scholarly journals Trimerization of the N-Terminal Tail of Zika Virus NS4A Protein: A Potential In Vitro Antiviral Screening Assay

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 335
Author(s):  
Janet To ◽  
Jaume Torres
Keyword(s):  
Zika Virus ◽  
Lipid Membranes ◽  
Transmembrane Domain ◽  
Protein A ◽  
Random Coil ◽  
Screening Assay ◽  
Vitro Assay ◽  
Helical Content ◽  
Membrane Morphogenesis

The nonstructural (NS) protein NS4A in flaviviruses is a membrane protein that is critical for virulence, and, among other roles, it participates in membrane morphogenesis. In dengue virus (DENV), the NS4A hydrophilic N–terminal tail, together with the first transmembrane domain, is involved in both homo-oligomerization and hetero–oligomerization with NS4B. In both DENV and Zika virus (ZIKV), this N-terminal tail (residues 1–48) forms a random coil in solution but becomes mostly α-helical upon interaction with detergents or lipid membranes. Herein, we show that a peptide from ZIKV NS4A that spans residues 4–58, which includes most of the N–terminal tail and a third of its first transmembrane domain, forms homotrimers in the absence of detergents or liposomes. After interaction with the latter, α–helical content increases, consistent with binding. The oligomeric size of NS4A is not known, as it has only been reported in SDS gels. Therefore, we propose that full-length NS4A forms homotrimers mediated by this region, and that disruption of the oligomerization of peptide ZIKV NS4A 4–58 in solution can potentially constitute the basis for an in vitro assay to discover antivirals.

PRTX-100 Inhibits Platelet Phagocytosis In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1081-1081 ◽  
Author(s):  
Chris Yatko ◽  
Christopher Herrem ◽  
Samia Siddiqui ◽  
Victor S. Sloan
Keyword(s):  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Staphylococcal Protein A ◽  
Human Monocytes ◽  
Peripheral Blood Mononuclear ◽  
Vitro Assay

Abstract Background: In idiopathic thrombocytopenic purpura (ITP), autoantibodies bind to platelets which are then phagocytosed by monocytes/macrophages and removed by the reticuloendothelial system. PRTX-100 (Staphylococcal protein A) is being investigated for the treatment of ITP. Objective: To assess the effect of PRTX-100 on phagocytosis of platelets in an in vitro assay. Methods: Human monocytes were isolated from whole blood peripheral blood mononuclear cells (PBMCs) by adherence and cultured for 6 days in RPMI + 5% human serum. 48 hours prior to phagocytosis assay, PRTX-100 was added at 250, 25, and 2.5ng/ml. Human platelets were labeled with a fluorescent (PerCP) lipophilic dye and opsonized with an antibody to MHC Class I (W632). 2×10−5 monocytes were co-cultured with 2×10−7 labeled platelets for 1 hour at 37 ° C. All conditions were performed in triplicate. After an hour, phycoerythrin (PE) labeled anti-CD61 antibody was added to assess surface bound platelets versus ingested platelets. Phagocytosis was determined by flow cytometric analysis. The monocyte population was gated upon by forward and side scatter properties, then verified by staining with CD14-FITC. Percent phagocytosis was calculated as the fraction of ingested platelets (PerCP +/CD61−) to the total PerCP population (PerCP +/CD61−) + ( PerCP+/CD61+) within the gated monocyte population. Results: PRTX-100 inhibits the phagocytosis of W632 opsonized platelets by human monocytes. Phagocytosis of W632 opsonized platelets was 40%, while phagocytosis in the presence of PRTX-100 at concentrations of 250, 25, and 2.5ng/ml was 18.3%, 23%, and 24.3%, respectively. Phagocytosis at 250ng/ml and 25ng/ml was significantly different from control phagocytosis with p values of 0.014 and 0.001 respectively by Student’s t test. Conclusions: PRTX-100 inhibits the phagocytosis of platelets by monocytes, the effector limb of ITP. Prevention of platelet phagocytosis is an important treatment goal in ITP. PRTX-100 has been shown to be generally safe and well-tolerated in a phase I study in healthy volunteers (J Clin Pharmacol, in press). PRTX -100 is a promising therapeutic option for ITP and deserves further study. Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets

DNA double-strand breaks induce formation of RP-A/Ku foci on in vitro reconstituted Xenopus sperm nuclei

2001 ◽  
Vol 114 (18) ◽  
pp. 3345-3357
Author(s):  
Paola Grandi ◽  
Michail Eltsov ◽  
Inga Nielsen ◽  
Ivan Raška
Keyword(s):  
Dna Replication ◽  
High Speed ◽  
Magnetic Beads ◽  
Protein A ◽  
Restriction Enzymes ◽  
Dna Double Strand Breaks ◽  
Vitro Assay ◽  
Xenopus Egg ◽  
Sperm Nuclei

Replication protein A (RP-A) is involved in DNA replication, repair and recombination. It has been demonstrated that RP-A clusters in foci prior to DNA replication and redistributes over chromatin during S-phase. Here, we show that RP-A foci also form in response to DNA double-strand (ds) breaks produced on Xenopus laevis sperm nuclei by restriction enzymes and then reconstituted with Xenopus egg high-speed extracts. Ku86 co-localizes with RP-A in the same foci. An unscheduled RP-A-dependent DNA synthesis takes place overlapping with RP-A and Ku86 foci. Immunoelectron-microscopy analysis reveals that these foci correspond to spherical bodies up to 300 nm in diameter, which contain RP-A, Ku86 and DNA. In an independent in vitro assay, we incubated linear dsDNA bound to magnetic beads with Xenopus egg extracts. Here, also RP-A and Ku cluster in foci as seen through immunofluorescence. Both proteins appear to enrich themselves in sequences near the ends of the DNA molecules and influence ligation efficiency of ds linear DNA to these ends. Thus, the Xenopus in vitro system allows for the generation of specific DNA ds breaks, RP-A and Ku can be used as markers for these lesions and the repair of this type of DNA damage can be studied under conditions of a normal nuclear environment.

scholarly journals Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation.

1995 ◽  
Vol 131 (6) ◽  
pp. 1377-1386 ◽  
Author(s):  
P Mayinger ◽  
V A Bankaitis ◽  
D I Meyer
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Protein A ◽  
Lipid Vesicles ◽  
Secretory Proteins ◽  
Transport Activity ◽  
Vitro Assay ◽  
Microsomal Membranes

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro-alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.

scholarly journals An efficient way to screen inhibitors of energy-coupling factor (ECF) transporters in bacteria uptake assay

2021 ◽  
Author(s):  
Spyridon Bousis ◽  
Steffen Winkler ◽  
Jörg Haupenthal ◽  
Francesco Fulco ◽  
Eleonora Diamanti ◽  
...  
Keyword(s):  
Cost Effective ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Screening Assay ◽  
Vitro Assay ◽  
Whole Cell ◽  
Fast Solution ◽  
Uptake Assay ◽  
Biological Environment

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships.

scholarly journals Regulated cleavage of glycan strands by the murein hydrolase SagB in S. aureus involves a direct interaction with LyrA (SpdC)

2021 ◽  
Author(s):  
Stephanie Willing ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Protein A ◽  
Direct Interaction ◽  
Staphylococcal Protein A ◽  
Lipid Ii ◽  
Molecular Ruler ◽  
Membrane Bound ◽  
Insight Into

LyrA (SpdC), a homologue of eukaryotic CAAX proteases that act on prenylated substrates, has been implicated in the assembly of several pathways of the envelope of Staphylococcus aureus. We described earlier the Lysostaphin resistance (Lyr) and Staphylococcal protein A display (Spd) phenotypes associated with loss of the lyrA (spdC) gene. However, a direct contribution to the assembly of pentaglycine crossbridges, the target of lysostaphin cleavage in S. aureus peptidoglycan, or of Staphylococcal protein A attachment to peptidoglycan could not be attributed directly to LyrA (SpdC). These two processes are catalyzed by the Fem factors and Sortase A, respectively. To gain insight into the function of LyrA (SpdC), here we use affinity chromatography and LC-MS/MS analysis and report that LyrA interacts with SagB. SagB cleaves glycan strands of peptidoglycan to achieve physiological length. Similar to sagB peptidoglycan, lyrA peptidoglycan contains extended glycan strands. Purified lyrA peptidoglycan can still be cleaved to physiological length by SagB in vitro. LyrA does not modify or cleave peptidoglycan, it also does not modify or stabilize SagB. The membrane bound domain of LyrA is sufficient to support SagB activity but predicted ‘CAAX enzyme’ catalytic residues in this domain are dispensable. We speculate that LyrA exerts its effect on bacterial prenyl substrates, specifically undecaprenol-bound peptidoglycan substrates of SagB, to help control glycan length. Such an activity also explains the Lyr and Spd phenotypes observed earlier. IMPORTANCE Peptidoglycan is assembled on the trans side of the plasma membrane from lipid II precursors into glycan chains that are crosslinked at stem peptides. In S. aureus, SagB, a membrane-associated N-acetylglucosaminidase, cleaves polymerized glycan chains to their physiological length. Deletion of sagB is associated with longer glycan strands in peptidoglycan, altered protein trafficking and secretion in the envelope, and aberrant excretion of cytosolic proteins. It is not clear whether SagB, with its single transmembrane segment, serves as the molecular ruler of glycan chains or whether other factors modulate its activity. Here, we show that LyrA (SpdC), a protein of the CAAX type II prenyl endopeptidase family, modulates SagB activity via interaction though its transmembrane domain.

scholarly journals Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases

2020 ◽  
Vol 26 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Sarah Wazir ◽  
Mirko M. Maksimainen ◽  
Heli I. Alanen ◽  
Albert Galera-Prat ◽  
Lari Lehtiö
Keyword(s):  
Drug Targets ◽  
Microtiter Plate ◽  
Post Translational Modification ◽  
Screening Assay ◽  
Fluorescent Compound ◽  
Vitro Assay ◽  
Adp Ribosylation ◽  
Compound Screening ◽  
A Chain

ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of β-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases.

scholarly journals Bidirectional FtsZ filament treadmilling transforms lipid membranes via torsional stress

2019 ◽  
Author(s):  
Diego A. Ramirez-Diaz ◽  
Adrian Merino-Salomon ◽  
Fabian Meyer ◽  
Michael Heymann ◽  
German Rivas ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Lipid Membranes ◽  
Force Production ◽  
Lipid Vesicles ◽  
Torsional Stress ◽  
Bacterial Cell Division ◽  
Giant Vesicles ◽  
Vitro Assay

AbstractFtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far however not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we designed an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ actively transformed these tubes into spring-like structures, where GTPase activity promoted spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, we found that FtsZ rings indeed exerts 0.14 – 1.09 pN forces upon GTP hydrolysis, through torsional stress induced by bidirectional treadmilling. These directional forces could further be demonstrated to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls.

scholarly journals Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E

2021 ◽  
Vol 22 (16) ◽  
pp. 8577
Author(s):  
Alon Ben David ◽  
Ada Barnea ◽  
Eran Diamant ◽  
Eyal Dor ◽  
Arieh Schwartz ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput Screening ◽  
Lethal Dose ◽  
Screening Assay ◽  
Vitro Assay ◽  
Time To Death ◽  
High Throughput Screening Assay ◽  
Binding Inhibitors

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

A short-term in vitro drug-screening assay for cancer chemotherapy.

1982 ◽  
Vol 28 (9) ◽  
pp. 1852-1856 ◽  
Author(s):  
S L Chen ◽  
J R Snoga ◽  
H Croft
Keyword(s):  
Tumor Cell ◽  
Dna Synthesis ◽  
Tumor Cells ◽  
Soft Agar ◽  
Cell Renewal ◽  
Short Term ◽  
Screening Assay ◽  
Vitro Assay ◽  
Drug Screening Assay

Abstract In this short-term in vitro assay for detection of tumor cell sensitivity to drugs, we have replaced the traditional soft-agar colony-forming assay with the measurement of DNA synthesis, to determine the cell renewal capability of the tumor cell population. The tumor cells are treated with drugs and cultured for five days. During the last 12--18 h of culture the cells are pulsed with [3H]thymidine, then harvested for scintillation counting. The effects of drugs are expressed as the percentage of DNA synthesis as compared with that of the control. When the % DNA synthesis is less than 40%, the drug is considered to be effective. So far we have studied tumor cells from multiple myeloma, non-Hodgkin's lymphoma, and carcinoma of lung, breast, ovary, stomach, and bladder. The overall negative predictive value is 1.0. The results are available within five days, compared with 21--28 days for completion of soft-agar assays; fewer cells are required; and the process is semiautomated.

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