Qiao, Xinxian
(2012)
Molecular Mechanisms of Secreting Vesicle Biogenesis and Secretion in Chronic Degenerative Diseases.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
Regulated trafficking and secretion of insulin by the β cell of the endocrine pancreas is critical to maintain our body energy homeostasis. Disruption of these processes typically leads to hyperglycemia and the complications of diabetes. Compared to methods using anti-insulin or C-peptide antibodies, the fluorescent protein labeling approaches provide many advantages in live-cell, real time format with dynamic spatial and temporal monitoring. Previous studies from our lab demonstrated that by fusing a GFP within the C peptide of mouse proinsulin (Ins-C-GFP) insulin secretory granule targeting, trafficking and exocytosis could be monitored in live cells. Confocal microscopy and western blot results showed over 85% of the Ins-C-GFP can be targeted to insulin granules, with highly efficient proteolytic processing to mature insulin and C-GFP.
Our present project aims to establish the minimum molecular determinants within human proinsulin required for its targeting to secretory granules. In order to do this, we designed a viral shuttle plasmid containing only the signal peptide, the first 5 residues of the B chain, followed by a monomeric GFP(B5), chemically synthesized with restriction sites for highly efficient and systematic chimeric and point mutagenesis. Confocal microscopy and 3-D reconstruction experiments revealed that the B5 vector was successfully expressed and nearly all of the fluorescent protein appeared within the ER(5 transfections; 72 cells), whereas the full-length hIns-C-emGFP vector efficiently targets insulin secretory granules. The results make it unlikely that the first five residues of the B chain are sufficient for human proinsulin targeting to secretory granules. The results also suggest that the middle of the C peptide is not necessary for human proinsulin targeting. We are presently characterizing a construct with the signal peptide alone without any insulin B chain residues (B0). By systematically adding back segments from hIns-C-emGFP to B5 or B0 and the following systematic point mutagenesis, we aim to establish the minimal segments and the precise residue(s) or motif(s) of human proinsulin required for targeting to secretory granules.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
19 December 2012 |
| Date Type: |
Publication |
| Defense Date: |
17 December 2012 |
| Approval Date: |
19 December 2012 |
| Submission Date: |
19 December 2012 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
31 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Cell Biology and Molecular Physiology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Proinsulin, Trafficking, motif, Ins-C-GFP,insulin granule, INS-1, Beta cell |
| Date Deposited: |
19 Dec 2012 14:17 |
| Last Modified: |
22 Apr 2024 19:06 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/17010 |
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