Oligomerization-driven MLKL ubiquitylation antagonizes necroptosis.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

CaV 2.1 α1 subunit motifs that control presynaptic CaV 2.1 subtype abundance are distinct from CaV 2.1 preference.

Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2 subtype levels are largely unsolved. Because the CaV 2 α1  subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1  subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1  subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1  subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1  subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1  subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2 subtype abundance and preference remain enigmatic. The CaV α1  subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1  subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1  subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2 subtype abundance but not preference, whereas the CaV 2.2 α1  subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2 subtype abundance or preference. The CaV 2 α1  subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.

Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca2+ signaling.

Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+ ) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

Histologic lesions of cestodiasis in octopuses.

Parasitism of cephalopods is common, including infection with Aggregata spp., Ichthyobodo spp., dicyemids, cestodes of the orders Tetraphyllidea and Trypanorhynchidea, and various crustaceans. Cestodiasis in octopuses is reported, although a full histologic description of lesions has not been previously described. Cestodiasis was identified in 10 octopuses of 4 different species, which included 4 common octopuses (Octopus vulgaris), 3 Caribbean reef octopuses (Octopus briareus), 2 two-spot octopuses (Octopus bimaculoides), and 1 giant Pacific octopus (Enteroctopus dofleini). Larval cestodes were present in the cecum (n = 5), intestines (n = 4), digestive gland (n = 3), chitinous alimentary tract (n = 2), renal appendage (n = 1), and salivary duct (n = 1). In 5 cases, larval cestodes invaded tissue and were associated with hemocytic inflammation and tracts of necrotic tissue in the intestines (n = 3), digestive gland (n = 3), and/or renal appendage (n = 1). When present in the chitinous alimentary tract (esophagus, stomach) or cecum, larval cestodes were in the central lumen and not associated with lesions. One adult cestode was identified in the mantle cavity and was not associated with lesions. Other common concurrent parasitic infections included enteric Aggregata spp. infection, branchial Rickettsia-like organism infection, enteric nematodiasis, and an arthropod-associated branchitis.

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies.

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 to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding 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 mediate membrane translocation, distinct from known phospholipid binding sites.

More Than Results: The Clinical and Research Relationship in the Evolving Detection and Surveillance of SARS-CoV-2.

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, real-time reverse transcription polymerase chain reaction (RT-PCR) became an essential tool for laboratories to provide high-sensitivity qualitative diagnostic testing for patients and real-time data to public health officials. Here we explore the predictive value of quantitative data from RT-PCR cycle threshold (Ct) values in epidemiological measures, symptom presentation, and variant transition. METHODS: To examine the association with hospitalizations and deaths, data from 74,479 patients referred to the Avera Institute for Human Genetics (AIHG) for COVID-19 testing in 2020 were matched by calendar week to epidemiological data reported by the South Dakota Department of Health. We explored the association between symptom data, patient age, and Ct values for 101 patients. We also explored changes in Ct values during variant transition detected by genomic surveillance sequencing of the AIHG testing population during 2021. RESULTS: Measures from AIHG diagnostic testing strongly explain variance in the South Dakota state positivity percentage (R2 = 0.758), a two-week delay in hospitalizations (R2 = 0.856), and a four-week delay in deaths (R2 = 0.854). Based on factor analysis of patient symptoms, three groups could be distinguished which had different presentations of age, Ct value, and time from collection. Additionally, conflicting Ct value results among SARSCoV- 2 variants during variant transition may reflect the community transmission dynamics. CONCLUSIONS: Measures of Ct value in RT-PCR diagnostic assays combined with routine screening have valuable applications in monitoring the dynamics of SARS-CoV-2 within communities.

The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.

Mixed lineage kinase domain-like (MLKL) is a component of the "necrosome," the multiprotein complex that triggers tumor necrosis factor (TNF)-induced cell death by necroptosis. To define the specific role and molecular mechanism of MLKL action, we generated MLKL-deficient mice and solved the crystal structure of MLKL. Although MLKL-deficient mice were viable and displayed no hematopoietic anomalies or other obvious pathology, cells derived from these animals were resistant to TNF-induced necroptosis unless MLKL expression was restored. Structurally, MLKL comprises a four-helical bundle tethered to the pseudokinase domain, which contains an unusual pseudoactive site. Although the pseudokinase domain binds ATP, it is catalytically inactive and its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation. Structure-guided mutation of the MLKL pseudoactive site resulted in constitutive, RIPK3-independent necroptosis, demonstrating that modification of MLKL is essential for propagation of the necroptosis pathway downstream of RIPK3.

SMCHD1's ubiquitin-like domain is required for N-terminal dimerization and chromatin localization.

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. Our current understanding of SMCHD1's molecular mechanism, and how substitutions within SMCHD1 lead to the diseases, facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS), are only emerging. Recent structural studies of its two component domains - the N-terminal ATPase and C-terminal SMC hinge - suggest that dimerization of each domain plays a central role in SMCHD1 function. Here, using biophysical techniques, we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal UBL (Ubiquitin-like) domain and ATP binding. We show that neither the dimerization event, nor the presence of a C-terminal extension past the transducer domain, affect SMCHD1's in vitro catalytic activity as the rate of ATP turnover remains comparable to the monomeric protein. We further examined the functional importance of the N-terminal UBL domain in cells, revealing that its targeted deletion disrupts the localization of full-length SMCHD1 to chromatin. These findings implicate UBL-mediated SMCHD1 dimerization as a crucial step for chromatin interaction, and thereby for promoting SMCHD1-mediated gene silencing.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Presynaptic voltage-gated calcium channels in the auditory brainstem.

Sound information encoding within the initial synapses in the auditory brainstem requires reliable and precise synaptic transmission in response to rapid and large fluctuations in action potential (AP) firing rates. The magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (CaV) in the presynaptic terminal are key determinants in triggering AP-mediated release. In the mammalian central nervous system (CNS), the CaV2.1 subtype is the critical subtype for CNS function, since it is the most efficient CaV2 subtype in triggering AP-mediated synaptic vesicle (SV) release. Auditory brainstem synapses utilize CaV2.1 to sustain fast and repetitive SV release to encode sound information. Therefore, understanding the presynaptic mechanisms that control CaV2.1 localization, organization and biophysical properties are integral to understanding auditory processing. Here, we review our current knowledge about the control of presynaptic CaV2 abundance and organization in the auditory brainstem and impact on the regulation of auditory processing.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA.

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

Suppression of cytokine signaling by SOCS3: characterization of the mode of inhibition and the basis of its specificity.

Janus kinases (JAKs) are key effectors in controlling immune responses and maintaining hematopoiesis. SOCS3 (suppressor of cytokine signaling-3) is a major regulator of JAK signaling and here we investigate the molecular basis of its mechanism of action. We found that SOCS3 bound and directly inhibited the catalytic domains of JAK1, JAK2, and TYK2 but not JAK3 via an evolutionarily conserved motif unique to JAKs. Mutation of this motif led to the formation of an active kinase that could not be inhibited by SOCS3. Surprisingly, we found that SOCS3 simultaneously bound JAK and the cytokine receptor to which it is attached, revealing how specificity is generated in SOCS action and explaining why SOCS3 inhibits only a subset of cytokines. Importantly, SOCS3 inhibited JAKs via a noncompetitive mechanism, making it a template for the development of specific and effective inhibitors to treat JAK-based immune and proliferative diseases.

Presynaptic Mitochondria Volume and Abundance Increase during Development of a High-Fidelity Synapse.

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse, which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semiautomated segmentation of mitochondria, high-throughput whole-terminal reconstruction, and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day (P)7 and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.SIGNIFICANCE STATEMENT Elucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

Presynaptic development is controlled by the core active zone proteins CAST/ELKS.

KEY POINTS: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held. CAST/ELKS regulate all three CaV 2 subtype channel levels in the presynaptic terminal and not just CaV 2.1. The half-life of ELKS is on the timescale of days and not weeks. Synaptic transmission was not impacted by the loss of CAST/ELKS. CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. ABSTRACT: Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z-stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV 2 channel subtype currents that correlated with a loss in presynaptic CaV 2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV 2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV 2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation.

Inhibitory Signaling to Ion Channels in Hippocampal Neurons Is Differentially Regulated by Alternative Macromolecular Complexes of RGS7.

The neuromodulatory effects of GABA on pyramidal neurons are mediated by GABAB receptors (GABABRs) that signal via a conserved G-protein-coupled pathway. Two prominent effectors regulated by GABABRs include G-protein inwardly rectifying K+ (GIRK) and P/Q/N type voltage-gated Ca2+ (CaV2) ion channels that control excitability and synaptic output of these neurons, respectively. Regulator of G-protein signaling 7 (RGS7) has been shown to control GABAB effects, yet the specificity of its impacts on effector channels and underlying molecular mechanisms is poorly understood. In this study, we show that hippocampal RGS7 forms two distinct complexes with alternative subunit configuration bound to either membrane protein R7BP (RGS7 binding protein) or orphan receptor GPR158. Quantitative biochemical experiments show that both complexes account for targeting nearly the entire pool of RGS7 to the plasma membrane. We analyzed the effect of genetic elimination in mice of both sexes and overexpression of various components of RGS7 complex by patch-clamp electrophysiology in cultured neurons and brain slices. We report that RGS7 prominently regulates GABABR signaling to CaV2, in addition to its known involvement in modulating GIRK. Strikingly, only complexes containing R7BP, but not GPR158, accelerated the kinetics of both GIRK and CaV2 modulation by GABABRs. In contrast, GPR158 overexpression exerted the opposite effect and inhibited RGS7-assisted temporal modulation of GIRK and CaV2 by GABA. Collectively, our data reveal mechanisms by which distinctly composed macromolecular complexes modulate the activity of key ion channels that mediate the inhibitory effects of GABA on hippocampal CA1 pyramidal neurons.SIGNIFICANCE STATEMENT This study identifies the contributions of distinct macromolecular complexes containing a major G-protein regulator to controlling key ion channel function in hippocampal neurons with implications for understanding molecular mechanisms underlying synaptic plasticity, learning, and memory.

Patterns of immune-cell infiltration in murine models of melanoma: roles of antigen and tissue site in creating inflamed tumors.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/ß-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/ß-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level.

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.

Ca2+ channel to synaptic vesicle distance accounts for the readily releasable pool kinetics at a functionally mature auditory synapse.

Precise regulation of synaptic vesicle (SV) release at the calyx of Held is critical for auditory processing. At the prehearing calyx of Held, synchronous and asynchronous release is mediated by fast and slow releasing SVs within the readily releasable pool (RRP). However, the posthearing calyx has dramatically different release properties. Whether developmental alterations in RRP properties contribute to the accelerated release time course found in posthearing calyces is not known. To study these questions, we performed paired patch-clamp recordings, deconvolution analysis, and numerical simulations of buffered Ca(2+) diffusion and SV release in postnatal day (P) 16-19 mouse calyces, as their release properties resemble mature calyces of Held. We found the P16-P19 calyx RRP consists of two pools: a fast pool (τ ≤ 0.9 ms) and slow pool (τ ∼4 ms), in which release kinetics and relative composition of the two pools were unaffected by 5 mm EGTA. Simulations of SV release from the RRP revealed that two populations of SVs were necessary to reproduce the experimental release rates: (1) SVs located close (∼5-25 nm) and (2) more distal (25-100 nm) to VGCC clusters. This positional coupling was confirmed by experiments showing 20 mm EGTA preferentially blocked distally coupled SVs. Lowering external [Ca(2+)] to in vivo levels reduced only the fraction SVs released from the fast pool. Therefore, we conclude that a dominant parameter regulating the mature calyx RRP release kinetics is the distance between SVs and VGCC clusters.