of a molecular framework emerging from recent results on the structure of voltage-gated channels