Summary of Study ST003174

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001819. The data can be accessed directly via it's Project DOI: 10.21228/M8KQ72 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003174
Study Title	O-GlcNAcase activity maintains stress granules for proximity-enhanced ATP production to ensure recovery from stress (Part 3)
Study Summary	Accurate disassembly of stress granules (SGs) after environmental stimuli release is essential for cells to maintain homeostasis , which requires ATP-consuming processes. However, the molecular mechanism whereby regulation of SGs programmatically disassembly and ATP restoration remain poorly understood in mammalian cells. Here we found that defect of OGA in cells leads to aggregates formation, severe autophagy and eventually apoptosis during stress recovery. OGA, which localized in SGs, had no effect on SGs formation but could protect SGs from rapid disassembly during stress recovery stage. Then the SGs localized glycolysis-related enzymes were reserved and concentrated in SGs during stress release for ATP production in a proximity manner, which was vital to guarantee cells resistant to stress and survival during recovery. Finally, supplementation of ATP to OGA knockdown cells during stress recovery significantly rescue cell from aggregates, autophagy and apoptosis. Together, these results describe a brand new mechanism on how OGA regulates the programmed disassembly of stress granules and restoration of ATP to safeguard cell viability in a very precisely programmed process, whose rate is rigorous regulated. This is a continuation of study ST002927 and ST002936 where an in vitro reaction was performed with 13C-glucose on purified stress granules, to validate the conclusion.
Institute	Zhejiang University
Department	Life Sciences Institute
Laboratory	Shixian Lin
Last Name	Chen
First Name	Yulin
Address	Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang province, China
Email	ychen209@qq.com
Phone	18868107794
Submit Date	2024-04-16
Raw Data Available	Yes
Raw Data File Type(s)	mzML, wiff
Analysis Type Detail	LC-MS
Release Date	2024-04-30
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP003300
Sampleprep Summary:	Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h were scraped off and centrifuged to collect cell pellets. The cell pellets were thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were centrifuge at 18000 × g for 20 min to collect pellets and the pellets were washed once with stress granule lysis buffer. Then, pellets were resuspended in stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were added for reaction at 37 'C for 0, 1 and 3 hr. The reaction was quenched by 3-fold methanol and centrifuge to collect the supernant for analysis.

Sampleprep ID:

SP003300

Sampleprep Summary:

Cells without stress treatment or treated with 0.6 M sorbitol stress for 1.5 h were scraped off and centrifuged to collect cell pellets. The cell pellets were thawed on ice, resuspended in stress granule lysis buffer (50 mM Tris, 100 mM K2OAc, 2 mM MgOAc, 0.5 mM DTT, 50 μg/mL heparin, 0.5% NP-40, 0.02% antifoam B, proteinase inhibitor cocktail, pH 7.4) and lysis by syringe. Lysates were centrifuged at 1000 × g for 5 min to collect supernatants. Supernatants were centrifuge at 18000 × g for 20 min to collect pellets and the pellets were washed once with stress granule lysis buffer. Then, pellets were resuspended in stress granule lysis buffer and centrifuged at 850 × g for 2 min to collect supernatants containing stress granule. Then NAD+, ATP and 13C-glucose were added for reaction at 37 'C for 0, 1 and 3 hr. The reaction was quenched by 3-fold methanol and centrifuge to collect the supernant for analysis.