Prior Functional Data Research Articles

Cryo-EM structures of the TolQ membrane protein in complex with inner membrane protein partner TolR from the opportunistic pathogen Acinetobacter baumannii provide information on structure and stoichiometry.

TolQ-TolR-TolA is an energy-transducing inner-membrane sub-complex conserved across Gram-negative bacteria. It maintains connection with periplasmic TolB-Pal sub-complex in an energy-dependent manner through TolA protein spanning the periplasm. Utilizing proton motive force (PMF), Tol-Pal complex contributes to the integrity of outer membrane and the whole cell envelope. TolQ remains the only part of Tol-Pal complex with unknown structure up to now. To address this, we determined 3.02 and 3.34 Å cryo-electron microscopy (cryo-EM) structures of the integral inner-membrane protein TolQ and in complex with TolR, respectively from Acinetobacter baumannii. A. baumannii is one of the most dreaded and clinically important Gram-negative pathogens causing a broad-spectrum nosocomial infection mainly among debilitated patients admitted to intensive care units. Our structural analysis revealed that TolQ forms a pentamer with a channel-like hydrophobic trans-membrane pore slightly shifted above the membrane opening into a large negatively charged cytoplasmic chamber which is in common with the closest TolQ-TolR paralogs ExbB-ExbD and MotA-MotB. We validated observed structural features of TolQ by prior functional data. According to our analysis the most functionally detrimental point mutations mapped on structure are localized inside the channel. Obtained structures suggest that two trans-membrane domains of TolR dimer fit inside TolQ channel in TolQ-TolR complex forming a proton-flow pathway for PMF utilization. Cryo-EM structures solved in this work were funded in whole or in part with Refeyn Travel Grant and U.S. federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract No. HHSN272201700060C (Center for Structural Genomics of Infectious Diseases (CSGID); http://csgid.org).

Barcode clonal tracking of tissue-resident immune cells in rhesus macaque highlights distinct clonal distribution pattern of tissue NK cells.

Tissue resident (TR) immune cells play important roles in facilitating tissue homeostasis, coordinating immune responses against infections and tumors, and maintaining immunological memory. While studies have shown these cells are distinct phenotypically and functionally from cells found in the peripheral blood (PB), the clonal relationship between these populations across tissues has not been comprehensively studied in primates or humans. We utilized autologous transplantation of rhesus macaque hematopoietic stem and progenitor cells containing high diversity barcodes to track the clonal distribution of T, B, myeloid and natural killer (NK) cell populations across tissues, including liver, spleen, lung, and gastrointestinal (GI) tract, in comparison with PB longitudinally post-transplantation, in particular we focused on NK cells which do not contain endogenous clonal markers and have not been previously studied in this context. T cells demonstrated tissue-specific clonal expansions as expected, both overlapping and distinct from blood T cells. In contrast, B and myeloid cells showed a much more homogeneous clonal pattern across various tissues and the blood. The clonal distribution of TR NK was more heterogenous between individual animals. In some animals, as we have previously reported, we observed large PB clonal expansions in mature CD56-CD16+ NK cells. Notably, we found a separate set of highly expanded PB clones in CD16-CD56- (DN) NK subset that were also contributing to TR NK cells in all tissues examined, both in TR CD56-CD16+ and DN populations but absent in CD56+16- TR NK across all tissues analyzed. Additionally, we observed sets of TR NK clones specific to individual tissues such as lung or GI tract and sets of TR NK clones shared across liver and spleen, distinct from other tissues. Combined with prior functional data that suggests NK memory is restricted to liver or other TR NK cells, these clonally expanded TR NK cells may be of interest for future investigation into NK cell tissue immunological memory, with implications for development of NK based immunotherapies and an understanding of NK memory.

Tissue-Resident Clonal Expansions of Rhesus Macaque NK Cells

Recent phenotypic, functional and transcriptomic analyses of natural killer (NK) cells in human and animals have established the presence of tissue resident NK(trNK) cells with specific characteristics and a central role in NK memory. The lack of endogenous clonal markers on NK cells impedes understanding the clonal genesis of trNKs. Transplantation of lentivirally-barcoded autologous hematopoietic stem and progenitor cells (HSPCs) has allowed tracking of NK cells at a clonal level in rhesus macaques (RM). We reported large KIR-restricted clonal expansions of mature CD56 -CD16 +NK in the peripheral blood (PB) of RM, clonally distinct from myeloid, T, B and CD56 +16 -NK, persisting for months to years, suggesting self-renewal independent of ongoing production from HSPCs (Wu et al, Cell Stem Cell, 2014 and Science Imm, 2018).We have now used this model to investigate the clonal distribution of NK cell populations in bone marrow (BM), liver, spleen, lymph nodes (LN), jejunum, colon and bronchoalveolar lavage (BAL). Serial of tissue biopsies were obtained from 3 barcoded monkeys by endoscopy and laparotomy overtime at steady state post transplantation , as well as tissues from necropsy. We compared clonal patterns between various trNKs and PB NKs collected from barcoded RM. Tissue or PB NK were defined as CD3-CD14-CD20-NKG2+ (NKG2A+ and NKG2C+), and NK subsets were further sorted for CD16, CD56, CD49a (putative liver tissue memory-like NK marker), and CXCR3 (critical for NK cell migration into tumor or normal tissues). The same expanded CD56 -CD16 +NK clones found in the PB were also detected at high abundance within BM, LN, liver and/or spleen CD56 -CD16 +NK, but not found in tissue CD56 +16 -NK and CD56 -16 -NK subsets. The liver and spleen bulk NK clonal patterns were highly correlated, and distinct from other tissues. We also observed tissue specific barcoded NK clones in BAL, jejunum and colon samples with no or very low abundance in other tissues and PB. Strikingly, a group of markedly expanded trNK clones, distinct from the expanded CD56 -CD16 +NK clones present concurrently or previously in PB, were present and shared across all tissues examined. These clones were enriched in CD56 -16 + trNK and absent in CD56 +16 - trNK. Notably, in both tissue and PB these clones were expanded in NKG2+ CD56-CD16- NK. These common expanded trNK cells were specifically enriched in both tissue and PB CD56 -16 -CXCR3 +NK, suggesting a role for this chemokine receptor and the ability of these clones to move between tissues. In contrast, CD49a expression did not enrich for these expanded clones.Clonally-expanded and persistent mature trNK cells, shared across multiple tissues but not present within PB mature CD56 -CD16 +NK subsets, combined with prior functional data suggesting NK memory is restricted to liver or other trNK cells, suggests these clonally-expanded trNK cells may be of interest. The pattern of shared clones across tissues, together with identification of a rare PB CD56-CD16-NK subpopulation harboring the clones, suggests preferential hematogenous homing of these clones to multiple tissues. Further analyses of gene expression and clonal dynamics are ongoing and should shed light on the ontology of trNK cells, with implications for the development of NK based immunotherapies and NK memory. DisclosuresNo relevant conflicts of interest to declare.

β11-12 linker isomerization governs acid-sensing ion channel desensitization and recovery.

Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the β11-12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process. To test this, we combined fast perfusion electrophysiology, molecular dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that both desensitization and recovery can be accelerated by orders of magnitude by mutating resides in this linker or the surrounding region. Furthermore, desensitization can be suppressed by trapping the linker in the resting state, indicating that isomerization of the β11-12 linker is not merely a consequence of, but a necessity for the desensitization process in ASICs.

Identification of lung cancer histology-specific variants applying Bayesian framework variant prioritization approaches within the TRICL and ILCCO consortia.

Large-scale genome-wide association studies (GWAS) have likely uncovered all common variants at the GWAS significance level. Additional variants within the suggestive range (0.0001> P > 5×10(-8)) are, however, still of interest for identifying causal associations. This analysis aimed to apply novel variant prioritization approaches to identify additional lung cancer variants that may not reach the GWAS level. Effects were combined across studies with a total of 33456 controls and 6756 adenocarcinoma (AC; 13 studies), 5061 squamous cell carcinoma (SCC; 12 studies) and 2216 small cell lung cancer cases (9 studies). Based on prior information such as variant physical properties and functional significance, we applied stratified false discovery rates, hierarchical modeling and Bayesian false discovery probabilities for variant prioritization. We conducted a fine mapping analysis as validation of our methods by examining top-ranking novel variants in six independent populations with a total of 3128 cases and 2966 controls. Three novel loci in the suggestive range were identified based on our Bayesian framework analyses: KCNIP4 at 4p15.2 (rs6448050, P = 4.6×10(-7)) and MTMR2 at 11q21 (rs10501831, P = 3.1×10(-6)) with SCC, as well as GAREM at 18q12.1 (rs11662168, P = 3.4×10(-7)) with AC. Use of our prioritization methods validated two of the top three loci associated with SCC (P = 1.05×10(-4) for KCNIP4, represented by rs9799795) and AC (P = 2.16×10(-4) for GAREM, represented by rs3786309) in the independent fine mapping populations. This study highlights the utility of using prior functional data for sequence variants in prioritization analyses to search for robust signals in the suggestive range.

AI-15 * EPIDERMAL GROWTH FACTOR-LIKE MODULE CONTAINING MUCIN-LIKE HORMONE RECEPTOR 2 ROLE IN PREDICTING SURVIVAL IN INVASIVE GLIOMAS

Epidermal growth factor (EGF) module-containing mucin-like receptor 2 (EMR2) is a member of the seven span transmembrane adhesion G-protein coupled receptor subclass. This protein is expressed in a subset of glioblastoma (GBM) cells and associated with an invasive phenotype. The expression pattern and functional significance of EMR2 in low grade or anaplastic astrocytomas is unknown and our goal was to expand and further define EMR2's role in gliomas with an aggressive invasive phenotype. Using the TCGA survival data we describe EMR2 expression patterns across histologic grades of gliomas and demonstrate an association between increased EMR2 expression and poor survival (p < 0.05). We support this data with qPCR of EMR2 expression in 3 separate tumor grades from our own brain tumor research center showing increase in EMR2 expression correlates with tumor grade (p < 0.05). This data supports our prior functional data depicting that EMR2-positive neoplasms possess a greater capacity for infiltrative and metastatic spread. Genomic analysis suggests that EMR2 overexpression is associated with the mesenchy mal GBM subtype(p < 0.0001). We also demonstrate that immunohistorchemistry (IHC) is a feasible method for screening GBM patients for EMR2 expression. Protein and mRNA analysis demonstrated variable expression of all isoforms of EMR2 in all glioma grades, however GBM displayed the most diverse isoforms expression pattern as well as the highest expression of the EGF1-5 isoform of EMR2. Finally, a correlation of an increase in EMR2 expression after bevacizumab treatment in glioma cells lines as well as in an animal model is identified (P < 0.05). This observation should serve as the impetus for future studies to determine if this up-regulation of EMR2 plays a role in the observation of the diffuse and increasingly invasive recurrence patterns witnessed in a subset of GBM patients after bevacizumab treatment.

Epidermal growth factor-like module containing mucin-like hormone receptor 2 expression in gliomas.

Epidermal growth factor (EGF) module-containing mucin-like receptor 2 (EMR2) is a member of the seven span transmembrane adhesion G-protein coupled receptor subclass. This protein is expressed in a subset of glioblastoma (GBM) cells and associated with an invasive phenotype. The expression pattern and functional significance of EMR2 in low grade or anaplastic astrocytomas is unknown and our goal was to expand and further define EMR2's role in gliomas with an aggressive invasive phenotype. Using the TCGA survival data we describe EMR2 expression patterns across histologic grades of gliomas and demonstrate an association between increased EMR2 expression and poor survival (p < 0.05). This data supports prior functional data depicting that EMR2-positive neoplasms possess a greater capacity for infiltrative and metastatic spread. Genomic analysis suggests that EMR2 overexpression is associated with the mesenchymal GBM subtype (p < 0.0001). We also demonstrate that immunohistorchemistry is a feasible method for screening GBM patients for EMR2 expression. Protein and mRNA analysis demonstrated variable expression of all isoforms of EMR2 in all glioma grades, however GBM displayed the most diverse isoforms expression pattern as well as the highest expression of the EGF1-5 isoform of EMR2. Finally, a correlation of an increased EMR2 expression after bevacizumab treatment in glioma cells lines is identified. This observation should serve as the impetus for future studies to determine if this up-regulation of EMR2 plays a role in the observation of the diffuse and increasingly invasive recurrence patterns witnessed in a subset of GBM patients after bevacizumab treatment.

Structural basis for activation of the complement system by component C4 cleavage

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.

Localization of the Nucleic Acid Channel Regulatory Subunit, Cytosolic Malate Dehydrogenase

NACh is a nucleic acid-conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK(1) cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or L-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK(1) cells. LLC-PK(1) cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK(1) cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.

Cardiac Sodium Channel Gene Variants and Sudden Cardiac Death in Women

Several cardiac ion channel genes have been implicated in monogenic traits with a high risk of sudden cardiac death (SCD). Mutations or rare variants in these genes have been proposed as potential contributors to more common forms of SCD, but this hypothesis has not been assessed systematically. We directly sequenced the entire coding region and splice junctions of 5 cardiac ion channel genes, SCN5A, KCNQ1, KCNH2, KCNE1, and KCNE2, in 113 SCD cases from 2 large prospective cohorts of women (Nurses' Health Study) and men (Health Professional Follow-Up Study). Controls from the same population were then screened for the presence of mutations or rare variants identified in cases, and sequence variants without prior functional data were expressed in Xenopus oocytes to assess their biophysical consequences. No mutations or rare variants were identified in any of the 53 subjects who were men. In contrast, in 6 of 60 women (10%), we identified 5 rare missense variants in SCN5A that either had been associated previously with long-QT syndrome (A572D and G615E), had been reported to alter sodium channel function (F2004L), or had not been reported previously in control populations (A572F and W1205C). Of the 4 variants without prior functional data, 3 variants were located in the I-II linker (A572D, A572F, and G615E), and all resulted in significantly shorter recovery times from inactivation. When compared with 733 control samples from the same population, the overall frequency of these rare variants in SCN5A was significantly higher in the SCD cases (6/60, 10.0%) than in controls (12/733, 1.6%; P=0.001). Functionally significant mutations and rare variants in SCN5A may contribute to SCD risk among women.