scholarly journals Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

2020 ◽  
Vol 21 (3) ◽  
pp. 1091
Author(s):  
Lorena Pochini ◽  
Gilda Pappacoda ◽  
Michele Galluccio ◽  
Francesco Pastore ◽  
Mariafrancesca Scalise ◽  
...  
Keyword(s):  
Optimal Transport ◽  
Protein A ◽  
Direct Interaction ◽  
Organic Cation Transporter ◽  
Total Lipids ◽  
Transport Activity ◽  
Activity Impairment ◽  
Uptake Kinetic ◽  
Lipid Mixture ◽  
E Coli

The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.

scholarly journals Surfactant protein D binding to alveolar macrophages

1994 ◽  
Vol 300 (1) ◽  
pp. 237-242 ◽  
Author(s):  
K Miyamura ◽  
L E A Leigh ◽  
J Lu ◽  
J Hopkin ◽  
A López Bernal ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Serum Proteins ◽  
Specific Binding ◽  
Lectin Binding ◽  
Protein A ◽  
Direct Interaction ◽  
Surfactant Protein ◽  
Lung Epithelial Cells ◽  
Surfactant Protein D ◽  
Apparent Dissociation Constant

Surfactant protein D (SP-D) is a lung-specific protein, synthesized and secreted by lung epithelial cells. It belongs to group III of the family of C-type lectins; each member of this group has an unusual overall structure consisting of multiple globular ‘head’ regions (which contain the C-type lectin domains) linked by triple-helical, collagen-like, strands. This group includes the surfactant protein A (SP-A) and the serum proteins mannan-binding protein, conglutinin and collectin-43, all of which have been shown to bind to the C1q receptor found on a wide variety of cells, including macrophages. Both SP-D and SP-A have been shown to enhance oxygen radical production by alveolar macrophages. Although this strongly suggests a direct interaction between SP-D and a specific receptor on alveolar macrophages, it is still unclear whether SP-D binds to the same receptor used by SP-A and/or C1q. Human SP-D was isolated from amniotic fluid and was radiolabelled using 125I. Alveolar macrophages were isolated from human bronchioalveolar lavage fluid, and also from bovine lung washings, by differential adhesion to 24-well tissue-culture plates. The study was carried out using EDTA-containing buffers, to eliminate Ca(2+)-dependent C-type lectin binding, and was also carried out at 4 degrees C to eliminate possible internalization by the cells. 125I-SP-D showed specific binding to alveolar macrophages in both a time- and concentration-saturable manner. The binding was inhibited, by approx. 90%, on addition of a 200-fold excess of unlabelled SP-D. The apparent dissociation constant (Kd) was (3.6 +/- 1.3) x 10(-11) M, based on the assumption that native SP-D is assembled as a dodecamer of 12 identical polypeptides of 43 kDa to yield a protein of 516 kDa. C1q was also shown to bind alveolar macrophages (Kd 3 x 10(-6) M), but addition of C1q did not show inhibition of the binding of 125I-SP-D to the macrophages. We conclude that SP-D binds specifically to alveolar macrophages and the receptor involved is different from that utilized by C1q.

scholarly journals AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

2021 ◽  
Vol 12 ◽  
Author(s):  
Rafael Ayerbe-Algaba ◽  
Nuria Bayó ◽  
Ester Verdú ◽  
Raquel Parra-Millán ◽  
Jesús Seco ◽  
...  
Keyword(s):  
Protein A ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Host Cells ◽  
Gram Negative ◽  
E Coli ◽  
Cyclic Hexapeptide ◽  
Diphenyltetrazolium Bromide ◽  
Peptide Derivatives

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.

scholarly journals Improved CUT&RUN chromatin profiling and analysis tools

2019 ◽  
Author(s):  
Michael P. Meers ◽  
Terri Bryson ◽  
Steven Henikoff
Keyword(s):  
Fusion Protein ◽  
High Efficiency ◽  
Low Cost ◽  
Protein A ◽  
Micrococcal Nuclease ◽  
Permeabilized Cells ◽  
E Coli ◽  
Carry Over ◽  
Chromatin Profiling ◽  
Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

Changes in protein composition of meiotic nodules during mammalian meiosis

1998 ◽  
Vol 111 (4) ◽  
pp. 413-423 ◽  
Author(s):  
A.W. Plug ◽  
A.H. Peters ◽  
K.S. Keegan ◽  
M.F. Hoekstra ◽  
P. de Boer ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Protein A ◽  
Reca Protein ◽  
Protein Composition ◽  
Homologous Chromosomes ◽  
E Coli ◽  
Recombination Nodules ◽  
Chromosome Synapsis ◽  
Repair Protein ◽  
Mismatch Repair Protein

Homologous chromosome synapsis and meiotic recombination are facilitated by several meiosis-specific structures: the synaptonemal complex (SC), and two types of meiotic nodules: (1) early meiotic nodules (MNs), also called zygotene nodules or early recombination nodules, and (2) late recombination nodules (RNs). The former are thought to be nucleoprotein complexes involved in the check for homology preceding, or accompanying synapsis, while the latter have been shown to be involved in reciprocal recombination. We have examined by immunocytochemistry the meiotic localization of a series of proteins at sites along the asynapsed axial elements prior to homologous synapsis and at sites along the SCs following synapsis. Several of the proteins examined have been implicated in repair/recombination and include RAD51, a mammalian homolog of the Escherichia coli RecA protein; Replication Protein-A (RPA), a single-strand DNA binding protein; and MLH1, a mismatch repair protein which is a homolog of the E. coli MutL protein. In addition two proteins were examined that have been implicated in meiotic checkpoints: ATM, the protein mutated in the human disease Ataxia Telangiectasia, and ATR, another member of the same family of PIK kinases. We present evidence that these proteins are all components of meiotic nodules and document changes in protein composition of these structures during zygonema and pachynema of meiotic prophase in mouse spermatocytes. These studies support the supposition that a subset of MNs are converted into RNs. However, our data also demonstrate changes in protein composition within the context of early MNs, suggesting a differentiation of these nodules during the process of synapsis. The same changes in protein composition occurred on both the normal X axis, which has no homologous pairing partner in spermatocytes, and on the axes of aberrant chromosomes that nonhomologously synapse during synaptic adjustment. These findings suggest that DNA sequences associated with MNs still must undergo an obligatory processing, even in the absence of interactions between homologous chromosomes.

Null mutation in sspA of Cronobacter sakazakii influences its tolerance to environmental stress

2021 ◽  
Author(s):  
Jie Zhan ◽  
Xin Tan ◽  
Xiaoyuan Wang
Keyword(s):  
Stress Tolerance ◽  
Protein A ◽  
Bacterial Chemotaxis ◽  
Water Concentration ◽  
Acid Tolerance ◽  
Opportunistic Pathogen ◽  
Cronobacter Sakazakii ◽  
Knockout Mutant ◽  
Cellular Survival ◽  
E Coli

Cronobacter sakazakii is a known foodborne opportunistic pathogen that can affect the intestinal health of infants. Despite undergoing complex manufacturing processes and low water concentration in the finished product, infant formula has been associated with Cronobacter infections, suggesting that C. sakazakii’s pathogenicity may be related to its tolerance to stress. In this study, the effect of the stringent starvation protein A (SspA), which plays an important role in E. coli cellular survival under environmental stresses, on the stress tolerance of C. sakazakii BAA894 was investigated by creating an sspA-knockout mutant. The effects of this mutation on the acid, desiccation and drug tolerance were assessed, and results showed that acid tolerance decreased, while desiccation tolerance increased in LB and decreased in M9. Moreover, the MICs of 10 antibiotics in LB medium and 8 antibiotics in M9 medium were determined and compared of the wild-type and ΔsspA. Transcriptome analysis showed that 27.21% or 37.78% of the genes in ΔsspA were significantly differentially expressed in LB or M9 media, the genes relevant to microbial metabolism in diverse environments and bacterial chemotaxis were detailed analyzed. The current study contributes towards an improved understanding of the role of SspA in C. sakazakii BAA894 stress tolerance.

A Synergistic Antimicrobial Mechanism of GO: Why Oxidative Stress Can Inactivate E. coli

NANO ◽  
2020 ◽  
Vol 15 (04) ◽  
pp. 2050054 ◽  
Author(s):  
Xin Li ◽  
Haoqi Zhao ◽  
Shidong Wang ◽  
Weiwu Zou ◽  
Peiyan Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Antibacterial Activity ◽  
Mammalian Cells ◽  
Water Solubility ◽  
Direct Interaction ◽  
Medical Application ◽  
Future Application ◽  
Sem Images ◽  
E Coli ◽  
Low Cytotoxicity

Graphene oxide (GO), a 2D nanomaterial, is a promising material for medical application, thanks to its water solubility, antibacterial activity and relatively low cytotoxicity. However, many factors, such as lateral dimension, purity and surface chemistry, may influence its antibacterial activity, its exact mechanism is still unknown. In this work, E. coli was used as model bacterium to determine the antibacterial activity of well-dispersed GO which was obtained by a modified Hummer method and dialyzed to remove the salts and acid used in the oxidation process. After co-culture with GO for 2[Formula: see text]h, up to 90% E. coli cells were inactivated when GO concentration at 8[Formula: see text][Formula: see text]g/mL. The direct interaction was not detected in FE-SEM images and the results of [Formula: see text] potential showed that the interaction between GO and E. coli are repulsive[Formula: see text] Our results showed that GO can produce ROS and inactivate SOD and CAT enzymes in low concentration after co-cultured with E. coli which explained the antibacterial activity of GO in aqueous solution. Meanwhile, GO, with high purity, showed low cytotoxicity towards mammalian cells and did not cause any observable hemoglobin after co-cultured with blood cells. The data presented here prove that GO is effectively inhibit E. coli through inactivating SOD, CAT enzymes and the oxidative stress produced by ROS. Furthermore, the good biocompatibility promised its future application.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

scholarly journals Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Minh Tan Nguyen ◽  
Musharrat Jahan Prima ◽  
Jung-A. Song ◽  
Julee Kim ◽  
Bich Hang Do ◽  
...  
Keyword(s):  
Metabolic Diseases ◽  
Protein A ◽  
Genetically Engineered ◽  
Oncostatin M ◽  
Glutathione S Transferase ◽  
Skeletal Tissue ◽  
E Coli ◽  
Th17 Cell Differentiation ◽  
Tissue Alteration

Abstract Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b‘a’ domain of PDI (PDIb‘a’), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

scholarly journals MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase in Escherichia coli

2002 ◽  
Vol 184 (19) ◽  
pp. 5323-5329 ◽  
Author(s):  
Junjie Zhang ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Kanamycin Resistance ◽  
Nucleoside Triphosphate ◽  
Cellular Functions ◽  
Genomic Libraries ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Nucleoside Triphosphate Pyrophosphohydrolase

ABSTRACT Era is an essential GTPase in Escherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using the era gene as bait in the yeast two-hybrid system to screen E. coli genomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPγS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi, with a preference for deoxynucleotides. A mazG deletion strain of E. coli was constructed by replacing the mazG gene with a kanamycin resistance gene. Unlike mutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, the mazG deletion did not result in a mutator phenotype in E. coli.

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