Overexpression of PTHrP in beta cells reveals key effects on endoplasmic reticulum stress
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ABSTRACT:
PROVIDER: PRJNA1254230 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33253968_1.fastq.gz | Fastqsanger.gz | |||
| SRR33253968_2.fastq.gz | Fastqsanger.gz | |||
| SRR33253969_1.fastq.gz | Fastqsanger.gz | |||
| SRR33253969_2.fastq.gz | Fastqsanger.gz | |||
| SRR33253970_1.fastq.gz | Fastqsanger.gz |
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Project description:Administration of parathyroid hormone-related protein (PTHrP) has been shown to increase insulin content and secretion in mice. PTHrP also increases beta-cell mass and proliferation. However, the mechanisms for these effects are unknown, and investigations have yet to examine PTHrP in the transcriptome. In this study, we transiently transfected a mouse beta-cell line (MIN6) with either full-length PTHrP or DSred vector. Insulin content and glucose-stimulated insulin secretion were measured, and RNA from the cells after incubating in 20mM glucose was collected. The results showed that PTHrP overexpression increased insulin content and the ratio of insulin secretion between low and high glucose (stimulation index). RNA-sequencing showed that PTHrP overexpression downregulated many genes associated with responses to endoplasmic reticulum (ER) stress such as Hspa40, Dnajc3 and Xbp1. Among enriched KEGG pathways, the ER stress gene pathway was the most strongly downregulated by far, and the most upregulated pathway was for biosynthesis of amino acids required for protein synthesis. These pathways suggest increased rates of protein biosynthesis. qPCR supported RNA-sequencing results for several ER stress genes (Xbp1, Bax, Bip). MIN6 cells transfected with PTHrP also had lower proinsulin-to-insulin ratio, indicting that PTHrP enhanced insulin processing in the ER. Our working hypothesis is that PTHrP augments insulin production and ER efficiency, which is consistent with observations of increased insulin content, decreased proinsulin-to-insulin ratio and reduced ER stress markers in MIN6 cells. In conclusion, our findings suggest a previously unknown role for PTHrP in beta-cell endoplasmic reticulum, which may have therapeutic implications for enhancing insulin production.
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Project description:In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a role for the ER in global protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression. HEK293 cells were fractionated between the cytosol and endoplasmic reticulum. Within each fraction, ribosome footprints were generated and sequenced. In parallel, total mRNA was sequenced.
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Project description:In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.
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Project description:We are currently studying how these information-carrying oligosaccharides are involved in cellular stress responses, particularly in human genetic diseases called Congenital Disorders Of Glycosylation in which oligosaccharide assembly is defective. We are interested in endoplasmic stress responses/unfolded protein responses. ER glycans and ER lectins are intimately involved in the folding of ER proteins. Therefore, ER lectins and ER glycosyltransferases may be controlled by these stress responses, to produce and/or recognize key glycans in the stress response. Our model system is the human dermal fibroblast. We have successfully calibrated the ER stress responses in these cells by determining the magnitudes of various stress effects (such as chaperone transcription) with different forms of ER stress. We are also completing a study in which activation the signaling molecules Ire1 and ATF6 are also calibrated. We prepared multiple identical aliquots of RNA from cells stressed under several of these calibrated conditions. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR): Study of the role of ER stress in the expression of genes for ER transferases and lectins that participate in ER folding and quality control using human skin fibroblasts. The UPR response in these cells has been calibrated using various readouts in response to different stresses: 1) 40 nM L-azetidine-2-carboxylate (AZC) 1 hour, 2) 0.2 mg/ml castanospermine, 24 hours, 3) 2 mM Dithiothreitol (DTT), 20 minutes, 4) 100nm Thapsigargin (TG), 30 minutes, 5) 5 ug/ml tunicamycin, 5 hours, 6) untreated control cells
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