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Purpose: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly., Methods: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test., Results: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in 3 consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the 4 families shared a founder homozygous variant, whereas the third and fourth had different homozygous variants in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays., Conclusion: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Necroptosis is a lytic and pro-inflammatory form of programmed cell death executed by the terminal effector, the MLKL (mixed lineage kinase domain-like) pseudokinase. Downstream of death and Toll-like receptor stimulation, MLKL is trafficked to the plasma membrane via the Golgi-, actin- and microtubule-machinery, where activated MLKL accumulates until a critical lytic threshold is exceeded and cell death ensues. Mechanistically, MLKL's lytic function relies on disengagement of the N-terminal membrane-permeabilising four-helix bundle domain from the central autoinhibitory brace helix: a process that can be experimentally mimicked by introducing the R30E MLKL mutation to induce stimulus-independent cell death. Here, we screened a library of 429 kinase inhibitors for their capacity to block R30E MLKL-mediated cell death, to identify co-effectors in the terminal steps of necroptotic signalling. We identified 13 compounds - ABT-578, AR-A014418, AZD1480, AZD5363, Idelalisib, Ipatasertib, LJI308, PHA-793887, Rapamycin, Ridaforolimus, SMI-4a, Temsirolimus and Tideglusib - each of which inhibits mammalian target of rapamycin (mTOR) signalling or regulators thereof, and blocked constitutive cell death executed by R30E MLKL. Our study implicates mTOR signalling as an auxiliary factor in promoting the transport of activated MLKL oligomers to the plasma membrane, where they accumulate into hotspots that permeabilise the lipid bilayer to cause cell death., (© 2024 The Author(s).)

Objective: To determine the ability of bacteria commonly isolated from equine limb wounds to survive in saltwater obtained from an equine hydrotherapy unit at different salinity concentrations and temperatures., Methods: Saltwater samples were obtained over a 2-week period (January 22, 2024 to February 2, 2024) from an equine hydrotherapy unit used for clinical patients, kept at either full salinity per manufacturer recommendations or diluted to half salinity to mimic the dilution that likely occurs in the clinical setting between cases when holding tanks are replenished with tap water only. Samples were then autoclaved to eliminate preexisting bacterial contamination before individual inoculation with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus zooepidemicus. Each inoculated sample was maintained at 2, 22, or 44 °C to represent typical holding tank conditions. The bacterial concentration was determined at each condition every 24 hours up to and including 96 hours. The lower limit of detection was set at 1 CFU/mL., Results: Salinity did not affect bacterial survival. Bacterial concentrations generally decreased with increasing temperature over time. Escherichia coli, S aureus, and S zooepidemicus concentrations decreased to the lower limit of detection at 44 °C by 24 to 48 hours, while P aeruginosa concentrations significantly decreased over 24 hours but remained well above the lower limit of detection., Conclusions: Common bacterial isolates of equine limb wounds can survive in typical saltwater hydrotherapy conditions., Clinical Relevance: Further investigation is warranted to determine the clinical relevance of these findings including protocols for hydrotherapy unit disinfection, wastage of treatment water, and case inclusion/exclusion criteria.

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators - Caspase-8, RIPK1, RIPK3 and MLKL - in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo., (© 2024. The Author(s).)

Steroid-associated laminitis remains a major concern with use of corticosteroids in horses. Individual case factors such as joint pathology, pre-existing endocrinopathies, or corticosteroid type, dose, and timing influencing steroid-induced laminitis risk have not been investigated. This study aimed to determine if systemic absorption of triamcinolone acetonide (TA) varies between intrasynovial (antebrachiocarpal) and extrasynovial (sacroiliac) injection sites, and to determine the effects of TA absorption on glucose, insulin, cortisol, and adrenocorticotropic hormone (ACTH). Twenty adult horses were randomized into antebrachiocarpal or sacroiliac joint injection groups, and each horse received bilateral injections with a total dose of 18 mg triamcinolone. Blood was collected prior to injection and at 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 60, and 72 h post-injection. Peak TA absorption occurred at 8 h in both groups, and was significantly higher in the intrasynovial group compared to the extrasynovial group (1.397 ng/mL, 0.672 ng/mL, p  < 0.05). Plasma TA levels were significantly higher in the intrasynovial group from 8 to 36 h post-injection ( p  < 0.05). There was no difference in glucose, insulin, cortisol, or ACTH between groups at any time point. Insulin and glucose were significantly increased from baseline at all timepoints from 10-72 h and 1-72 h post-injection, respectively. Horses with elevated baseline insulin values (>20 μU/mL) from both groups experienced a more marked hyperinsulinemia, reaching a mean peak insulin of 197.5 μU/mL as compared to 90.06 μU/mL in those with normal baseline insulin. Cortisol and ACTH were significantly decreased from baseline at timepoints from 4-72 h post-injection in both groups. This study is the first to evaluate drug absorption from the sacroiliac site and demonstrates that drug absorption varies between intrasynovial and extrasynovial injection sites. TA absorption causes metabolic derangements, most notably a marked hyperinsulinemia that is more severe in horses with elevated baseline insulin values. The influence of baseline endocrinopathies on response to corticosteroid administration as well as the effect of corticosteroid-induced metabolic derangements warrant further investigation as risk factors for corticosteroid-associated laminitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hallowell, Dembek, Horne, Knych, Messenger and Schnabel.)

Tripartite ATP-independent periplasmic (TRAP) transporters are nutrient-uptake systems found in bacteria and archaea. These evolutionary divergent transporter systems couple a substrate-binding protein (SBP) to an elevator-type secondary transporter, which is a first-of-its-kind mechanism of transport. Here, we highlight breakthrough TRAP transporter structures and recent functional data that probe the mechanism of transport. Furthermore, we discuss recent structural and biophysical studies of the ion transporter superfamily (ITS) members and highlight mechanistic principles that are relevant for further exploration of the TRAP transporter system., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Continual advancements in diagnostic imaging have allowed for more accurate and complete diagnoses of injuries in the performance horse. The use of several different imaging tools has further allowed the equine sports medicine clinician to more carefully direct treatment options, monitor response to therapy and guide rehabilitation recommendations. The advancements in diagnostic imaging and novel treatment options have led to the improvement in the overall prognosis of many injuries that affect the horse and their performance. The purpose of this section is to review the advancements made in diagnostic imaging of the horse and to aid the practitioner in the selection of the appropriate modality and how best to use them to guide treatment and monitoring decisions., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Current pharmacological treatments for bipolar disorder are inadequate and based on serendipitously discovered drugs often with limited efficacy, burdensome side-effects, and unclear mechanisms of action. Advances in drug development for the treatment of bipolar disorder remain incremental and have come largely from repurposing drugs used for other psychiatric conditions, a strategy that has failed to find truly revolutionary therapies, as it does not target the mood instability that characterises the condition. The lack of therapeutic innovation in the bipolar disorder field is largely due to a poor understanding of the underlying disease mechanisms and the consequent absence of validated drug targets. A compelling new treatment target is the Ca 2+ -calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme. CaMKK2 is highly enriched in brain neurons and regulates energy metabolism and neuronal processes that underpin higher order functions such as long-term memory, mood, and other affective functions. Loss-of-function polymorphisms and a rare missense mutation in human CAMKK2 are associated with bipolar disorder, and genetic deletion of Camkk2 in mice causes bipolar-like behaviours similar to those in patients. Furthermore, these behaviours are ameliorated by lithium, which increases CaMKK2 activity. In this review, we discuss multiple convergent lines of evidence that support targeting of CaMKK2 as a new treatment strategy for bipolar disorder., (© 2023. The Author(s).)

The necroptosis pathway is a lytic, pro-inflammatory mode of cell death that is widely implicated in human disease, including renal, pulmonary, gut and skin inflammatory pathologies. The precise mechanism of the terminal steps in the pathway, where the RIPK3 kinase phosphorylates and triggers a conformation change and oligomerization of the terminal pathway effector, MLKL, are only emerging. Here, we structurally identify RIPK3-mediated phosphorylation of the human MLKL activation loop as a cue for MLKL pseudokinase domain dimerization. MLKL pseudokinase domain dimerization subsequently drives formation of elongated homotetramers. Negative stain electron microscopy and modelling support nucleation of the MLKL tetramer assembly by a central coiled coil formed by the extended, ~80 Å brace helix that connects the pseudokinase and executioner four-helix bundle domains. Mutational data assert MLKL tetramerization as an essential prerequisite step to enable the release and reorganization of four-helix bundle domains for membrane permeabilization and cell death., (© 2023. The Author(s).)

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. Here we show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKL S132P in biological membranes and MLKL S132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-β induced death in non-hematopoietic cells. In vivo, Mlkl S131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease., (© 2023. Springer Nature Limited.)

The past decade has seen the emergence of the necroptosis programmed cell death pathway as an important contributor to the pathophysiology of myriad diseases. The receptor interacting protein kinase (RIPK)1 and RIPK3, and the pseudokinase executioner protein, mixed lineage kinase domain-like (MLKL), have grown to prominence as the core pathway components. Depending on cellular context, these proteins also serve as integrators of signals, such as post-translational modifications and protein or metabolite interactions, adding layers of complexity to pathway regulation. Here, we describe the emerging picture of the web of proteins that tune necroptotic signal transduction and how these events have diverged across species, presumably owing to selective pressures of pathogens upon the RIPK3-MLKL protein pair., Competing Interests: Declaration of interests C.R.H, A.L.S. and J.M.M. contribute to a project developing necroptosis inhibitors in collaboration with Anaxis Pty Ltd., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Limited information exists regarding associations between distal interphalangeal joint (DIPJ) abnormalities and synovial invagination changes in the distal sesamoid (navicular) bone. This retrospective, analytical study aimed to measure specific characteristics of the synovial invaginations of the navicular bone to determine whether any single characteristic was associated with abnormalities in the DIPJ or navicular apparatus (NA) using high field MRI and a sample of 200 horses' feet. The DIPJ and NA were graded independently by three scorers. The grades were averaged, creating a global pathology score for the DIPJ, NA, and synovial invaginations. Higher global scores represented more severe pathology. The number of invaginations, depth of penetration, invagination shape, and cross-sectional area (CSA) of the largest invagination were recorded. Interobserver agreement was measured using Cohen's Kappa. Associations of global scores of the DIPJ and NA with individual invagination characteristics were assessed using linear mixed modeling. A significant relationship was found between the number of invaginations and global DIPJ score, with higher invagination numbers associated with higher DIPJ scores. For invagination depth and CSA, a significant relationship was noted with global scores of both the DIPJ and NA. Reliable relationships between the shape of synovial invaginations and global scores of DIPJ and NA were not found, likely due to poor interobserver scoring (0.305). These findings suggest that primary DIPJ disease and NA pathology should be considered when noticing alterations to navicular synovial invaginations on MRI. This contrasts traditional views that synovial invagination abnormalities are indicative solely of NA pathology., (© 2022 The Authors. Veterinary Radiology & Ultrasound published by Wiley Periodicals LLC on behalf of American College of Veterinary Radiology.)

Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL., (© 2022. The Author(s).)

Transcription is the principal control point for bacterial gene expression, and it enables a global cellular response to an intracellular or environmental trigger. Transcriptional regulation is orchestrated by transcription factors, which activate or repress transcription of target genes by modulating the activity of RNA polymerase. Dissecting the nature and precise choreography of these interactions is essential for developing a molecular understanding of transcriptional regulation. While the contribution of X-ray crystallography has been invaluable, the 'resolution revolution' of cryo-electron microscopy has transformed our structural investigations, enabling large, dynamic and often transient transcription complexes to be resolved that in many cases had resisted crystallisation. In this review, we highlight the impact cryo-electron microscopy has had in gaining a deeper understanding of transcriptional regulation in bacteria. We also provide readers working within the field with an overview of the recent innovations available for cryo-electron microscopy sample preparation and image reconstruction of transcription complexes., (© 2021 The Author(s).)

Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling., (© 2021. The Author(s).)

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., (© 2021. The Author(s).)

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021 The Author(s).)

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., (© 2021 The Author(s).)

Necroptosis is a lytic, inflammatory cell death pathway that is dysregulated in many human pathologies. The pathway is executed by a core machinery comprising the RIPK1 and RIPK3 kinases, which assemble into necrosomes in the cytoplasm, and the terminal effector pseudokinase, MLKL. RIPK3-mediated phosphorylation of MLKL induces oligomerization and translocation to the plasma membrane where MLKL accumulates as hotspots and perturbs the lipid bilayer to cause death. The precise choreography of events in the pathway, where they occur within cells, and pathway differences between species, are of immense interest. However, they have been poorly characterized due to a dearth of validated antibodies for microscopy studies. Here, we describe a toolbox of antibodies for immunofluorescent detection of the core necroptosis effectors, RIPK1, RIPK3, and MLKL, and their phosphorylated forms, in human and mouse cells. By comparing reactivity with endogenous proteins in wild-type cells and knockout controls in basal and necroptosis-inducing conditions, we characterise the specificity of frequently-used commercial and recently-developed antibodies for detection of necroptosis signaling events. Importantly, our findings demonstrate that not all frequently-used antibodies are suitable for monitoring necroptosis by immunofluorescence microscopy, and methanol- is preferable to paraformaldehyde-fixation for robust detection of specific RIPK1, RIPK3, and MLKL signals.

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.

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA) 3 -repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA) 3 -repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

In this issue of Structure, Lange et al. (2020) report the structure of the pseudokinase domain of IRAK3, a negative regulator of Myddosome inflammatory signaling. The IRAK3 pseudokinase domain forms a head-to-head dimer, suggesting a new mode of kinase/pseudokinase allostery by which IRAK3 could attenuate the activity of IRAK4 in cells., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The life cycle of Baculoviridae family insect viruses depends on the viral protein kinase, PK-1, to phosphorylate the regulatory protein, p6.9, to induce baculoviral genome release. Here, we report the crystal structure of Cydia pomenella granulovirus PK-1, which, owing to its likely ancestral origin among host cell AGC kinases, exhibits a eukaryotic protein kinase fold. PK-1 occurs as a rigid dimer, where an antiparallel arrangement of the αC helices at the dimer core stabilizes PK-1 in a closed, active conformation. Dimerization is facilitated by C-lobe:C-lobe and N-lobe:N-lobe interactions between protomers, including the domain-swapping of an N-terminal helix that crowns a contiguous β-sheet formed by the two N-lobes. PK-1 retains a dimeric conformation in solution, which is crucial for catalytic activity. Our studies raise the prospect that parallel, side-to-side dimeric arrangements that lock kinase domains in a catalytically-active conformation could function more broadly as a regulatory mechanism among eukaryotic protein kinases.

Body size is central to ecology at levels ranging from organismal fecundity to the functioning of communities and ecosystems. Understanding temperature-induced variations in body size is therefore of fundamental and applied interest, yet thermal responses of body size remain poorly understood. Temperature-size (T-S) responses tend to be negative (e.g. smaller body size at maturity when reared under warmer conditions), which has been termed the temperature-size rule (TSR). Explanations emphasize either physiological mechanisms (e.g. limitation of oxygen or other resources and temperature-dependent resource allocation) or the adaptive value of either a large body size (e.g. to increase fecundity) or a short development time (e.g. in response to increased mortality in warm conditions). Oxygen limitation could act as a proximate factor, but we suggest it more likely constitutes a selective pressure to reduce body size in the warm: risks of oxygen limitation will be reduced as a consequence of evolution eliminating genotypes more prone to oxygen limitation. Thus, T-S responses can be explained by the 'Ghost of Oxygen-limitation Past', whereby the resulting (evolved) T-S responses safeguard sufficient oxygen provisioning under warmer conditions, reflecting the balance between oxygen supply and demands experienced by ancestors. T-S responses vary considerably across species, but some of this variation is predictable. Body-size reductions with warming are stronger in aquatic taxa than in terrestrial taxa. We discuss whether larger aquatic taxa may especially face greater risks of oxygen limitation as they grow, which may be manifested at the cellular level, the level of the gills and the whole-organism level. In contrast to aquatic species, terrestrial ectotherms may be less prone to oxygen limitation and prioritize early maturity over large size, likely because overwintering is more challenging, with concomitant stronger end-of season time constraints. Mechanisms related to time constraints and oxygen limitation are not mutually exclusive explanations for the TSR. Rather, these and other mechanisms may operate in tandem. But their relative importance may vary depending on the ecology and physiology of the species in question, explaining not only the general tendency of negative T-S responses but also variation in T-S responses among animals differing in mode of respiration (e.g. water breathers versus air breathers), genome size, voltinism and thermally associated behaviour (e.g. heliotherms)., (© 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H 2 O 2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H 2 O 2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H 2 O 2 . This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding how proteins interact with DNA, and particularly the stoichiometry of a protein-DNA complex, is key information needed to elucidate the biological role of the interaction, e.g. transcriptional regulation. Here, we present an emerging analytical ultracentrifugation method that features multi-wavelength detection to characterise complex mixtures by deconvoluting the spectral signals of the interaction partners into separate sedimentation profiles. The spectral information obtained in this experiment provides direct access to the molar stoichiometry of the interacting system to complement traditional hydrodynamic information. We demonstrate this approach by characterising a multimeric assembly process between the transcriptional repressor of bacterial sialic acid metabolism, NanR and its DNA-binding sequence. The method introduced in this study can be extended to quantitatively analyse any complex interaction in solution, providing the interaction partners have different optical properties.

While humans lack the biosynthetic pathways for meso -diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum ( Vs DapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso -diaminopimelate. Kinetic studies show that Vs DapL follows a Michaelis-Menten mechanism, with a K M app of 4.0 mM toward its substrate l,l-diaminopimelate. The k cat (0.46 s -1 ) and the k cat / K M (115 s -1 M -1 ) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that Vs DapL exists in a monomer-dimer self-association, with a K D 2-1 of 7.4 μM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in Vs DapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

Human pathogenic and commensal bacteria have evolved the ability to scavenge host-derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known-here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N-acetyl-d-glucosamine, N-acetyl-d-galactosamine and N-acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N-acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate-a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism., (© 2019 Wiley Periodicals, Inc.)

In environments where glucose is limited, some pathogenic bacteria metabolize host-derived sialic acid as a nutrient source. N -Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N -acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, ORF, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding E X GH motif is altered to E X GY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK ( Sa NanK), which is the first characterization of a Gram-positive NanK. We report the kinetic activity of Sa NanK along with the ligand-free, N -acetylmannosamine-bound and substrate analog GlcNAc-bound crystal structures (2.33, 2.20, and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that Sa NanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the E X GY motif reveals that the tyrosine binds to the N -acetyl group to select for the "boat" conformation of N -acetylmannosamine. Moreover, Sa NanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterization provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections., (© 2020 Coombes et al.)

Variation in the degree of sexual size dimorphism (SSD) among taxa is generally considered to arise from differences in the relative intensity of male-male competition and fecundity selection. One might predict, therefore, that SSD will vary systematically with (1) the intensity of sexual selection for increased male size, and (2) the intensity of fecundity selection for increased female size. To test these two fundamental hypotheses, we conducted a phylogenetic comparative analysis of SSD in fish. Specifically, using records of body length at first sexual maturity from FishBase, we quantified variation in the magnitude and direction of SSD in more than 600 diverse freshwater and marine fish species, from sticklebacks to sharks. Although female-biased SSD was common, and thought to be driven primarily by fecundity selection, variation in SSD was not dependent on either the allometric scaling of reproductive energy output or fecundity in female fish. Instead, systematic patterns based on habitat and life-history characteristics associated with varying degrees of male-male competition and paternal care strongly suggest that adaptive variation in SSD is driven by the intensity of sexual selection for increased male size.

Body size at maturity often varies with environmental conditions, as well as between males and females within a species [termed Sexual Size Dimorphism (SSD)]. Variation in body size clines between the sexes can determine the degree to which SSD varies across environmental gradients. We use a meta-analytic approach to investigate whether major biogeographical and temporal (intra-annually across seasons) body size clines differ systematically between the sexes in arthropods. We consider 329 intra-specific environmental gradients in adult body size across latitude, altitude and with seasonal temperature variation, representing 126 arthropod species from 16 taxonomic orders. On average, we observe greater variability in male than female body size across latitude, consistent with the hypothesis that, over evolutionary time, directional selection has acted more strongly on male than female size. In contrast, neither sex exhibits consistently greater proportional changes in body size than the other sex across altitudinal or seasonal gradients, akin to earlier findings for plastic temperature-size responses measured in the laboratory. Variation in the degree to which body size gradients differ between the sexes cannot be explained by a range of potentially influential factors, including environment type (aquatic vs. terrestrial), voltinism, mean species' body size, degree of SSD, or gradient direction. Ultimately, if we are to make better sense of the patterns (or lack thereof) in SSD across environmental gradients, we require a more detailed understanding of the underlying selective pressures driving clines in body size. Such understanding will provide a more comprehensive hypothesis-driven approach to explaining biogeographical and temporal variation in SSD.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn 2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules., (© 2018 Federation of European Biochemical Societies.)

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

Major biological and biogeographical rules link body size variation with latitude or environmental temperature, and these rules are often studied in isolation. Within multivoltine species, seasonal temperature variation can cause substantial changes in adult body size, as subsequent generations experience different developmental conditions. Yet, unlike other size patterns, these common seasonal temperature-size gradients have never been collectively analysed. We undertake the largest analysis to date of seasonal temperature-size gradients in multivoltine arthropods, including 102 aquatic and terrestrial species from 71 global locations. Adult size declines in warmer seasons in 86% of the species examined. Aquatic species show approximately 2.5-fold greater reduction in size per °C of warming than terrestrial species, supporting the hypothesis that greater oxygen limitation in water than in air forces aquatic species to exhibit greater plasticity in body size with temperature. Total percentage change in size over the annual cycle appears relatively constant with annual temperature range but varies between environments, such that the overall size reduction in aquatic-developing species (approx. 31%) is almost threefold greater than in terrestrial species (approx. 11%). For the first time, we show that strong correlations exist between seasonal temperature-size gradients, laboratory responses and latitudinal-size clines, suggesting that these patterns share common drivers., (© 2017 The Author(s).)