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41.
Dietmar Weichert Andrew C. Kruse Aashish Manglik Christine Hiller Cheng Zhang Harald Hübner Brian K. Kobilka Peter Gmeiner 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(29):10744-10748
Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.One of the major obstacles to the investigation of the structural basis of G protein-coupled receptor (GPCR) activation is the flexibility of their seven-transmembrane core, particularly in the active state (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β2-adrenergic receptor (β2AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7–16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-containing compounds is chemoselective for cysteine and enforced by the affinity of the ligand-pharmacophore rather than by the electrophilicity of the cross-linking function (19). We refer to the described ligands as covalent rather than irreversible agonists because cleavage may be promoted by reducing agents and the disulfide transfer process is a reversible chemical reaction in general.Structural information on the target protein facilitates the development of covalent ligand-receptor pairs. Mutation of H932.64 in the β2AR to cysteine introduced an anchor for the disulfide-based covalent agonist FAUC50, which does not perturb ligand binding or the activation of the receptor, and thus enabled, to our knowledge, the first agonist-bound GPCR structure (20). Taking advantage of the high structural homology among aminergic GPCRs, we reasoned that the introduction of cysteine into position X2.64 should also result in a covalently binding receptor mutant for other aminergic GPCRs.We here report a methodology to generate disulfide-based covalent ligand-receptor pairs to promote structural and functional studies on GPCRs. We demonstrate that even the low-affinity endogenous agonists noradrenaline, dopamine, and serotonin can be converted into efficient covalently binding molecular tools for the β2AR, the dopamine D2 receptor (D2R), and the 5-hydroxytryptamine 2A (5-HT2A) serotonergic subtype representing Gs-, Gi-, and Gq-coupled GPCRs, respectively. Analogous studies were conducted starting from histamine and the receptor subtype H1. We applied this strategy to obtain an active-state crystal structure of the β2ARH93C and a covalent (nor)adrenaline analog. 相似文献
42.
Felix Schnarwiler Moritz Niemann Nicholas Doiron Anke Harsman Sandro K?ser Jan Mani Astrid Chanfon Caroline E. Dewar Silke Oeljeklaus Christopher B. Jackson Mascha Pusnik Oliver Schmidt Chris Meisinger Sebastian Hiller Bettina Warscheid Achim C. Schnaufer Torsten Ochsenreiter André Schneider 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(21):7624-7629
Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.Mitochondria are a hallmark of all eukaroytic cells. They derive from an endosymbiontic event between a free-living bacterium and a presumably prokaryotic host cell. More than 1.5 billion years of evolution resulted in a great diversification of mitochondria. As a consequence, the shape and number of organelles per cell as well as size, content, copy number, and organization of their genomes vary greatly between different taxons (1). However, all eukaryotes must be able to faithfully transmit mitochondria to their offspring (2, 3).Unlike most other eukaryotes, the parasitic protozoa Trypanosoma brucei has a single mitochondrion throughout its life and its cell cycle. Due to the single-unit nature of the mitochondrion, its duplication must be coordinated with the duplication of the nucleus (4). The mitochondrial genome of T. brucei, termed kinetoplast DNA (kDNA), is essential for growth of both the procyclic insect stage and the bloodstream form of the parasite (5). It consists of a disk-shaped single-unit kDNA network that localizes to a distinct region within the mitochondrial matrix (6). The kDNA is physically connected with the cytosolic basal body, the organizing center of the eukaryotic flagellum, via a high-order transmembrane structure termed tripartite attachment complex (TAC) (7) of which only few components have been identified (8–10). Replication of the kDNA network occurs at a defined stage of the cell cycle shortly before the onset of the nuclear S phase. After replication, the kDNA networks need to be correctly positioned so that during cell and mitochondrial division each daughter cell receives a single organelle with a single kDNA network. This process requires an intact TAC and is mediated by the movement of the basal body: one kDNA network remains connected to the basal body of the old flagellum whereas the other one segregates with the basal body of the new flagellum (7, 11).Unlike trypanosomes, Saccharomyces cerevisiae propagates by budding and contains highly dynamic mitochondria that constantly divide and fuse (12, 13). Mitochondrial inheritance in budding yeast therefore requires a mechanism to move mitochondria and their genomes from the mother cell into the growing bud. The protein-associated mitochondrial genomes of S. cerevisiae, termed nucleoids, localize to dozens of globular foci that are distributed all over the organelles. Most actively replicating nucleoids are associated with a protein complex that includes the outer membrane (OM) protein MDM10 as a central unit, as well as the proteins MDM12, MDM34, and MMM1 (14–16). The protein complex forms the endoplasmic reticulum (ER)–mitochondria encounter structure (ERMES) tethering the ER to the mitochondrion (17). The ERMES has also been suggested to connect to cytosolic actin fibers that mediate the movement of mitochondria to the bud of dividing yeast cells (14, 18, 19). Besides its role in mitochondrial inheritance, the ERMES has been implicated in maintenance of mitochondrial morphology and in phospholipid and calcium exchange as well as in the assembly of the protein translocase of the mitochondrial OM (TOM) (20, 21). Some of the proposed ERMES functions are controversial and there is evidence that some of them might be due to secondary effects caused by the drastically altered mitochondrial morphology (22).The central ERMES subunit, the β-barrel protein MDM10 belongs to the mitochondrial porin superfamily, which comprises the three members voltage-dependent anion channel (VDAC), Tom40, and MDM10. Whereas VDAC and Tom40 have so far been found in all eukaryotes, including T. brucei (23, 24), MDM10 is specific to the fungal clade.In this study we identify a mitochondrial OM protein of T. brucei as a novel component of the TAC. We show that the protein defines a novel subclass of the mitochondrial porin superfamily that is specialized in mitochondrial DNA inheritance. 相似文献
43.
Marianne Federlin Karl-Anton Hiller Gottfried Schmalz 《Clinical oral investigations》2014,18(8):1975-1984
Objectives
This study was conducted to evaluate a self-adhesive resin luting cement [RelyX Unicem 3MESPE–RXU] for luting partial ceramic crowns (PCCs) with and without selective enamel etching in a prospective, randomized clinical trial.Materials and methods
Thirty-four patients had received the intended treatment. Two PCCs (Vita Mark II; Cerec 3D; Sirona) had been placed in a split-mouth design: one with RXU without enamel etching (RXU), the other with RXU with selective enamel etching (RXU?+?E). Restorations were evaluated at baseline (BL) and after 12, 24, and 36 months (USPHS criteria). For statistical analysis, the Chi-square test was applied (α?=?0.05). Clinical survival of all restorations (n?=?68) after 3 years was determined using Kaplan–Meier analysis.Results
Twenty three patients (12 male/11 female) were available for clinical evaluation after 3 years. 19 RXU-PCCs were placed in molars, four in premolars, 18 RXU?+?E–PCCs in molars, five in premolars. Concerning clinical changes, no significant differences were found between luting strategies RXU/RXU?+?E at all recalls. Statistically significant changes over time were observed for marginal adaptation and marginal discoloration between BL and 36 m for RXU and RXU?+?E. For RXU?+?E, postoperative hypersensitivities decreased significantly from BL (n?=?6) to 36 m (n?=?0). Of the 68 restorations originally included, eight RXU and four RXU?+?E restorations failed. At 3 years, Kaplan–Meier survival of RXU was 72.9 %, that of RXU?+?E 87.6 %. Survival rates were not statistically significant different.Conclusions
Although clinical survival of RXU?+?E is slightly better at 3 years, restorations of both groups perform similar with respect to clinical changes over time as evaluated by modified USPHS criteria.Clinical relevance
The self-adhesive resin cement RXU can be used in conjunction with selective enamel etching, because survival rates of PCCs in the RXU?+?E group were not lower but, as a trend, even better than without enamel etching. 相似文献44.
45.
46.
Teresa Thim Konstantin Johannes Scholz Karl-Anton Hiller Wolfgang Buchalla Christian Kirschneck Jonathan Fleiner Johan Peter Woelber Fabian Cieplik 《Nutrients》2022,14(4)
The influence of patient-specific factors such as medical conditions, low-density lipoprotein cholesterol (LDL-C) or levels of 25-hydroxyvitamin D (25OHD) on periodontal diseases is frequently discussed in the literature. Therefore, the aim of this retrospective cross-sectional study was to evaluate potential associations between radiographic bone loss (RBL) and patient-specific risk factors, particularly LDL-C and 25OHD levels. Patients from a dental practice, who received full-mouth cone beam CTs (CBCTs) and blood-sampling in the course of implant treatment planning, were included in this study. RBL was determined at six sites per tooth from CBCT data. LDL-C and 25OHD levels were measured from venous blood samples. Other patient-specific risk factors were assessed based on anamnesis and dental charts. Statistical analysis was performed applying non-parametric procedures (Mann–Whitney U tests, error rates method). Data from 163 patients could be included in the analysis. RBL was significantly higher in male patients, older age groups, smokers, patients with high DMFT (decayed/missing/filled teeth) score, lower number of teeth, and high LDL-C levels (≥160 mg/dL). Furthermore, patients with high 25OHD levels (≥40 ng/mL) exhibited significantly less RBL. In summary, RBL was found to be associated with known patient-specific markers, particularly with age and high LDL-C levels. 相似文献
47.
Anke de Haan Markus A. Landolt Eiko I. Fried Kristian Kleinke Eva Alisic Richard Bryant Karen Salmon Sue-Huei Chen Shu-Tsen Liu Tim Dalgleish Anna McKinnon Alice Alberici Jade Claxton Julia Diehle Ramón Lindauer Carlijn de Roos Sarah L. Halligan Rachel Hiller Christian H. Kristensen Beatriz O.M. Lobo Nicole M. Volkmann Meghan Marsac Lamia Barakat Nancy Kassam-Adams Reginald D.V. Nixon Susan Hogan Raija-Leena Punamäki Esa Palosaari Elizabeth Schilpzand Rowena Conroy Patrick Smith William Yule Richard Meiser-Stedman 《Journal of child psychology and psychiatry, and allied disciplines》2020,61(1):77-87
48.
49.
50.
Leah Standeven Julie Price Hiller Karen Mulder George Zhu Sunita Ghosh Jennifer L. Spratlin 《Clinical colorectal cancer》2013,12(2):103-112
BackgroundOur aims were to evaluate adherence to guidelines on colorectal cancer surveillance and outcomes for patients enrolled in an innovative follow-up program at our cancer center.Patients and MethodsA retrospective chart review was conducted at the Cross Cancer Institute in Edmonton, Canada. Patients with stage II/III colorectal cancer who completed treatment and who entered into the program from December 1, 2007, to December 31, 2009, were identified. The minimum standard of care follow-up was defined as (1) carcinoembryonic antigen (CEA) testing every 120 days for 3 years; (2) computed tomography of chest, abdomen, and pelvis at 10 to 14 months and 22 to 26 months after surgery; and (3) colonoscopy within 14 months of surgery.ResultsA total of 408 patients met inclusion criteria. Two hundred (49.0%) patients were adherent to all 3 components of surveillance. Among all patients, 57 (14.0%) were nonadherent to computed tomography imaging, 135 (33.1%) were nonadherent to colonoscopy, and 96 (23.5%) were nonadherent to CEA testing. Determinants of nonadherence are described. In total, 192 (47.2%) patients had an abnormal surveillance investigation that led to 307 follow-up events. After a median of 1.6 years, 69 (16.9%) patients had documented tumor recurrence. Sixty-one (88.4%) of these 69 patients had recurrence diagnosed via surveillance, and 31 (44.9%) patients were considered potentially resectable.ConclusionsOur study demonstrated an improvement in CEA testing since the program began; however, adherence rates for all components are not yet optimal. Alterations to surveillance program management are outlined. Further investigation will determine whether intense follow-up improves patient survival locally. 相似文献