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PI3Kα, consisting of the p110α isoform of the catalytic subunit of PI 3-kinase (encoded by PIK3CA) and the p85α regulatory subunit (encoded by PI3KR1) is activated by growth factor receptors. The identification of common oncogenic mutations in PIK3CA has driven the development of many inhibitors that bind to the ATP-binding site in the p110α subunit. Upon activation, PI3Kα undergoes conformational changes that promote its membrane interaction and catalytic activity, yet the effects of ATP-site directed inhibitors on the PI3Kα membrane interaction are unknown. Using FRET and Biolayer Interferometry assays, we show that a class of ATP-site directed inhibitors represented by GSK2126458 block the growth factor activated PI3KαWT membrane interaction, an activity dependent on the ligand forming specific ATP-site interactions. The membrane interaction for hot spot oncogenic mutations that bypass normal p85α regulatory mechanisms was insensitive to GSK2126458, while GSK2126458 could regulate mutations found outside of these hot spot regions. Our data show that the effect of GSK126458 on the membrane interaction requires the enzyme to revert from its growth factor activated state to a basal state. We find that an ATP substrate analogue can increase the wild type PI3Kα membrane interaction, uncovering a substrate based regulatory event that can be mimicked by different inhibitor chemotypes. Our findings, together with the discovery of small molecule allosteric activators of PI3Kα illustrate that PI3Kα membrane interactions can be modulated by factors related to ligand binding both within the ATP site and at allosteric sites., (Copyright 2024 The Author(s).)

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family., Competing Interests: Declaration of interests K.M.S., J.M., and L.Z. are inventors on patents owned by University of California, San Francisco, covering GTPase-targeting small molecules. K.M.S. has consulting agreements for the following companies, which involve monetary and/or stock compensation: AperTOR, BioTheryX, BridGene Biosciences, Erasca, Exai, G Protein Therapeutics, Genentech, Initial Therapeutics, Kumquat Biosciences, Kura Oncology, Lyterian, Merck, Montara Therapeutics, Nested, Nextech, Revolution Medicines, Rezo, Totus, Type6 Therapeutics, Vevo, Vicinitas, and Wellspring Biosciences (Araxes Pharma). J.E.B. has consulting agreements for the following companies, which involve monetary and/or stock compensation: Reactive Biosciences, Scorpion Therapeutics, and Olema Oncology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Stroke-like migraine attacks after radiation therapy (SMART) syndrome is a rare complication of craniospinal irradiation (CSI). Patients commonly present with headaches, seizures, and paroxysmal focal neurological deficits. There is a dearth of studies reported in the literature with an estimated fewer than 100 cases described since it was initially defined in the mid-1990s. The authors present the case of a 23-year-old patient with a history of childhood medulloblastoma and prior ventriculoperitoneal shunt (VPS), chemotherapy, and CSI who presented with headaches and new-onset seizures. Magnetic resonance imaging (MRI) of the brain showed diffuse left temporoparietal and occipital leptomeningeal and cortical enhancement. However, cerebrospinal fluid (CSF) analysis was unremarkable for neoplastic, infectious, or inflammatory etiology. Initiation of systemic steroid therapy resulted in drastic improvement of the patient's symptoms and prompted antiepileptic drug (AED) wean and persistent resolution of leptomeningeal and cortical contrast enhancement on brain MRI. When evaluating MRI evidence of leptomeningeal enhancement, neurosurgeons should consider SMART syndrome in the differential diagnosis, especially when extensive workup rules out more common causes of this finding such as leptomeningeal disease (LMD). Proper identification of SMART syndrome can lead to timely treatment, avoidance of invasive procedures such as tissue biopsy, and improved clinical outcomes., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Adereti et al.)

Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNAβ1 isoform of the phosphatase calcineurin. We previously identified that CNAβ1 directly binds to FAM126A. Here, we report a cryoelectron microscopic (cryo-EM) structure of a truncated PI4KA complex bound to calcineurin, revealing a unique direct interaction between PI4KA and calcineurin. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and computational analysis show that calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KA's horn domain. We also characterized conserved LTLT and PSISIT calcineurin binding sequences in the C terminus of FAM126A. These dual sites in PI4KA and FAM126A are both in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how calcineurin can regulate PI4KA activity., Competing Interests: Declaration of interests J.E.B. reports personal fees from Scorpion Therapeutics and Reactive therapeutics and research contracts from Novartis and Calico Life Sciences., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIa/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease., Competing Interests: Competing Interests J.E.B. reports personal fees from Scorpion Therapeutics and Reactive therapeutics; and research contracts from Novartis and Calico Life Sciences.

The gene PIK3CA , encoding the catalytic subunit p110α of PI3Kα, is the second most frequently mutated gene in cancer, with the highest frequency oncogenic mutants occurring in the C-terminus of the kinase domain. The C-terminus has a dual function in regulating the kinase, playing a putative auto-inhibitory role for kinase activity and being absolutely essential for binding to the cell membrane. However, the molecular mechanisms by which these C-terminal oncogenic mutations cause PI3Kα overactivation remain unclear. To understand how a spectrum of C-terminal mutations of PI3Kα alter kinase activity compared to the WT, we perform unbiased and biased Molecular Dynamics simulations of several C-terminal mutants and report the free energy landscapes for the C-terminal "closed-to-open" transition in the WT, H1047R, G1049R, M1043L and N1068KLKR mutants. Results are consistent with HDX-MS experimental data and provide a molecular explanation why H1047R and G1049R reorient the C-terminus with a different mechanism compared to the WT and M1043L and N1068KLKR mutants. Moreover, we show that in the H1047R mutant, the cavity, where the allosteric ligands STX-478 and RLY-2608 bind, is more accessible contrary to the WT. This study provides insights into the molecular mechanisms underlying activation of oncogenic PI3Kα by C-terminal mutations and represents a valuable resource for continued efforts in the development of mutant selective inhibitors as therapeutics., Competing Interests: JEB reports personal fees from Scorpion Therapeutics, and Reactive therapeutics, and research contracts from Calico Life Sciences., (© 2024 Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome., Competing Interests: TB, MT, XN, AA, SG, IM, SD, AS, AG, MP, JI, JB, HP, FT No competing interests declared, EB JEB reports personal fees from Scorpion Therapeutics, Reactive therapeutics and Olema Oncology, US HLP serves as an advisor to and owns stock in Cerberus Therapeutics. HLP serves as a consultant to Johnson and Johnson, Immatics Therapeutics, Cue Biopharma, Revela Therapeutics, and Tiba Bio, (© 2023, Bates et al.)

Management of venous thromboembolism (VTE) in patients with primary and metastatic brain tumors (BT) is challenging because of the risk of intracranial hemorrhage (ICH). There are no prospective clinical trials evaluating safety and efficacy of direct oral anticoagulants (DOACs), specifically in patients with BT, but they are widely used for VTE in this population. A group of neuro-oncology experts convened to provide practical clinical guidance for the off-label use of DOACs in treating VTE in patients with BT. We searched PubMed for the following terms: BTs, glioma, glioblastoma (GBM), brain metastasis, VTE, heparin, low-molecular-weight heparin (LWMH), DOACs, and ICH. Although prospective clinical trials are needed, the recommendations presented aim to assist clinicians in making informed decisions regarding DOACs for VTE in patients with BT., (© 2024 American Cancer Society.)

Motor performance is monitored continuously by specialized brain circuits and used adaptively to modify behavior on a moment-to-moment basis and over longer time periods. During vocal behaviors, such as singing in songbirds, internal evaluation of motor performance relies on sensory input from the auditory and vocal-respiratory systems. Sensory input from the auditory system to the motor system, often referred to as auditory feedback, has been well studied in singing zebra finches ( Taeniopygia guttata ), but little is known about how and where nonauditory sensory feedback is evaluated. Here we show that brief perturbations in air sac pressure cause short-latency neural responses in the higher-order song control nucleus HVC (used as proper name), an area necessary for song learning and song production. Air sacs were briefly pressurized through a cannula in anesthetized or sedated adult male zebra finches, and neural responses were recorded in both nucleus parambigualis (PAm), a brainstem inspiratory center, and HVC, a cortical premotor nucleus. These findings show that song control nuclei in the avian song system are sensitive to perturbations directly targeted to vocal-respiratory, or viscerosensory, afferents and support a role for multimodal sensory feedback integration in modifying and controlling vocal control circuits. NEW & NOTEWORTHY This study presents the first evidence of sensory input from the vocal-respiratory periphery directly activating neurons in a motor circuit for vocal production in songbirds. It was previously thought that this circuit relies exclusively on sensory input from the auditory system, but we provide groundbreaking evidence for nonauditory sensory input reaching the higher-order premotor nucleus HVC, expanding our understanding of what sensory feedback may be available for vocal control.

Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover., Competing Interests: Competing interests statement:J.E.B. reports personal fees from Scorpion Therapeutics, Reactive therapeutics, and Olema Oncology; and contracts from Novartis and Calico Life Sciences.

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a K d of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The type III Phosphatidylinositol 4-kinase alpha (PI4KA) is an essential lipid kinase that is a master regulator of phosphoinositide signalling at the plasma membrane (PM). It produces the predominant pool of phosphatidylinositol 4-phosphate (PI4P) at the PM, with this being essential in lipid transport and in regulating the PLC and PI3K signalling pathways. PI4KA is essential and is highly conserved in all eukaryotes. In yeast, the PI4KA ortholog stt4 predominantly exists as a heterodimer with its regulatory partner ypp1. In higher eukaryotes, PI4KA instead primarily forms a heterotrimer with a TTC7 subunit (ortholog of ypp1) and a FAM126 subunit. In all eukaryotes PI4KA is recruited to the plasma membrane by the protein EFR3, which does not directly bind PI4KA, but instead binds to the TTC7/ypp1 regulatory partner. Misregulation in PI4KA or its regulatory partners is involved in myriad human diseases, including loss of function mutations in neurodevelopmental and inflammatory intestinal disorders and gain of function in human cancers. This review describes an in-depth analysis of the structure function of PI4KA and its regulatory partners, with a major focus on comparing and contrasting the differences in regulation of PI4KA throughout evolution., Competing Interests: Declaration of competing interest JEB reports personal fees from Scorpion Therapeutics, Reactive therapeutics, and Olema Oncology; and contracts from Novartis and Calico Life Sciences. Other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: To investigate the clinical findings, treatment strategies, and outcomes in dogs with confirmed hepatic abscessation., Design: Retrospective cohort study from 2010 to 2019., Setting: Multicenter study., Animals: Fifty-six client-owned dogs with hepatic abscessation confirmed by culture, cytology, or histopathology., Measurements and Main Results: Dogs were presented for lethargy (39/56), hyporexia (31/56), and vomiting (26/56). Abnormal physical examination findings included increased temperature (41/56) and abdominal pain (22/54). CBCs revealed neutrophilia (31/49), toxic changes (25/49), anemia (28/49), and thrombocytopenia (23/49). Biochemical analyses revealed increased alkaline phosphatase (45/50), increased alanine aminotransferase (40/50), hypoalbuminemia (25/48), and hyperbilirubinemia (19/49). Hypoglycemia was found in 13 of 49 dogs. Hepatic abscesses ranging from 0.5 to 15 cm in diameter were identified ultrasonographically in 37 of 48 dogs; 19 of 37 had solitary abscesses, and 18 of 37 had multifocal abscessation. Escherichia coli was the most commonly cultured organism, isolated in 18 of 42 cases. Histopathology revealed underlying hepatic neoplasia in 10 of 47 dogs. Surgical management was performed in 41 of 49 dogs, and 35 of 41 survived to discharge. Medical management was performed in 8 of 49 dogs, and 5 of 8 survived to discharge. With univariate analysis, hypoglycemia and multifocal abscessation were associated with decreased odds of survival (odds ratio [OR]: 0.2, 95% confidence interval [CI]: 0.03-0.9, P = 0.04; OR: 0.07, 95% CI: 0.01-0.6, P = 0.02, respectively). With multivariate analysis, only multifocal abscessation was associated with decreased odds of survival (OR: 0.09, 95% CI: 0.01-0.87, P = 0.04)., Conclusions: Hepatic abscessation, although rare, should remain a differential diagnosis for dogs presenting with nonspecific clinical signs and increase liver enzyme activities, especially with concurrent increased temperature and neutrophilia. Rate of survival to discharge for dogs in this study was consistent with previously reported survival rates, with 40 of 56 (71%) of the total population surviving to discharge. No variables assessed were able to predict survival to discharge; however, hypoglycemia and multifocal abscessation should be assessed in larger populations to determine prognostic significance., (© Veterinary Emergency and Critical Care Society 2023.)

Lipid phosphoinositides are master regulators of multiple cellular functions. Misregulation of the activity of the lipid kinases that generate phosphoinositides is causative of human diseases, including cancer, neurodegeneration, developmental disorders, immunodeficiencies, and inflammatory disease. This review will present a summary of recent discoveries on the roles of two phosphoinositide kinases (PI4KA and PIKfyve), which have emerged as targets for therapeutic intervention. Phosphatidylinositol 4-kinase alpha (PI4KA) generates PI4P at the plasma membrane and PIKfyve generates PI(3,5)P 2 at endo-lysosomal membranes. Both of these enzymes exist as multi-protein mega complexes that are under myriad levels of regulation. Human disease can be caused by either loss or gain-of-function of these complexes, so understanding how they are regulated will be essential in the design of therapeutics. We will summarize insight into how these enzymes are regulated by their protein-binding partners, with a major focus on the unanswered questions of how their activity is controlled., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JEB reports personal fees from Scorpion Therapeutics, Reactive therapeutics, and Olema Oncology; and contracts from Novartis and Calico Life Sciences. Other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention., Competing Interests: NH, MJ, SN, MR, MP, HR, UD, BM, ES, SH, CY No competing interests declared, JB JEB reports personal fees from Scorpion Therapeutics, Reactive therapeutics and Olema Oncology; and research grants from Novartis, (© 2023, Harris, Jenkins et al.)

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding., (© 2023. The Author(s).)

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Lipid phosphoinositides are master regulators of almost all aspects of a cell's life and death and are generated by the tightly regulated activity of phosphoinositide kinases. Although extensive efforts have focused on drugging class I phosphoinositide 3-kinases (PI3Ks), recent years have revealed opportunities for targeting almost all phosphoinositide kinases in human diseases, including cancer, immunodeficiencies, viral infection and neurodegenerative disease. This has led to widespread efforts in the clinical development of potent and selective inhibitors of phosphoinositide kinases. This Review summarizes our current understanding of the molecular basis for the involvement of phosphoinositide kinases in disease and assesses the preclinical and clinical development of phosphoinositide kinase inhibitors., (© 2022. Springer Nature Limited.)

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gβγ subunits, p110γ-p84 is weakly recruited to membranes by Gβγ subunits alone and requires recruitment by Ras to allow for Gβγ activation. We mapped two distinct Gβγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gβγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated., Competing Interests: Declaration of interests J.E.B. reports personal fees from Scorpion Therapeutics and Olema Oncology and research grants from Novartis., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Akt is a master regulator of pro-growth signaling in the cell. Akt is activated by phosphoinositides that disrupt the autoinhibitory interface between the kinase and pleckstrin homology (PH) domains and then is phosphorylated at T308 and S473. Akt hyperactivation is oncogenic, which has spurred development of potent and selective inhibitors as therapeutics. Using hydrogen deuterium exchange mass spectrometry (HDX-MS), we interrogated the conformational changes upon binding Akt ATP-competitive and allosteric inhibitors. We compared inhibitors against three different states of Akt1. The allosteric inhibitor caused substantive conformational changes and restricts membrane binding. ATP-competitive inhibitors caused extensive allosteric conformational changes, altering the autoinhibitory interface and leading to increased membrane binding, suggesting that the PH domain is more accessible for membrane binding. This work provides unique insight into the autoinhibitory conformation of the PH and kinase domain and conformational changes induced by Akt inhibitors and has important implications for the design of Akt targeted therapeutics., Competing Interests: Declaration of interests J.E.B. reports personal fees from Scorpion Therapeutics and Olema Oncology and research grants from Novartis., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4-kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4-phosphate (PI4P)-enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein-binding partners and post-translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection., (© 2022 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Phosphorylation is a ubiquitous mechanism by which signals are transduced in cells. Protein kinases, enzymes that catalyze the phosphotransfer reaction are, themselves, often regulated by phosphorylation. Paradoxically, however, a substantial fraction of more than 500 human protein kinases are capable of catalyzing their own activation loop phosphorylation. Commonly, these kinases perform this autophosphorylation reaction in trans , whereby transient dimerization leads to the mutual phosphorylation of the activation loop of the opposing protomer. In this study, we demonstrate that protein kinase D (PKD) is regulated by the inverse mechanism of dimerization-mediated trans -autoinhibition, followed by activation loop autophosphorylation in cis . We show that PKD forms a stable face-to-face homodimer that is incapable of either autophosphorylation or substrate phosphorylation. Dissociation of this trans -autoinhibited dimer results in activation loop autophosphorylation, which occurs exclusively in cis . Phosphorylation serves to increase PKD activity and prevent trans -autoinhibition, thereby switching PKD on. Our findings not only reveal the mechanism of PKD regulation but also have profound implications for the regulation of many other eukaryotic kinases.

Pleasurable touch is paramount during social behavior, including sexual encounters. However, the identity and precise role of sensory neurons that transduce sexual touch remain unknown. A population of sensory neurons labeled by developmental expression of the G protein-coupled receptor Mrgprb4 detects mechanical stimulation in mice. Here, we study the social relevance of Mrgprb4-lineage neurons and reveal that these neurons are required for sexual receptivity and sufficient to induce dopamine release in the brain. Even in social isolation, optogenetic stimulation of Mrgprb4-lineage neurons through the back skin is sufficient to induce a conditioned place preference and a striking dorsiflexion resembling the lordotic copulatory posture. In the absence of Mrgprb4-lineage neurons, female mice no longer find male mounts rewarding: sexual receptivity is supplanted by aggression and a coincident decline in dopamine release in the nucleus accumbens. Together, these findings establish that Mrgprb4-lineage neurons initiate a skin-to-brain circuit encoding the rewarding quality of social touch., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

PIK3CA encoding the phosphoinositide 3-kinase (PI3K) p110α catalytic subunit is frequently mutated in cancer, with mutations occurring widely throughout the primary sequence. The full set of mechanisms underlying how PI3Ks are activated by all oncogenic mutations on membranes are unclear. Using a synergy of biochemical assays and hydrogen deuterium exchange mass spectrometry (HDX-MS), we reveal unique regulatory mechanisms underlying PI3K activation. Engagement of p110α on membranes leads to disengagement of the ABD of p110α from the catalytic core, and the C2 domain from the iSH2 domain of the p85 regulatory subunit. PI3K activation also requires reorientation of the p110α C-terminus, with mutations that alter the inhibited conformation of the C-terminus increasing membrane binding. Mutations at the C-terminus (M1043I/L, H1047R, G1049R, and N1068KLKR) activate p110α through distinct mechanisms, with this having important implications for mutant selective inhibitor development. This work reveals unique mechanisms underlying how PI3K is activated by oncogenic mutations, and explains how double mutants can synergistically increase PI3K activity., (© 2023. The Author(s).)

The formation of complexes between Rab11 and its effectors regulates multiple aspects of membrane trafficking, including recycling and ciliogenesis. WD repeat-containing protein 44 (WDR44) is a structurally uncharacterized Rab11 effector that regulates ciliogenesis by competing with prociliogenesis factors for Rab11 binding. Here, we present a detailed biochemical and biophysical characterization of the WDR44-Rab11 complex and define specific residues mediating binding. Using AlphaFold2 modeling and hydrogen/deuterium exchange mass spectrometry, we generated a molecular model of the Rab11-WDR44 complex. The Rab11-binding domain of WDR44 interacts with switch I, switch II, and the interswitch region of Rab11. Extensive mutagenesis of evolutionarily conserved residues in WDR44 at the interface identified numerous complex-disrupting mutations. Using hydrogen/deuterium exchange mass spectrometry, we found that the dynamics of the WDR44-Rab11 interface are distinct from the Rab11 effector FIP3, with WDR44 forming a more extensive interface with the switch II helix of Rab11 compared with FIP3. The WDR44 interaction was specific to Rab11 over evolutionarily similar Rabs, with mutations defining the molecular basis of Rab11 specificity. Finally, WDR44 can be phosphorylated by Sgk3, with this leading to reorganization of the Rab11-binding surface on WDR44. Overall, our results provide molecular detail on how WDR44 interacts with Rab11 and how Rab11 can form distinct effector complexes that regulate membrane trafficking events., Competing Interests: Conflict of interest J. E. B. reports personal fees from Scorpion Therapeutics and Olema Oncology and research grants from Novartis. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A large amount of the human proteome is composed of highly dynamic regions that do not adopt a single static conformation. These regions are defined as intrinsically disordered, and they are found in a third of all eukaryotic proteins. They play instrumental roles in many aspects of protein signaling, but can be challenging to characterize by biophysical methods. Intriguingly, many of these regions can adopt stable secondary structure upon interaction with a variety of binding partners, including proteins, lipids, and ligands. This review will discuss the application of Hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a powerful biophysical tool that is particularly well suited for structural and functional characterization of intrinsically disordered regions in proteins. A focus will be on the theory of hydrogen exchange, and its practical application to identify disordered regions, as well as characterize how they participate in protein-protein and protein-membrane interfaces. A particular emphasis will be on how HDX-MS data can be presented specifically tailored for analysis of intrinsically disordered regions, as well as the technical aspects that are critical to consider when designing HDX-MS experiments for proteins containing intrinsically disordered regions., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The OpenNotes (ON) mandate in the 21st Century Cures Act requires that patients or their legally authorized representatives be able to access their medical information in their electronic medical record (EMR) in real time. Ethics notes fall under the domain of this policy. We argue that ethics notes are unique from other clinical documentation in a number of ways: they lack best-practice guidelines, are written in the context of common misconceptions surrounding the purpose of ethics consultation, and often answer questions of a different nature than other documentation. Thus, we believe the clinical ethics community would benefit from clarification on when the withholding of ethics notes is justified. We provide recommendations for excluding information from ethics documentation based on the likelihood and magnitude of harm that may occur with particular disclosures and suggest approaches to decrease the potential harms that may occur. We define and explain six types of reasons to exclude information from ethics notes based on significant harms that are not addressed in the ON policy: (1) harmful revelations from a protected chart note; (2) negative emotional effects on patients or families; (3) the purpose of the consultation is undermined by harmful consequences; (4) avoidable negative impact on interpersonal dynamics; (5) inappropriate labeling or disclosure of medical, social, or financial information; and (6) inclusion of biasing or otherwise unfair information. We also suggest approaches to mitigate harm when excluding, including, reframing, or delaying release of information that is perceived to be relevant to an ethics case. Overall, we hope our analysis and recommendations will initiate a much-needed discussion about the impact of the ON mandate on clinical ethics documentation., (Copyright 2022 The Journal of Clinical Ethics. All rights reserved.)

T rimer I ndependent of N uA4 involved in T ranscription I nteractions with N ucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.

Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC_1438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC_1438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i.e., the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP 2 ), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP 2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes., Competing Interests: ES, JS, RH, BT, QZ, NH, MJ No competing interests declared, JB Burke reports consulting fees from Scorpion Therapeutics and Olema Oncology, and research grants from Novartis, which are all outside the scope of this work, JS Partial ownership of KXTbio, Inc which licenses the production of WH-15, (© 2022, Siraliev-Perez et al.)

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5'-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity ( k chem ), rate of covalent bond formation and proximity ( k inact and reaction space volume V r ), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of k chem , k inact , K i , and V r , which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes.

3-phosphoinositide-dependent kinase 1 (PDK1) is an essential serine/threonine protein kinase, which plays a crucial role in cell growth and proliferation. It is often referred to as a 'master' kinase due to its ability to activate at least 23 downstream protein kinases implicated in various signaling pathways. In this study, we have elucidated the mechanism of phosphoinositide-driven PDK1 auto-activation. We show that PDK1 trans-autophosphorylation is mediated by a PIP 3 -mediated face-to-face dimer. We report regulatory motifs in the kinase-PH interdomain linker that allosterically activate PDK1 autophosphorylation via a linker-swapped dimer mechanism. Finally, we show that PDK1 is autoinhibited by its PH domain and that positive cooperativity of PIP 3 binding drives switch-like activation of PDK1. These results imply that the PDK1-mediated activation of effector kinases, including Akt, PKC, Sgk, S6K and RSK, many of whom are not directly regulated by phosphoinositides, is also likely to be dependent on PIP 3 or PI(3,4)P 2 ., (© 2022. The Author(s).)

PIK3CA , which encodes the p110α catalytic subunit of PI3Kα, is one of the most frequently genetically activated kinases in solid tumors. In this issue of Cancer Discovery , Song and colleagues report that the related PI3Kα inhibitors taselisib and inavolisib trigger receptor tyrosine kinase (RTK)-dependent degradation of the mutant p110α protein in breast cancer cells that are positive for HER2 RTK, limiting feedback-mediated drug resistance and potentially widening the therapeutic index of PI3Kα inhibition. See related article by Song et al., p. 204 ., (©2022 American Association for Cancer Research.)

Objective: To compare effectiveness of maropitant and ondansetron in preventing preoperative vomiting and nausea in healthy dogs premedicated with a combination of hydromorphone, acepromazine, and glycopyrrolate., Animals: 88 dogs owned by rescue organizations., Procedures: Dogs received maropitant (n = 29) or ondansetron (28) PO 2 hours prior to premedication or did not receive an antiemetic (31; control). Dogs were evaluated for vomiting, nausea, and severity of nausea (scored for 6 signs) for 15 minutes following premedication with hydromorphone, acepromazine, and glycopyrrolate., Results: A significantly lower percentage of dogs vomited after receiving maropitant (3/29 [10%]), compared with control dogs (19/31 [62%]) and dogs that received ondansetron (15/28 [54%]). A significantly lower percentage of dogs appeared nauseated after receiving maropitant (3/29 [10%]), compared with control dogs (27/31 [87%]) and dogs that received ondansetron (14/28 [50%]), and a significantly lower percentage of dogs appeared nauseated after receiving ondansetron, compared with control dogs. Nausea severity scores for hypersalivation, lip licking, hard swallowing, and hunched posture were significantly lower for dogs that received maropitant than for control dogs, and scores for hypersalivation, lip licking, and hard swallowing were significantly lower for dogs that received ondansetron than for control dogs., Conclusions and Clinical Relevance: Oral administration of maropitant 2 hours prior to premedication with hydromorphone reduced the incidence of vomiting and the incidence and severity of nausea in healthy dogs. Oral administration of ondansetron reduced the incidence and severity of nausea but not the incidence of vomiting.

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Calcineurin, the conserved protein phosphatase and target of immunosuppressants, is a critical mediator of Ca 2+ signaling. Here, to discover calcineurin-regulated processes we examined an understudied isoform, CNAβ1. We show that unlike canonical cytosolic calcineurin, CNAβ1 localizes to the plasma membrane and Golgi due to palmitoylation of its divergent C-terminal tail, which is reversed by the ABHD17A depalmitoylase. Palmitoylation targets CNAβ1 to a distinct set of membrane-associated interactors including the phosphatidylinositol 4-kinase (PI4KA) complex containing EFR3B, PI4KA, TTC7B and FAM126A. Hydrogen-deuterium exchange reveals multiple calcineurin-PI4KA complex contacts, including a calcineurin-binding peptide motif in the disordered tail of FAM126A, which we establish as a calcineurin substrate. Calcineurin inhibitors decrease PI4P production during Gq-coupled GPCR signaling, suggesting that calcineurin dephosphorylates and promotes PI4KA complex activity. In sum, this work discovers a calcineurin-regulated signaling pathway which highlights the PI4KA complex as a regulatory target and reveals that dynamic palmitoylation confers unique localization, substrate specificity and regulation to CNAβ1., (© 2021. The Author(s).)

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1., (© 2021. The Author(s).)

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gβγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gβγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gβγ activation. A nanobody that specifically binds to the p101-Gβγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P 2 ] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P 3 - or PI(3,4)P 2 -containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins., (© 2021. The Author(s).)

Background: Previous studies have demonstrated success in using autogenous bone graft for arthrodesis in patients with failed surgeries of the hallux. These patients have several causes for pain and dysfunction preoperatively, including a shortened first ray, nonunion, and poor hallux alignment., Methods: In this study, a consecutive series of 36 patients (38 procedures) were treated with a patellar wedge interposition structural allograft to salvage bone loss from great toe arthrodesis malunion, painful joint replacement, failed osteotomy, or infection of the great toe metatarsophalangeal (MP) joint with shortening of the first ray. The goals of the surgery were to restore length to the first ray and provide a stable MP joint fusion to relieve pain. The 38 treated toes were evaluated for preoperative and postoperative American Orthopaedic Foot & Ankle Society (AOFAS) MP scores, subjective patient outcome scores, and clinically successful fusion of the hallux., Results: At a minimum 1-year follow-up (mean, 3.2 years), all but 2 feet healed with a solid fusion, and all healed patients reported good or excellent outcomes. AOFAS MP scores averaged 43.5 preoperatively and 77.2 postoperatively. Three patients with infection as cause for nonunion of the initial procedure were treated with staged procedures, including the use of a temporary antibiotic spacer and mini external fixator; all 3 healed without recurrent infection. One patient had a fracture of her allograft following her interposition arthrodesis, but it fused successfully after a second interposition arthrodesis surgery. Two patients developed a nonunion of the revision arthrodesis., Conclusion: The use of an interposition patellar wedge allograft can restore length to the first ray and provide successful salvage of arthrodesis nonunions and bone loss from failed hemiarthroplasty and total joint implants of the great toe MP joint., Level of Evidence: Level IV, retrospective case series.

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer., Competing Interests: Conflict of interests 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.)