Work Description
Title: Sec61A2 Translocon Insulin Biosynthesis Data Open Access Deposited
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(2024). Sec61A2 Translocon Insulin Biosynthesis Data [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/k4gj-7423
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Files (Count: 2; Size: 70.7 MB)
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Deep_Blue_Data_Sec61A2_Readme.txt | 2024-11-12 | 2024-12-12 | 8.71 KB | Open Access |
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Sec61A2_translocon_insulin_biosy...a.zip | 2024-07-12 | 2024-08-11 | 70.7 MB | Open Access |
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Date: 1 November, 2024
Dataset Title: Role of Sec61a2 translocon in insulin biosynthesis
Dataset Creators: Xiaoxi Xu, Thomas W Bell, Truc Le, Ivy Zhao, Emily Walker, Yiqing Wang, Ning Xu, Scott A Soleimanpour, Holger A Russ, Ling Qi, Billy Tsai, Ming Liu, Peter Arvan
Dataset Contact: Peter Arvan, M.D, Ph.D., email: [email protected]
Funding: Described in publication: Xiaoxi Xu, Thomas W. Bell, Truc Le, Ivy Zhao, Emily Walker, Yiqing Wang, Ning Xu, Scott A. Soleimanpour, Holger A. Russ, Ling Qi, Billy Tsai, Ming Liu*, Peter Arvan*: Role of Sec61α2 translocon in insulin biosynthesis. Diabetes, 2024 Sep 26:db240115. Online ahead of print.
Research Overview:
Translocational regulation of proinsulin biosynthesis in pancreatic β-cells is unknown, although several studies have reported an important accessory role for the Translocon-Associated Protein complex to assist preproinsulin delivery into the endoplasmic reticulum via the heterotrimeric Sec61 translocon (comprised of α, β and γ subunits). The actual protein-conducting channel is the α-subunit encoded either by Sec61A1 or its paralog Sec61A2. Although the underlying channel selectivity for preproinsulin translocation is unknown, almost all studies of Sec61α to date have focused on Sec61α1. There is currently no evidence to suggest that this gene product plays a major role in proinsulin production, whereas genome-wide association studies indicate linkage of Sec61A2 with diabetes. Here, we report that evolutionary differences in mouse preproinsulin signal peptides affect proinsulin biosynthesis. Moreover, we find that although some preproinsulin translocation can proceed through Sec61α1, Sec61α2 has a greater impact on proinsulin biosynthesis in pancreatic β-cells. Remarkably, Sec61α2-translocon deficiency exerts a significant inhibitory effect on the biosynthesis of preproinsulin itself, including a disproportionate increase of full-length nascent chain unreleased from ribosomes. This study not only reveals novel translocational regulation of proinsulin biosynthesis, but also provides a rationale for genetic evidence suggesting an important role of Sec61α2 in maintaining blood glucose homeostasis.
Methodology:
The role of Sec61a2 translocon in insulin biosynthesis was studied in mouse pancreatic β-cell lines and primary mouse islets.
Date Coverage: 2021-2024
Instrument and/or Software specifications: Adobe Photoshop CC 2017, ImageJ， GraphPad Prism 8 software.
Files contained here:
The folder contains 27 files organized according to date and the experiments conducted over 4 years. The files are described below:
2021
1- 21 03 25 hPro signal peptide efficiency.jpg: Impact of the preproinsulin signal peptide on proinsulin biosynthesis.
2- 21 11 15.pdf: a. Structural and functional information of Sec61α2. b. Impact of Sec61α2 on proinsulin levels in pancreatic β-cells. c. Impact of Sec61α2 on proinsulin biosynthesis (pulse-chase analysis).
3- 21 11 16.pdf: a. Structural and functional information of Sec61α2. b. Impact of Sec61α2 on proinsulin levels in pancreatic β-cells.
2022
4- 22 01 13.pdf: a. Impact of overexpression of exogenous Sec61α2 on proinsulin levels in pancreatic β-cells.
5- 22 01 27 Sec61a2KD copy.pdf: Sec61α2 deficiency reduced proinsulin levels in pancreatic β-cells.(MG132 treated)
6- 22 02 24.pdf: Impact of overexpression of exogenous Sec61α2 or SSR3 in 293T cells.(293T cells were transfected with untagged human proinsulin plasmid and replated first).
7- 22 03 30.pdf: CHX washout experiment in pancreatic β-cells by immunofluorescence staining (MG132 treated).
8- 22 04 28.pdf: Collective data about CHX washout experiment in pancreatic β-cells (by immunofluorescence).
9- 22 06 02 KDslide14 INS1E slide17Min6.pdf: a. CHX washout experiment in primary mouse islets (by immunofluorescence). b. Information of the 'ribo-tagged'(Flag-RPL22) Min6 cell pool and the subclone used in the ribosome pull-down assay. c. Preliminary data of the impact of Sec61α2 translocon on insulin biosynthesis. d. Generation of Sec61α2-KO Min6 cells.
10- 22 06 30 Sec61a2 + CHXwashout.pdf: a. Impact of Sec61α2 on proinsulin levels in pancreatic β-cells. b. CHX washout experiment in primary mouse islets (db/db mice, conducted by immunofluorescence). c. Proteasomal degradation of WT proinsulin in pancreatic β-cells.
11- 22 07 28.pdf: a.Re-expression of Sec61α2 rescued proinsulin levels in Sec61α2-KO Min6 cells. b. CHX washout experiment in primary mouse islets (db/db mice, conducted by immunofluorescent). Protocol included. c. Proteasomal degradation of WT proinsulin in pancreatic β-cells.
12- 22 08 25 Sec61A1+A2 Proins+Ins (PKRi LastSlide).pdf: a. Immunofluorescence data showing the expression of Flag-RPL22 in the 'ribo-tagged' Min6 cells. b. Comparison of the impact of Sec61α2 with Sec61α1 on proinsulin levels in pancreatic β-cells.
13- 22 09 29.pdf: a. Impact of Sec61α2 on TRAP(translocon-associated protein)/SSR(signal sequence receptor) subunits in pancreatic β-cells. b. Pharmacological study shows the effects of acute blocking of translocon in pancreatic β-cells.
2023
14- 23 03 23.pdf: a. Preliminary data of ribosome pull-down assay in 'ribo-tagged' Min6 cells. b. Western blot data shows a new method to detect preproinsulin biosynthesis by CADA derivative: TL033 (rather than pulse-chase assay).
15- 23 03 30.pdf: a. Impact of Sec61α2 on proinsulin biosynthesis in pancreatic β-cells (ribosome pull-down assay, pharmacological inhibition). b. The relationship of Sec61α2 and TRAP/SSR subunit. c. Impact of the preproinsulin signal peptide on proinsulin biosynthesis in pancreatic β-cells.
16- 23 05 04 KO slide1 KDslide15.pdf: a. Impact of Sec61α2 on proinsulin biosynthesis in pancreatic β-cells (ribosome pull-down assay, pharmacological inhibition). b. Overexpression of Sec61α1, Sec61α2 or SSR3 in Sec61α2-KO Min6 cells. c. Impact of siRNA mediated Sec61α2 knockdown on proinsulin levels at different timepoint after transfection. d. Sec61α2 knockdown in rat INS1E cells and human EndoC-βH3 cells. e.Impact of the preproinsulin signal peptide on proinsulin biosynthesis in pancreatic β-cells.
17- 23 05 24.pdf: Impact of Sec61α2 on proinsulin biosynthesis in pancreatic β-cells (ribosome pull-down assay, pharmacological inhibition).
18- 23 06 29 v4.pdf: a. Impact of Sec61α2 on proinsulin biosynthesis in pancreatic β-cells (data were collected from Sec61α2-KO Min6 cells and Sec61α2-KD Min6 cells at 48/72h post transection). b. Detection of preproinsulin biosynthesis by ribosome pull-down assay. Cells treated with protein synthesis inhibitors served as negative control.
19- 23 08 13 sec61 effect on SSR3.pdf: a. Impact of Sec61α2 on SSR3 in pancreatic β-cells.
20- 23 08 17.pdf: Summarized data about the role of Sec61α2 translocon in insulin biosynthesis.
21- 23 12 31 Fig2 quantification+original picture of Fig 4A (1).pdf: a. Quantification of preproinsulin/proinsulin levels in Figure 2. b. Information of the CRISPR/Cas12a plasmid used to generate Sec61α2-KO Min6 cells.
2024
22- 24 04 16 mouse ins1 and ins2.pdf: Impact of the preproinsulin signal peptide on proinsulin biosynthesis. Data were collected from Min6 cells and 293T cells transfected with human proinsulin plasmids.
23- 24 05 08.pdf: a. The effects of pharmacological inhibition of translocon and/or proteasome in wild type and Sec61α2 β-KO mice. b. Impact of the preproinsulin signal peptide on proinsulin biosynthesis (Quantification).
24- 24 06 27.pdf: a. Impact of the preproinsulin signal peptide on proinsulin biosynthesis (Quantification). b. Collective data shows that exogenous Sec61α2 overexpression rescues proinsulin levels in Sec61α2-KD Min6 cells. c. Lentivirus mediated Sec61α2 knockdown in mouse primary islets.
25- 24 07 05.pdf: a. siRNA mediated Sec61α2 knockdown in mouse primary islets. b. Collective data shows that exogenous Sec61A2 overexpression rescues proinsulin levels in Sec61α2-KD Min6 cells. Anti-myc blotting, proinsulin secretion in low/high glucose and the quantification of mature insulin levels in the cell lysates were added.
26- 24 07 11.pdf: Collective data about the siRNA or lentivirus mediated Sec61α2 knockdown in dispersed or intact mouse islets.
27- 24 07 12.pdf: Representative data about the impact of Sec61α2 knockdown in mouse islets.
Related publication(s):
Xiaoxi Xu, Thomas W. Bell, Truc Le, Ivy Zhao, Emily Walker, Yiqing Wang, Ning Xu, Scott A. Soleimanpour, Holger A. Russ, Ling Qi, Billy Tsai, Ming Liu*, Peter Arvan*: Role of Sec61α2 translocon in insulin biosynthesis. Diabetes, 2024 Sep 26:db240115. Online ahead of print.
Use and Access:
This data set is made available under a Creative Commons Attribution-Noncommercial license (CC BY-NC 4.0).
To Cite Data:
Arvan, P. Sec61A2 translocon insulin biosynthesis data [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/k4gj-7423