Structure of the Thiostrepton Resistance Methyltransferase��S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal “L30-like” putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr·rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.RNA modifications and the enzymes that catalyze their formation are critical for cellular viability. Certain RNA modifications are extremely well characterized, such as CCA addition and amino acylation of the 3′-ends of tRNA, and the contributions of some nucleotide modifications to the creation of specific functional tRNA structures (1–3). Although the single most common nucleotide modification is pseudouridine, by far the most abundant type of RNA chemical modification is methylation (4). A vast array of unique mono-, di-, and trimethylations of each RNA base and/or ribose sugar 2′-OH is possible, and important new functions for these modifications continue to emerge. In ribosomal RNA (rRNA),² for example, modifications cluster in functionally critical regions where methylation may act as a checkpoint in ribosome subunit assembly (5), influence the process of translation (6), and alter resistance to certain antibiotics (7, 8).RNA methylation is catalyzed by members of two classes (I and IV) of S-adenosyl-l-methionine (AdoMet)-dependent RNA methyltransferase (MTase) enzymes (9). In bacteria, rRNA methylations are incorporated by both “housekeeping” MTases and those that confer resistance to antibiotics. Although members of the former group are often highly conserved, the latter are generally only found in the antibiotic-producing strain as one mechanism of defense against self-intoxication (10). However, several instances of antibiotic resistance MTase genes in non-producer strains, including pathogenic bacteria, have been identified, and it is clear that these genes are mobile resistance determinants, usually obtained by lateral gene transfer.Several classes of antibiotics target the conserved centers on the ribosome, altering or blocking critical steps in translation such as decoding and peptidyl transfer, to exert their bactericidal effect (11). RNA MTases have been identified as clinically significant resistance determinants to a number of these, including the aminoglycoside (Arm MTase) and erythromycin (Erm MTase) antibiotics (12, 13). Another functionally critical ribosome domain, the factor binding site (or “GTPase” center), is also the target for a family of thiazole-containing peptide antibiotics (14), which includes thiostrepton. These antibiotics have been important biochemical tools for studies of ribosome function but are of limited clinical use due to their poor aqueous solubility. Thiostrepton is, however, used in veterinary medicine, and recent studies suggest it may have application in development of novel antimalarial and anticancer strategies (15, 16). The minimal rRNA sequence for interaction of thiostrepton is a highly conserved, independently folded 58-nucleotide rRNA domain that is also bound by ribosomal protein L11. Resistance to thiostrepton can result from mutations in the N-terminal domain of L11 or its entire absence, whereas mutation of the target nucleoside (A1067) confers far greater resistance (17–19). In the thiostrepton producer Streptomyces azureus the thiostrepton resistance MTase (Tsr) catalyzes the 2′-O-methylation of A1067 resulting in specific and total resistance to thiostrepton (20).Here we present the crystal structure of Tsr in complex with AdoMet. The structure definitively places Tsr into the SpoU/TrmD (SPOUT) family of enzymes and provides the basis for modeling the Tsr·rRNA recognition process.     

Improved Binding of Raf to Ras��GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.     

A Slow ATP-induced Conformational Change Limits the Rate of DNA Binding but Not the Rate of �� Clamp Binding by the Escherichia coli �� Complex Clamp Loader

In Escherichia coli, the γ complex clamp loader loads the β-sliding clamp onto DNA. The β clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ complex binds β and DNA to form a ternary complex. Binding to primed template DNA triggers γ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of γ complex binding β and DNA. A fluorescence intensity-based β binding assay was developed in which the fluorescence of pyrene covalently attached to β increases when bound by γ complex. Using this assay, an association rate constant of 2.3 × 10⁷ m⁻¹ s⁻¹ for γ complex binding β was determined. The rate of β binding was the same in experiments in which γ complex was preincubated with ATP before adding β or added directly to β and ATP. In contrast, when γ complex is preincubated with ATP, DNA binding is faster than when γ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote β binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for γ complex to bind β before DNA during the clamp loading reaction cycle.     

Tumor Necrosis Factor �� Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-��B

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.     

Absorption of Naphthalene by Leaves and Its Interaction with Chlorophyll–Protein Complexes of Pea Plants

Russian Journal of Plant Physiology - Mechanisms of photosynthesis inhibition by vaporous naphthalene, its permeation into thylakoids, and interactions with chlorophyll–protein complexes were...     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Molecular Probes: What Is the Range of Their Interaction with the Environment?

We performed pressure-tuning hole-burning experiments on a modified cytochrome c protein in a glycerol/buffer glass. The shift and the broadening of the holes were investigated for various frequencies within the inhomogeneous band. On the basis of a simple model, we were able to estimate the interaction range between chromophore and protein. It is ~4.5 Å. The parameters that enter the model are the compressibility, the static mean-square displacement, the inhomogeneous width, and the average spectral shift per pressure. From this result and from our experiments on pressure-induced denaturing, we conclude that water molecules have to be brought very close to the chromophore during the denaturation process.     

��-Secretase Inhibitor Potency Is Decreased by Aberrant ��-Cleavage Location of the ��Swedish Mutant�� Amyloid Precursor Protein

The amyloid-β (Aβ) peptide, widely known as the causative molecule of Alzheimer disease (AD), is generated by the sequential cleavage of amyloid precursor protein (APP) by the aspartyl proteases BACE1/β-secretase and presenilin/γ-secretase. Inhibition of BACE1, therefore, is a promising strategy for preventing the progression of AD. However, β-secretase inhibitors (BSIs) exhibit unexpectedly low potency in cells expressing “Swedish mutant” APP (APPswe) and in the transgenic mouse Tg2576, an AD model overexpressing APPswe. The Swedish mutation dramatically accelerates β-cleavage of APP and hence the generation of Aβ; this acceleration has been assumed to underlie the poor inhibitory activity of BSI against APPswe processing. Here, we studied the mechanism by which the Swedish mutation causes this BSI potency decrease. Surprisingly, decreased BSI potency was not observed in an in vitro assay using purified BACE1 and substrates, indicating that the accelerated β-cleavage resulting from the Swedish mutation is not its underlying cause. By focusing on differences between the cell-based and in vitro assays, we have demonstrated here that the potency decrease is caused by the aberrant subcellular localization of APPswe processing and not by accelerated β-cleavage or the accumulation of the C-terminal fragment of β-cleaved APP. Because most patients with sporadic AD express wild type APP, our findings suggest that the wild type mouse is superior to the Tg2576 mouse as a model for determining the effective dose of BSI for AD patients. This work provides novel insights into the potency decrease of BSI and valuable suggestions for its development as a disease-modifying agent.     

Electroendocytosis Is Driven by the Binding of Electrochemically Produced Protons to the Cell’s Surface

Electroendocytosis involves the exposure of cells to pulsed low electric field and is emerging as a complementary method to electroporation for the incorporation of macromolecules into cells. The present study explores the underlying mechanism of electroendocytosis and its dependence on electrochemical byproducts formed at the electrode interface. Cell suspensions were exposed to pulsed low electric field in a partitioned device where cells are spatially restricted relative to the electrodes. The cellular uptake of dextran-FITC was analyzed by flow cytometery and visualized by confocal microscopy. We first show that uptake occurs only in cells adjacent to the anode. The enhanced uptake near the anode is found to depend on electric current density rather than on electric field strength, in the range of 5 to 65 V/cm. Electrochemically produced oxidative species that impose intracellular oxidative stress, do not play any role in the stimulated uptake. An inverse dependence is found between electrically induced uptake and the solution’s buffer capacity. Electroendocytosis can be mimicked by chemically acidifying the extracellular solution which promotes the enhanced uptake of dextran polymers and the uptake of plasmid DNA. Electrochemical production of protons at the anode interface is responsible for inducing uptake of macromolecules into cells exposed to a pulsed low electric field. Expanding the understanding of the mechanism involved in electric fields induced drug-delivery into cells, is expected to contribute to clinical therapy applications in the future.     

Interaction of Myocilin with γ-Synuclein Affects Its Secretion and Aggregation

Summary Mutations in the gene encoding human myocilin are associated with some cases of juvenile and early-onset glaucoma. Glaucomatous mutations prevent myocilin from being secreted. The analysis of the defects associated with mutations point to the existence of factor(s) in addition to mutations that might be implicated in the development of glaucoma. In the present paper, we found that interaction of myocilin with one of the members of the synuclein family alters its properties, including its ability to be secreted. Results of immunoprecipitation show that myocilin is a γ-synuclein-interacting protein. Further analysis demonstrated that both myocilin and γ-synuclein are expressed in human TM cells, immortalized rat ganglion (RGC-5) cells, and HT22 hippocampal neurons. According to Western blotting, in addition to monomeric form with molecular weight 17 kDa γ-synuclein is present as higher molecular weight forms (∼35 and 68 KDa), presumably dimer and tetramer. Myocilin and γ-synuclein have partially overlapping perinuclear localization. Dexamethasone upregulates myocilin expression in RGC-5 cells and HT22 hippocampal neurons. We found alterations of myocilin properties as a result of its interaction with γ-synuclein. In cultured cells, γ-synuclein upregulates myocilin expression, inhibits its secretion and prevents the formation of high molecular weight forms of myocilin. Although both α-synuclein and γ-synuclein are expressed in HTM cells, only γ-synuclein interacts with myocilin and alters its properties. We conclude that myocilin and γ-synuclein interact and as a result, myocilin's properties are changed. Since myocilin and γ-synuclein have partially overlapping intracellular localization in cell types that are implicated in glaucoma development, their interaction may play an important role in glaucoma.     

Interaction of Tumor with Its Micro-environment: A Mathematical Model

This paper is concerned with early development of transformed epithelial cells (TECs) in the presence of fibroblasts in the tumor micro-environment. These two types of cells interact by means of cytokines such as transforming growth factor (TGF-β) and epidermal growth factor (EGF) secreted, respectively, by the TECs and the fibroblasts. As this interaction proceeds, TGF-β induces fibroblasts to differentiate into myofibroblasts which secrete EGF at a larger rate than fibroblasts. We monitor the entire process in silico, in a setup which mimics experiments in a Tumor Chamber Invasion Assay, where a semi-permeable membrane coated by extracellular matrix (ECM) is placed between two chambers, one containing TECs and another containing fibroblasts. We develop a mathematical model, based on a system of PDEs, that includes the interaction between TECs, fibroblasts, myofibroblasts, TGF-β, and EGF, and we show how model parameters affect tumor progression. The model is used to generate several hypotheses on how to slow tumor growth and invasion. In an Appendix, it is proved that the mathematical model has a unique global in-time solution.     

Is the Image Quality of I-124-PET Impaired by an Automatic Correction of Prompt Gammas?

Objectives

The aim of this study is to evaluate the quality of I-124 PET images with and without prompt gamma compensation (PGC) by comparing the recovery coefficients (RC), the signal to noise ratios (SNR) and the contrast to F-18 and Ga-68. Furthermore, the influence of the PGC on the quantification and image quality is evaluated.

Methods

For measuring the image quality the NEMA NU2-2001 PET/SPECT-Phantom was used containing 6 spheres with a diameter between 10 mm and 37 mm placed in water with different levels of background activity. Each sphere was filled with the same activity concentration measured by an independently cross-calibrated dose calibrator. The “hot” sources were acquired with a full 3D PET/CT (Biograph mCT®, Siemens Medical USA). Acquisition times were 2 min for F-18 and Ga-68, and 10 min for I-124. For reconstruction an OSEM algorithm was applied. For I-124 the images were reconstructed with and without PGC. For the calculation of the RCs the activity concentrations in each sphere were determined; in addition, the influence of the background correction was studied.

Results

The RCs of Ga-68 are the smallest (79%). I-124 reaches similar RCs (87% with PGC, 84% without PGC) as F-18 (84%). showing that the quantification of I-124 images is similar to F-18 and slightly better than Ga-68. With background activity the contrast of the I-124 PGC images is similar to Ga-68 and F-18 scans. There was lower background activity in the I-124 images without PGC, which probably originates from an overcorrection of the scatter contribution. Consequently, the contrast without PGC was much higher than with PGC. As a consequence PGC should be used for I-124.

Conclusions

For I-124 there is only a slight influence on the quantification depending on the use of the PGC. However, there are considerable differences with respect to I-124 image quality.