Proinsulin Trafficking through the Secretory Pathway By Gautam Rajpal A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2012 Doctoral Committee Professor Peter Arvan Professor James Bardwell Professor Randal J. Kaufman Professor Ram K. Menon Associate Professor Martin G. Myers Jr. Acknowledgements I want to thank Dr. Arvan for his consistent support throughout my formative years in graduate school. My committee members, Dr. James Bardwell, Dr. Cunming Duan, Dr. Randal Kaufman, Dr. Ram Menon, and Dr. Martin Myers for their feedback. Linda Brush and Alice Koshy for their help with insulin analysis. Dr. Ming Liu for my initial training in the lab. And all the lab members past and present for their continued support and encouragement. ii Table of Contents Acknowledgments……………………………………………………………………….ii List of Figures…………………………………………………………………………...vi List of Tables…………………………………………………………………………....ix Abstract………………………………………………………………………………..…x Chapter 1 INTRODUCTION .............................................................................. 1 Chapter 2 SINGLE-CHAIN INSULINS AS RECEPTOR AGONISTS ............... 11 2.1 Abstract ................................................................................................. 11 2.2 Introduction ............................................................................................ 12 2.3 Results .................................................................................................. 17 2.4 Discussion ............................................................................................. 30 2.5 Methods ................................................................................................. 34 2.6 Figures .................................................................................................. 44 iii CHAPTER 3 ENDOPLASMIC RETICULUM OXIDOREDUCTASES AND PROINSULIN FOLDING ..................................................................................... 53 3.1 Abstract: ................................................................................................ 53 3.2 Introduction ............................................................................................ 56 3.2.1 The Concept of Secretory Pathway Catalysis of Disulfide Bond Formation .................................................................................................... 57 3.2.2 Transfer of Reducing Equivalents from the ER Lumen to the Cytosol 61 3.2.3 Proper Disulfide Bond Formation in the Secretory Pathway may also Include Disulfide Reduction and Isomerization ............................................ 63 3.2.4 The Family of PDI-like ER Oxidoreductases ................................... 64 3.2.5 Selective Activities of Oxidoreductases within the ER Environment 67 3.2.6 Oxidation of Proinsulin Within the ER ............................................. 69 3.3 Results .................................................................................................. 73 3.3.1 PDI & WT-Proinsulin interaction ..................................................... 73 iv 3.3.2 ERp72 & WT-Proinsulin Interaction ................................................ 78 3.4 Discussion ............................................................................................. 82 3.5 Methods ................................................................................................. 85 3.6 Figures .................................................................................................. 88 CHAPTER 4 CONCLUSIONS ..................................................................... 117 REFERENCES ................................................................................................. 130 ‘ v List of Figures Figure 1-1. Crystal structure of insulin. ............................................................... 10 Figure 2-1. Comparison of SCI levels measured by RIA to that measured by steady state metabolic labeling........................................................................... 44 Figure 2-2. Cleavage of SCIs in the secretory granule environment of AtT20/PC2 cells. ................................................................................................................... 45 Figure 2-3. Extraction of SCIs, or insulin standard, from media bathing CHO-IR cells (overexpressing human insulin receptors). ................................................. 46 Figure 2-4. Competitive binding of recombinant SCIs (produced in transfected 293T cells) with 125iodoinsulin. ............................................................................ 47 Figure 2-5. Kinase activation of insulin receptors and IGF1 receptors by recombinant SCIs (produced in transfected 293T cells). .................................... 48 Figure 2-6. 3H-deoxyglucose uptake in 3T3L1 adipocytes. ................................ 49 Figure 2-7. A single-chain insulin bearing a -QRGGGGGQR- linker (Table 1 line 27) suppresses gluconeogenesis in primary rat hepatocytes. ............................ 50 Figure 3-1. Oxidoreductase-catalyzed disulfide bond formation. ....................... 88 vi Figure 3-2. Domain structure of selected ER oxidoreductases. .......................... 91 Figure 3-3. Recovery of newly synthesized proinsulin with PDI and ERO1 beta knockdown.......................................................................................................... 92 Figure 3-4. Measurement of ER Oxidoreductases and Stress Markers. ............. 93 Figure 3-5. Recovery of newly synthesized proinsulin with PDI overexpression. 94 Figure 3-6. ER Exit of proinsulin, PDI-KD (Ins-1 Cells). ...................................... 95 Figure 3-7. Steady-State Measurements of total insulin in Ins-1 cells, PDI-KD. 96 Figure 3-8. Steady-State Measurements of Human Insulin in Ins 832/13 cells. 97 Figure 3-9. PDI-KD effects on enhanced ER export are selective for proinsulin as a substrate .......................................................................................................... 98 Figure 3-10. Steady-State Levels of Proinsulin, 293 and HepG2 cells. ............. 99 Figure 3-11. ER Exit of AAT, PDI-KD. .............................................................. 100 Figure 3-12. IAPP Processing, PDI-KD (Ins-1 Cells). ....................................... 101 Figure 3-13. Catalytic domain is required for retention of proinsulin when overexpressing PDI. ......................................................................................... 102 vii Figure 3-14a,b. The catalytic domain of PDI is required to restore the retentive function of after knockdown. ............................................................................. 103 Figure 3-15. The A domain is required for efficient retention of proinsulin by PDI. ......................................................................................................................... 105 Figure 3-16. PDI directly interacts with proinsulin via its catalytic domain. ....... 106 Figure 3-17. Quantification of relative transcription levels of ER Oxidoreductases ......................................................................................................................... 107 Figure 3-18a,b. Expression of ER oxidoreductases in response to high glucose. ......................................................................................................................... 109 Figure 3-19. Recovery of newly synthesized proinsulin with ERp72 knockdown. ......................................................................................................................... 111 Figure 3-20. ER Exit of newly synthesized proinsulin, ERp72-KD. ................... 112 Figure 3-21. ER Exit of AAT, ERp72-KD. ......................................................... 113 Figure 3-22. Steady-State Measurements of total insulin in Ins-1 cells, ERp72- KD. ................................................................................................................... 114 Figure 4-1. PDI-KD increases secretion of mutant A6/A11 proinsulin. ............. 129 viii List of Tables Table 1. SCI constructs prepared for this study. ................................................. 51 Table 3-2. Mammalian PDI-like family members. ............................................. 115 Table 3-3. PDX-1 occupancy in Mouse and Human Islets.. ............................. 116 ix ABSTRACT Proinsulin Trafficking through the Secretory Pathway By Gautam Rajpal Chair: Peter Arvan Proinsulin, the insulin precursor protein, is synthesized in the Endoplasmic Reticulum (ER) of pancreatic beta cells, where it folds to the native state, involving the formation of three evolutionarily conserved disulfide bonds. Once folded, proinsulin exits the ER, traverses the secretory pathway to the trans-Golgi Network (TGN) and into budding secretory granules. Proinsulin is then processed by endopeptidases that excise the connecting C-peptide that links the x B- and A-chains, leading to the creation of mature, two-chain insulin. Upon secretion to the bloodstream, insulin binds to cell surface insulin receptors on target tissues, resulting in the activation of signaling cascades that promote metabolic homeostasis. This thesis aims to look at two distinct aspects of the proinsulin maturation process. In the first part of my thesis work, I have designed a proinsulin with a shortened linker peptide with the intent to create a bioengineered protein