Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3–mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.Programmed necrosis or “necroptosis” has emerged in the past 5 years as a cell death mechanism that complements the conventional cell death pathway, apoptosis, in multicellular organisms. In contrast to apoptosis, necroptosis does not appear to serve an important role in multicellular organism development (1–3) but participates in the defense against pathogens and is a likely culprit in destructive inflammatory conditions (4–7). Receptor Interacting Protein Kinase-3 (RIPK3) was identified as a key effector of necroptosis in 2009 (4, 5) and its substrate, the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL), in 2012 (8, 9), but the molecular events following RIPK3-mediated phosphorylation of MLKL required to induce cell death are unclear. The RIPK1/RIPK3/MLKL necrosome has been proposed to activate PGAM5 (phosphoglycerate mutase 5) and Drp1 (Dynamin-related protein 1) to cause mitochondrial fragmentation and cell death (10), but the requirement for PGAM5, Drp1, and mitochondria for necroptosis has been questioned (1, 11–13).We described the structure of mouse MLKL revealing that MLKL contains a C-terminal pseudokinase domain and an N-terminal four-helix bundle (4HB) domain connected by a two-helix linker (the “brace” helices) (1). Based on our mutational and biochemical analyses, we proposed that the catalytically inactive pseudokinase domain functions as a molecular switch and that RIPK3-mediated phosphorylation triggers this switch by inducing a conformational change in MLKL (1, 14).Recently it has been proposed that the 4HB domain is the death effector domain within MLKL and that the killing function of MLKL relies on its oligomerization and plasma membrane association (15–18). The stoichiometry of the oligomer is, however, contentious and has been reported to contain three (15), four (16), and possibly six (17) MLKL protomers. Furthermore, several mechanisms for how this oligomer causes cell death have been proposed: Cai et al. proposed it activates the calcium channel protein Tprm7 and promotes calcium influx (15), Chen et al. showed it increased sodium influx (16), and Wang et al. proposed that the oligomerized form of MLKL has the ability to bind negatively charged lipids, including phosphoinositides and cardiolipin, which facilitates its disruption of membrane integrity (17), a model supported by a subsequent paper (18).Here, we show that the MLKL 4HB domain is sufficient to induce necroptosis and identify several charged residues clustered on two faces that are required for this function. Surprisingly the polarity of several of these charged residues is not conserved between mouse and human MLKL, and alanine substitution of negatively charged residues on the α4 helix of the 4HB domain disrupted function. This finding challenges the importance of phospholipid binding to the killing activity of the 4HB domain and illustrates that membrane association cannot solely be attributed to the interaction of poorly conserved basic residues within the MLKL 4HB domain. Intriguingly, mutation of a second cluster of residues on the 4HB domain did not preclude membrane localization or oligomerization but did prevent cell death, illustrating that additional function(s) beyond membrane translocation are required for the 4HB domain to induce cell death. MLKL oligomerization and membrane translocation were also inhibited by a small molecule, compound 1, which we identified on the basis of its affinity for the nucleotide binding site of the MLKL pseudokinase domain. These data support a model for MLKL function whereby the pseudokinase domain of MLKL holds the 4HB domain in check until phosphorylated by RIPK3, which causes a conformational change in the pseudokinase domain to unleash the 4HB domain to oligomerize and associate with membranes. Activation of MLKL can be thwarted by a small MLKL binding molecule, indicating the feasibility of targeting the nucleotide binding or “pseudoactive” sites of pseudokinases, a hitherto unexplored class of therapeutic targets.     

Endoscopic Internal Drainage with Enteral Nutrition (EDEN) for Treatment of Leaks Following Sleeve Gastrectomy

Background

Endoscopic treatment of gastric leaks (GL) following sleeve gastrectomy (SG) involves different techniques; however, standard management is not yet established. We report our experience about endoscopic internal drainage of leaks using pigtail stents coupled with enteral nutrition (EDEN) for 4 to 6 weeks until healing is achieved.

Methods

In 21 pts (18 F, 41 years), one or two plastic pigtail stents were delivered across the leak 25.6 days (4–98) post-surgery. In all patients, nasojejunal tube was inserted. Check endoscopy was done at 4 to 6 weeks with either restenting if persistent leak, or removal if no extravasation of contrast in peritoneal cavity, or closure with an Over-the-Scope Clip® (OTSC®) if contrast opacifying the crossing stent without concomitant peritoneal extravasation.

Results

Twenty-one out of 21 (100 %) patients underwent check endoscopy at average of 30.15 days (26–45) from stenting. In 7/21 (33.3 %) patients leak sealed, 2/7 needed OTSC®. Second check endoscopy, 26.7 days (25–42) later, showed sealed leak in 10 out 14; 6/10 had OTSC®. Four required restenting. One patient, 28 days later, needed OTSC®. One healed at 135 days and another 180 days after four and seven changes, respectively. One patient is currently under treatment. In 20/21 (95.2 %), GL have healed with EID treatment of 55.5 days (26–?180); all are asymptomatic on a normal diet at average follow-up of 150.3 days (20–276).

Conclusions

EDEN is a promising therapeutic approach for treating leaks following SG. Multiple endoscopic sessions may be required.     

Reinfection risk of novel coronavirus (COVID-19): A systematic ‎review of current evidence

BACKGROUNDThere is recently a concern regarding the reinfection and reactivation of previously reCoVered coronavirus disease 2019 (CoVID-19) patients.AIMTo summarize the recent findings and reports of CoVID-19 reinfection in patients previously reCoVered from the disease.METHODSThis study was a systematic review of current evidence conducted in August 2020. The authors studied the probable reinfection risk of novel coronavirus (CoVID-19). We performed a systematic search using the keywords in online databases. The investigation adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist to ensure the reliability and validity of this study and results.RESULTSWe reviewed 31 studies. Eight studies described reCoVered patients with reinfection. Only one study reported reinfected patients who died. In 26 studies, there was no information about the status of the patients. Several studies indicated that reinfection is not probable and that post-infection immunity is at least temporary and short.CONCLUSIONBased on our review, we concluded that a positive polymerase chain reaction retest could be due to several reasons and should not always be considered as reinfection or reactivation of the disease. Most relevant studies in positive retest patients have shown relative and probably temporary immunity after the reCoVery of the disease.     

The use of external mesh reinforcement to reduce intimal hyperplasia and preserve the structure of human saphenous veins

The saphenous vein is the conduit of choice in bypass graft procedures. Haemodynamic factors play a major role in the development of intimal hyperplasia (IH), and subsequent bypass failure. To evaluate the potential protective effect of external reinforcement on such a failure, we developed an ex vivo model for the perfusion of segments of human saphenous veins under arterial shear stress. In veins submitted to pulsatile high pressure (mean pressure at 100 mmHg) for 3 or 7 days, the use of an external macroporous polyester mesh 1) prevented the dilatation of the vessel, 2) decreased the development of IH, 3) reduced the apoptosis of smooth muscle cells, and the subsequent fibrosis of the media layer, 4) prevented the remodelling of extracellular matrix through the up-regulation of matrix metalloproteinases (MMP-2, MMP-9) and plasminogen activator type I. The data show that, in an experimental ex vivo setting, an external scaffold decreases IH and maintains the integrity of veins exposed to arterial pressure, via increase in shear stress and decrease wall tension, that likely contribute to trigger selective molecular and cellular changes.     

Developmental profile of GABAA-receptors in the marmoset monkey: Expression of distinct subtypes in pre- and postnatal brain

Gamma aminobutyric acid (GABA)_A-receptors are expressed in fetal mammalian brain before the onset of synaptic inhibition, suggesting their involvement in brain development. In this study, we have analyzed the maturation of the GABA_A-receptor in the marmoset monkey forebrain to determine whether distinct receptor subtypes are expressed at particular stages of pre- and postnatal ontogeny. The distribution of the subunits α1, α2, and β2,3 was investigated immunohistochemically between embryonic day 100 (6 weeks before birth) and adulthood. Prenatally, the α2- and β2,3-subunit-immunoreactivity (-IR) was prominent throughout the forebrain, whereas the α1-subunit-IR appeared in selected regions shortly before birth. The α2-subunit-IR disappeared gradually to become restricted to a few regions in adult forebrain. By contrast, the α1-subunit-IR increased dramatically after birth and replaced the α2-subunit in the basal forebrain, pallidum, thalamus, and most of the cerebral cortex. Staining for the β2,3-subunits was ubiquitous at every age examined, indicating their association with either the α1- or the α2-subunit in distinct receptor subtypes. In neocortex, the α1-subunit-IR was first located selectively to layers IV and VI of primary somatosensory and visual areas. Postnatally, it increased throughout the cortex, with the adult pattern being established only during the second year. The switch in expression of the α1- and α2-subunits indicates that the subunit composition of major GABA_A-receptor subtypes changes during ontogeny. This change coincides with synaptogenesis, suggesting that the emergence of α1-GABA_A-receptors parallels the formation of inhibitory circuits. A similar pattern has been reported in rat, indicating that the developmental regulation of GABA_A-receptors is conserved across species, possibly including man. However, the marmoset brain is more mature than the rat brain at the onset of α1-subunit expression, suggesting that α1-GABA_A-receptors are largely dispensable in utero, but may be required for information processing after birth. © 1996 Wiley-Liss, Inc.