A high‐throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase ‘inserted’ state

FEBS Journal ◽  
2020 ◽  
Author(s):  
Yang Liu ◽  
Jiayu Yu ◽  
Mengyuan Wang ◽  
Qingfang Zeng ◽  
Xinmiao Fu ◽  
...  
Keyword(s):  
Atp Synthase ◽  
High Throughput ◽  
Protein Crosslinking ◽  
Physiological Relevance

scholarly journals High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the “inserted” state of the ATP synthase ε-subunit

2019 ◽  
Author(s):  
Yang Liu ◽  
Jiayu Yu ◽  
Mengyuan Wang ◽  
Qingfang Zeng ◽  
Xinmiao Fu ◽  
...  
Keyword(s):  
Atp Synthase ◽  
High Throughput ◽  
Living Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Unnatural Amino Acid ◽  
Fine Tuning ◽  
Cell Protein ◽  
Experimental Conditions ◽  
Physiological Relevance ◽  
In Cells

AbstractATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδεab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδε) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ε-subunit could adopt either an “inserted” or “non-inserted” state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an “inserted” state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the “inserted” and “non-inserted” states of the ε-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

scholarly journals A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Henrik Renner ◽  
Martha Grabos ◽  
Katharina J Becker ◽  
Theresa E Kagermeier ◽  
Jie Wu ◽  
...  
Keyword(s):  
High Throughput ◽  
Three Dimensional ◽  
3D Culture ◽  
Drug Effects ◽  
Global Gene Expression ◽  
Automated Generation ◽  
Chemical Screening ◽  
Physiological Relevance ◽  
Composition And Structure ◽  
High Throughput Screens

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates (‘organoids’) or 3D structures with less physiological relevance (‘spheroids’). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

scholarly journals High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

2015 ◽  
Vol 20 (5) ◽  
pp. 597-605 ◽  
Author(s):  
Helen Plant ◽  
Clare Stacey ◽  
Choi-Lai Tiong-Yip ◽  
Jarrod Walsh ◽  
Qin Yu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Compound Library ◽  
Viral Inhibition ◽  
The United Kingdom ◽  
Physiological Relevance ◽  
Syncytial Virus ◽  
Infant Hospitalization ◽  
Screening Cascade

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives

2021 ◽  
Vol 26 (2) ◽  
pp. 168-191
Author(s):  
David G. McLaren ◽  
Vinit Shah ◽  
Thomas Wisniewski ◽  
Lucien Ghislain ◽  
Chang Liu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Direct Detection ◽  
Label Free ◽  
Physiological Relevance ◽  
Wide Range ◽  
And Performance ◽  
Sensitivity Specificity

For nearly two decades mass spectrometry has been used as a label-free, direct-detection method for both functional and affinity-based screening of a wide range of therapeutically relevant target classes. Here, we present an overview of several established and emerging mass spectrometry platforms and summarize the unique strengths and performance characteristics of each as they apply to high-throughput screening. Multiple examples from the recent literature are highlighted in order to illustrate the power of each individual technique, with special emphasis given to cases where the use of mass spectrometry was found to be differentiating when compared with other detection formats. Indeed, as many of these examples will demonstrate, the inherent strengths of mass spectrometry—sensitivity, specificity, wide dynamic range, and amenability to complex matrices—can be leveraged to enhance the discriminating power and physiological relevance of assays included in screening cascades. It is our hope that this review will serve as a useful guide to readers of all backgrounds and experience levels on the applicability and benefits of mass spectrometry in the search for hits, leads, and, ultimately, drugs.

Advances in Microprobe Analysis Applied to Muscle, Nerve and Gland

1982 ◽  
Vol 40 ◽  
pp. 404-407
Author(s):  
T. E. Hutchinson ◽  
D. E. Johnson ◽  
A. C. Lee ◽  
E. Y. Wang
Keyword(s):  
Biological Tissue ◽  
Microprobe Analysis ◽  
Physiological State ◽  
External Environment ◽  
Physiological Relevance ◽  
Muscle Nerve ◽  
Technique Development

Microprobe analysis of biological tissue is now in the end phase of transition from instrumental and technique development to applications pertinent to questions of physiological relevance. The promise,implicit in early investigative efforts, is being fulfilled to an extent much greater than many had predicted. It would thus seem appropriate to briefly report studies exemplifying this, ∿. In general, the distributions of ions in tissue in a preselected physiological state produced by variations in the external environment is of importance in elucidating the mechanisms of exchange and regulation of these ions.

154: Integration of TPSA and High-Throughput Mass Spectrometry Data Improves Prostate Cancer Prediction

2007 ◽  
Vol 177 (4S) ◽  
pp. 52-53
Author(s):  
Stefano Ongarello ◽  
Eberhard Steiner ◽  
Regina Achleitner ◽  
Isabel Feuerstein ◽  
Birgit Stenzel ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Mass Spectrometry ◽  
High Throughput ◽  
Mass Spectrometry Data ◽  
Cancer Prediction
Sign in / Sign up

Export Citation Format

Share Document