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UV-inducible crosslinking IP-MS to elucidate the role of nuclear Hsp104 during quiescence in Saccharomyces cerevisiae

ABSTRACT: In this study, we utilize high-content microscopy screening and quantitative proteomics to establish a function of nuclear Hsp104 during aging. We find that the cytosolic-nuclear partitioning of Hsp104 is critical to maintain nuclear proteostasis in quiescent cells. Whereas high global translation rates in rapidly growing cells constrain Hsp104 to the cytosol, a decrease in protein biosynthesis re-directs the disaggregase to the nucleus dependent on a specific C-terminal motif. Nuclear Hsp104 interacts with latent eIF2 translation initiation subcomplexes and suppresses protein aggregation. This protects the dormant translation machinery from age-induced damage, enabling the rapid resumption of protein synthesis upon re-entry into the cell cycle. In this project two separate IP-MS experiments are included, KGSBVK (IP-MS, 9 samples) and GMCAVK (UV-inducible crosslinking IP-MS, 12 samples).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Georgios Mermelekas

LAB HEAD: Janne Lehtiö

PROVIDER: PXD043093 | Pride | 2024-01-12

REPOSITORIES: Pride

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			Action	DRS
	GMCAVK1-12_genes_table.txt	Txt
	GMCAVK1_12_20220216_5out15_3hgrad_sample1.raw	Raw
	GMCAVK1_12_20220216_5out15_3hgrad_sample10.raw	Raw
	GMCAVK1_12_20220216_5out15_3hgrad_sample11.raw	Raw
	GMCAVK1_12_20220216_5out15_3hgrad_sample12.raw	Raw

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Project description:Prenatal exposure to neurotoxicants such as lead (Pb) may cause stable changes in the DNA methylation (5mC) profile of the fetal genome. However few studies have examined its effect on the DNA de-methylation pathway, specifically the dynamic changes of the 5-hydroxymethylcytosine (5hmC) profile. Therefore, in this study, we investigate the relationship between Pb exposure and 5mC and 5hmC modifications during early development. To study the changes in the 5hmC profile, we use a novel modification of the Infinium™ Human methylation 450K assay (Illumina, Inc.), which we named HMeDIP-450K assay, in an in vitro human embryonic stem cell model of Pb-exposure. We model Pb-exposure associated 5hmC changes as clusters of correlated, adjacent CpG sites, which are co-responding to Pb. We further extend our study to look at Pb-dependent changes in high density 5hmC regions in umbilical cord blood DNA from 48 mother-infant pairs from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) cohort. For our study, we randomly selected UCB from 24 male and 24 female children from the 1st and 4th quartiles of Pb levels. Our data show that Pb-associated changes in the 5hmC and 5mC profiles can be divided into sex-dependent and sex-independent categories. Interestingly, differential 5mC sites are better markers of Pb-associated sex-dependent changes compared to differential 5hmC sites. In this study we identified several 5hmC and 5mC genomic loci, which we believe might have some potential as early biomarkers of prenatal Pb-exposure. Human Methylation 450K array coupled with 5-hydroxymethylcytosine (5hmC) IP or HMeDIP-450K array, was used to determine the high density 5hmC profile of 46 Umbilical cord blood samples from the ELLEMENT cohort. HM450K array, without IP was used to determine the 5-methylcytosine(5mC) plus 5hmC profile. The 5hmC region detected from the HMeDIP-450K array was subtracted from the HM450K(without IP) to get putative high density 5mC regions.

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UV-inducible crosslinking IP-MS to elucidate the role of nuclear Hsp104 during quiescence in Saccharomyces cerevisiae

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