PLCG2-associated immune dysregulation (PLAID) comprises broad and distinct clinical presentations related to functional classes of genetic variants.

BACKGROUND: Pathogenic variants of phospholipase C gamma 2 (PLCG2) cause 2 related forms of autosomal-dominant immune dysregulation (ID), PLCγ2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammatory PLAID (APLAID). Since describing these conditions, many PLCG2 variants of uncertain significance have been identified by clinical sequencing of patients with diverse features of ID. OBJECTIVE: We sought to functionally classify PLCG2 variants and explore known and novel genotype-function-phenotype relationships. METHODS: Clinical data from patients with PLCG2 variants were obtained via standardized questionnaire. PLCG2 variants were generated by mutagenesis of enhanced green fluorescent protein (EGFP)-PLCG2 plasmid, which was overexpressed in Plcg2-deficient DT-40 B cells. B-cell receptor-induced calcium flux and extracellular signal-regulated kinase phosphorylation were assayed by flow cytometry. In some cases, stimulation-induced calcium flux was also measured in primary patient cells. RESULTS: Three-fourths of PLCG2 variants produced functional alteration of B-cell activation, in vitro. Thirteen variants led to gain of function (GOF); however, most functional variants defined a new class of PLCG2 mutation, monoallelic loss of function (LOF). Susceptibility to infection and autoinflammation were common with both GOF and LOF variants, whereas a new phenotypic cluster consisting of humoral immune deficiency, autoinflammation, susceptibility to herpesvirus infection, and natural killer cell dysfunction was observed in association with multiple heterozygous LOF variants detected in both familial and sporadic cases. In some cases, PLCG2 variants produced greater effects in natural killer cells than in B cells. CONCLUSIONS: This work expands the genotypic and phenotypic associations with functional variation in PLCG2, including a novel form of ID in carriers of heterozygous loss of PLCG2 function. It also demonstrates the need for more diverse assays for assessing the impact of PLCG2 variants on human disease.

Structural Basis and Functional Role of Intramembrane Trimerization of the Fas/CD95 Death Receptor.

Fas (CD95, Apo-1, or TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. We determined the NMR structures of mouse and human Fas TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex.

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

The spacing effect in remote information-integration category learning.

The present experiments examined whether the temporal distribution of procedural category learning experiences would impact learning outcomes. Participants completed the remote category learning experiments on a smartphone in one of two learning conditions: massed or distributed. Consistent with expectations, distributed learners in both experiments reached higher accuracy levels than massed learners. In Experiment 1 the effect disappeared after accounting for reaction time differences, suggesting that it was driven by attentional mechanisms. In Experiment 2, the spacing advantage was only present for previously studied items during a post-learning test, suggesting a role of consolidation. In both experiments, it seems likely that temporal spacing helped participants discover the optimal information-integration categorization strategy. These results suggest that adult category learning is facilitated by temporal spacing. Future work may further explore the effects of temporal and contextual distinctiveness of learning experiences on category learning outcomes.

Cross-infectivity of Vorticella sp. across genera of mosquitoes for development of biological mosquito control strategies.

Protists in general comprise about one-third of the parasitic species infecting arthropod vectors, the role of free-living and epibiotic ciliates on mosquitoes have been insufficiently studied either due to their low pathogenicity or facultative parasites. Studies have shown that exposure of Paramecium ciliate protists, like Vorticella species, to first instar Culex nigripalpus Theobald, larvae delayed larval development and reduced biomass of emerged adults due to competition for food sources like bacteria and other microbes essential to mosquito growth and survival. Thus, we report on the capacity of a Vorticella sp. protist's ability to cross-infect host species and parasitize multiple mosquito larvae. The unique adapted behavior with the ability to remain on the exuviae in tree hole habitats provide a novel delivery system to develop products for target species-specific mosquitocides, larvicides, or viricides to be applied and sustained in aquatic systems.

Uphyloplot2: visualizing phylogenetic trees from single-cell RNA-seq data.

BACKGROUND: Recent advances in single cell sequencing technologies allow for greater resolution in assessing tumor clonality using chromosome copy number variations (CNVs). While single cell DNA sequencing technologies are ideal to identify tumor sub-clones, they remain expensive and in contrast to single cell RNA-seq (scRNA-seq) methods are more limited in the data they generate. However, CNV data can be inferred from scRNA-seq and bulk RNA-seq, for which several tools have been developed, including inferCNV, CaSpER, and HoneyBADGER. Inferences regarding tumor clonality from CNV data (and other sources) are frequently visualized using phylogenetic plots, which previously required time-consuming and error-prone, manual analysis. RESULTS: Here, we present Uphyloplot2, a python script that generates phylogenetic plots directly from inferred RNA-seq data, or any Newick formatted dendrogram file. The tool is publicly available at https://github.com/harbourlab/UPhyloplot2/ . CONCLUSIONS: Uphyloplot2 is an easy-to-use tool to generate phylogenetic plots to depict tumor clonality from scRNA-seq data and other sources.

An online repository of solvation thermodynamic and structural maps of SARS-CoV-2 targets.

SARS-CoV-2 recently jumped species and rapidly spread via human-to-human transmission to cause a global outbreak of COVID-19. The lack of effective vaccine combined with the severity of the disease necessitates attempts to develop small molecule drugs to combat the virus. COVID19_GIST_HSA is a freely available online repository to provide solvation thermodynamic maps of COVID-19-related protein small molecule drug targets. Grid inhomogeneous solvation theory maps were generated using AmberTools cpptraj-GIST, 3D reference interaction site model maps were created with AmberTools rism3d.snglpnt and hydration site analysis maps were created using SSTMap code. The resultant data can be applied to drug design efforts: scoring solvent displacement for docking, rational lead modification, prioritization of ligand- and protein- based pharmacophore elements, and creation of water-based pharmacophores. Herein, we demonstrate the use of the solvation thermodynamic mapping data. It is hoped that this freely provided data will aid in small molecule drug discovery efforts to defeat SARS-CoV-2.

Testing inhomogeneous solvation theory in structure-based ligand discovery.

Binding-site water is often displaced upon ligand recognition, but is commonly neglected in structure-based ligand discovery. Inhomogeneous solvation theory (IST) has become popular for treating this effect, but it has not been tested in controlled experiments at atomic resolution. To do so, we turned to a grid-based version of this method, GIST, readily implemented in molecular docking. Whereas the term only improves docking modestly in retrospective ligand enrichment, it could be added without disrupting performance. We thus turned to prospective docking of large libraries to investigate GIST's impact on ligand discovery, geometry, and water structure in a model cavity site well-suited to exploring these terms. Although top-ranked docked molecules with and without the GIST term often overlapped, many ligands were meaningfully prioritized or deprioritized; some of these were selected for testing. Experimentally, 13/14 molecules prioritized by GIST did bind, whereas none of the molecules that it deprioritized were observed to bind. Nine crystal complexes were determined. In six, the ligand geometry corresponded to that predicted by GIST, for one of these the pose without the GIST term was wrong, and three crystallographic poses differed from both predictions. Notably, in one structure, an ordered water molecule with a high GIST displacement penalty was observed to stay in place. Inclusion of this water-displacement term can substantially improve the hit rates and ligand geometries from docking screens, although the magnitude of its effects can be small and its impact in drug binding sites merits further controlled studies.

Heterozygosity for transmembrane activator and calcium modulator ligand interactor A144E causes haploinsufficiency and pneumococcal susceptibility in mice.

BACKGROUND: The B-cell receptor transmembrane activator and calcium modulator ligand interactor (TACI) is important for T-independent antibody responses. One in 200 blood donors are heterozygous for the TACI A181E mutation. OBJECTIVE: We sought to investigate the effect on B-cell function of TACI A181E heterozygosity in reportedly healthy subjects and of the corresponding TACI A144E mutation in mice. METHODS: Nuclear factor κB (NF-κB) activation was measured by using the luciferase assay in 293T cells cotransfected with wild-type and mutant TACI. TACI-driven proliferation, isotype switching, and antibody responses were measured in B cells from heterozygous TACI A144E knock-in mice. Mouse mortality was monitored after intranasal pneumococcal challenge. RESULTS: Levels of natural antibodies to the pneumococcal polysaccharide component phosphocholine were significantly lower in A181E-heterozygous than TACI-sufficient Swedish blood donors never immunized with pneumococcal antigens. Although overexpressed hTACI A181E and mTACI A144E acted as dominant-negative mutations in transfectants, homozygosity for A144E in mice resulted in absent TACI expression in B cells, indicating that the mutant protein is unstable when naturally expressed. A144E heterozygous mice, such as TACI+/- mice, expressed half the normal level of TACI on their B cells and exhibited similar defects in a proliferation-inducing ligand-driven B-cell activation, antibody responses to TNP-Ficoll, production of natural antibodies to phosphocholine, and survival after intranasal pneumococcal challenge. CONCLUSION: These results suggest that TACI A181E heterozygosity results in TACI haploinsufficiency with increased susceptibility to pneumococcal infection. This has important implications for asymptomatic TACI A181E carriers.

The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

The gamma-butyrolactone receptors BulR1 and BulR2 of Streptomyces tsukubaensis: tacrolimus (FK506) and butyrolactone synthetases production control.

Streptomyces tsukubaensis is a well-established industrial tacrolimus producer strain, but its molecular genetics is very poorly known. This information shortage prevents the development of tailored mutants in the regulatory pathways. A region (named bul) contains several genes involved in the synthesis and control of the gamma-butyrolactone autoregulator molecules. This region contains ten genes (bulA, bulZ, bulY, bulR2, bulS2, bulR1, bulW, bluB, bulS1, bulC) including two γ-butyrolactone receptor homologues (bulR1, bulR2), two putative gamma-butyrolactone synthetase homologues (bulS1, bulS2) and two SARP regulatory genes (bulY, bulZ). Analysis of the autoregulatory element (ARE)-like sequences by electrophoretic mobility shift assays and footprinting using the purified BulR1 and BulR2 recombinant proteins revealed six ARE regulatory sequences distributed along the bul cluster. These sequences showed specific binding of both BulR1 (the gamma-butyrolactone receptor) and BulR2, a possible pseudo γ-butyrolactone receptor. The protected region in all cases covered a 28-nt sequence with a palindromic structure. Optimal docking area analysis of BulR1 proved that this protein can be presented as either monomer or dimer but not oligomers and that it binds to the conserved ARE sequence in both strands. The effect on tacrolimus production was analysed by deletion of the bulR1 gene, which resulted in a strong decrease of tacrolimus production. Meanwhile, the ΔbulR2 mutation did not affect the biosynthesis of this immunosuppressant.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

Concerted action of wild-type and mutant TNF receptors enhances inflammation in TNF receptor 1-associated periodic fever syndrome.

TNF, acting through p55 tumor necrosis factor receptor 1 (TNFR1), contributes to the pathogenesis of many inflammatory diseases. TNFR-associated periodic syndrome (TRAPS, OMIM 142680) is an autosomal dominant autoinflammatory disorder characterized by prolonged attacks of fevers, peritonitis, and soft tissue inflammation. TRAPS is caused by missense mutations in the extracellular domain of TNFR1 that affect receptor folding and trafficking. These mutations lead to loss of normal function rather than gain of function, and thus the pathogenesis of TRAPS is an enigma. Here we show that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade.

1H, 15N, 13C backbone and Cß resonance assignments for UBQLN1 UBA and UBAA domains.

UBQLN1 functions in autophagy and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), and a flexible central region which functions as a chaperone to prevent protein aggregation. Here, we report the 1H, 15N, and 13C resonance assignments for the backbone (NH, N, C', Cα, and Hα) and sidechain Cß atoms of the UBQLN1 UBA and an N-terminally adjacent segment called the UBA-adjacent domain (UBAA). We find a subset of the resonances corresponding to the UBAA to have concentration-dependent chemical shifts, likely due to self-association. We also find the backbone amide nitrogen of T572 to be shifted upfield relative to the average value for a threonine amide nitrogen, a phenomenon likely caused by T572 Hγ1 engagement in a hydrogen bond with adjacent backbone carbonyl atoms. The assignments described in this manuscript can be used to study the protein dynamics of the UBQLN1 UBA and UBAA as well as the interaction of these domains with other proteins.

E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure.

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates in vitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1 UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.

Modeling nonaqueous proton wires built from helical peptides: biased proton transfer driven by helical dipoles.

We report gas-phase electronic structure calculations on helical peptides that act as scaffolds for imidazole-based hydrogen-bonding networks (proton wires). We have modeled various 21-residue polyalanine peptides substituted at regular intervals with histidines (imidazole-bearing amino acids), using a hybrid approach with a semiempirical method (AM1) for peptide scaffolds and density functional theory (B3LYP) for proton wires. We have computed energy landscapes including barriers for Grotthuss-shuttling-type proton motions though wires supported on 3(10)-, α- and π-helical structures, showing the 3(10)- and α-helices to be attractive targets in terms of high proton affinities, low Grotthuss shuttling barriers, and high stabilities. Moreover, bias forces provided by the helical dipole moments were found to promote unidirectional proton translocation.

Using a reduced dimensionality model to compute the thermodynamic properties of finite polypeptide aggregates.

By implementing a simple reduced dimensionality model to describe the interactions in finite systems composed of two seven-amino-acid peptides, the thermodynamic properties of ordered and disordered aggregates were computed. Within this model, the hydrophobicity of each amino acid was varied, and the stability of the systems computed. Accurate averages in the canonical ensemble were obtained using various replica exchange Monte Carlo algorithms. Low and high temperature regions were encountered where the ordered and disordered aggregates were stabilized. It was observed that as the degree of hydrophobicity increased, the stability of the aggregates increased, with a significant energetic stabilization obtained for the ordered aggregates. Upon decreasing the concentration of the solution, the stability of the amorphous aggregates increased when compared to the ordered systems.

Understanding Proton Transfer in Non-aqueous Biopolymers based on Helical Peptides: A Quantum Mechanical Study.

Histidine (an imidazole-based amino acid) is a promising building block for short aromatic peptides containing a proton donor/acceptor moiety. Previous studies have shown that polyalanine helical peptides substituted at regular intervals with histidine residues exhibit both structural stability as well as high proton affinity and high conductivity. Here, we present first-principle calculations of non-aqueous histidine-containing 310-, α- and π-helices and show that they are able to form hydrogen-bonded networks mimicking proton wires that have the ability to shuttle protons via the Grotthuss shuttling mechanism. The formation of these wires enhances the stability of the helices, and our structural characterizations confirm that the secondary structures are conserved despite distortions of the backbones. In all cases, the helices exhibit high proton affinity and proton transfer barriers on the order of 1~4 kcal/mol. Zero-point energy calculations suggest that for these systems, ground state vibrational energy can provide enough energy to cross the proton transport energy barrier. Additionally, ab initio molecular dynamics results suggests that the protons are transported unidirectionally through the wire at a rate of approximately 2 Å every 20 fs. These results demonstrate that efficient deprotonation-controlled proton wires can be formed using non-aqueous histidine-containing helical peptides.