Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The ...precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway.
The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting ...the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.
Base excision repair (BER) initiated by alkyladenine DNA glycosylase (AAG) is essential for removal of aberrantly methylated DNA bases. Genome instability and accumulation of aberrant bases accompany ...multiple diseases, including cancer and neurological disorders. While BER is well studied on naked DNA, it remains unclear how BER efficiently operates on chromatin. Here, we show that AAG binds to chromatin and forms complex with RNA polymerase (pol) II. This occurs through direct interaction with Elongator and results in transcriptional co-regulation. Importantly, at co-regulated genes, aberrantly methylated bases accumulate towards the 3'end in regions enriched for BER enzymes AAG and APE1, Elongator and active RNA pol II. Active transcription and functional Elongator are further crucial to ensure efficient BER, by promoting AAG and APE1 chromatin recruitment. Our findings provide insights into genome stability maintenance in actively transcribing chromatin and reveal roles of aberrantly methylated bases in regulation of gene expression.
The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage ...Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation tomembranes where activatedMLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobodybinding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediatemembrane translocation, distinct from known phospholipid binding sites.
Although emotions are reported in a large majority of dreams, little is known about the factors that account for night-to-night and person-to-person variations in people's experience of dream affect. ...We investigated the relationship between waking trait and state variables and dream affect by testing multilevel models intended to predict the affective valence of people's everyday dreams. Participants from the general population completed measures of personality and trauma history followed by a three-week daily journal in which they noted dream recall, valence of dreamed emotions and level of perceived stress for the day as well as prior to sleep onset. Within-subject effects accounted for most of the explained variance in the reported valence of dream affect. Trait anxiety was the only variable that significantly predicted dream emotional valence at the between-subjects level. In addition to highlighting the need for more fine-grained measures in this area of research, our results point to methodological limitations and biases associated with retrospective estimates of general dream affect and bring into focus state variables that may best explain observed within-subject variance in emotions experienced in everyday dreams.
A fast-growing field of research focuses on microbial biocontrol in the phyllosphere. Phyllosphere microorganisms possess a wide range of adaptation and biocontrol factors, which allow them to adapt ...to the phyllosphere environment and inhibit the growth of microbial pathogens, thus sustaining plant health. These biocontrol factors can be categorized in direct, microbe-microbe, and indirect, host-microbe, interactions. This review gives an overview of the modes of action of microbial adaptation and biocontrol in the phyllosphere, the genetic basis of the mechanisms, and examples of experiments that can detect these mechanisms in laboratory and field experiments. Detailed insights in such mechanisms are key for the rational design of novel microbial biocontrol strategies and increase crop protection and production. Such novel biocontrol strategies are much needed, as ensuring sufficient and consistent food production for a growing world population, while protecting our environment, is one of the biggest challenges of our time.
Necroptosis is a lytic programmed cell death pathway with origins in innate immunity that is frequently dysregulated in inflammatory diseases. The terminal effector of the pathway, MLKL, is licensed ...to kill following phosphorylation of its pseudokinase domain by the upstream regulator, RIPK3 kinase. Phosphorylation provokes the unleashing of MLKL's N-terminal four-helix bundle (4HB or HeLo) domain, which binds and permeabilizes the plasma membrane to cause cell death. The precise mechanism by which the 4HB domain permeabilizes membranes, and how the mechanism differs between species, remains unclear. Here, we identify the membrane binding epitope of mouse MLKL using NMR spectroscopy. Using liposome permeabilization and cell death assays, we validate K69 in the α3 helix, W108 in the α4 helix, and R137/Q138 in the first brace helix as crucial residues for necroptotic signaling. This epitope differs from the phospholipid binding site reported for human MLKL, which comprises basic residues primarily located in the α1 and α2 helices. In further contrast to human and plant MLKL orthologs, in which the α3-α4 loop forms a helix, this loop is unstructured in mouse MLKL in solution. Together, these findings illustrate the versatility of the 4HB domain fold, whose lytic function can be mediated by distinct epitopes in different orthologs.
In a series of three visual perspective-taking experiments, we asked adult participants to judge their own or someone else's visual perspective in situations where both perspectives were either the ...same or different. We found that participants could not easily ignore what someone else saw when making self-perspective judgments. This was observed even when participants were only required to take their own perspective within the same block of trials (Experiment 2) or even within the entire experiment (Experiment 3), i.e. under conditions which gave participants a clear opportunity to adopt a strategy of ignoring the other person's irrelevant perspective. Under some circumstances, participants were also more efficient at judging the other person's perspective than at judging their own perspective. Collectively, these results suggest that adults make use of rapid and efficient processes to compute what other people can see.
The ongoing global pandemic of coronavirus disease 2019 (COVID-19) has rapidly disrupted traditional modes of operation in health care and education. In March 2020, institutions in the United States ...began to implement a range of policies to discourage direct contact and encourage social distancing. These measures have placed us in an unprecedented position where education can no longer occur at close quarters-most notably, around a multiheaded microscope-but must instead continue at a distance. This guide is intended to be a resource for pathologists and pathologists-in-training who wish to leverage technology to continue collaboration, teaching, and education in this era. The article is focused mainly on anatomic pathology; however, the technologies easily lend themselves to clinical pathology education as well. Our aim is to provide curated lists of various online resources that can be used for virtual learning in pathology, provide tips and tricks, and share our personal experience with these technologies. The lists include videoconferencing platforms; pathology Web sites; free online educational resources, including social media; and whole slide imaging collections. We are currently living through a unique situation without a precedent or guidebook, and we hope that this guide will enable the community of pathology educators worldwide to embrace the opportunities that 21st century technology provides.
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