This application focuses on a globular protein of central medical importance - human proinsulin - and investigates sequence determinants of its foldability. Proper folding of proinsulin is fundamental to the function of the pancreatic beta cell and maintenance of metabolic homeostasis in vertebrates. Little is known about the baseline structure of proinsulin due to long-standing difficulties in obtaining crystals and the intractability of its NMR spectrum due to aggregation.
In Aims 1 and 2 we will overcome these limitations through heteronuclear NMR studies of an engineered monomer. We seek to compare the structure of the insulin moiety to that of insulin itself and to evaluate the extent of local order in the connecting peptide.
Aim 3 investigates sequence determinants of foldability in the endoplasmic reticulum of mammalian secretory cell lines. These cell-biological studies will test the hypothesis that residues in a specific folding nucleus are required for proper folding independently of effects of mutations on the stability or receptor-binding affinity of the folded protein. We hypothesize that """"""""folding mutations"""""""" will be found that block kinetic accessibility to the ground state.
Aim 4 builds on these results to test whether putative in vivo folding mutations indeed impair oxidative folding of proinsulin in vitro. Because structural studies of nonfolding variants might be infeasible using biosynthetic material, we propose to employ total chemical synthesis to prepare the corresponding mutant insulins for functional, thermodynamic, and structural studies. Structures of """"""""unfoldable"""""""" variants are sought in Aim 5. Can specific side chains in a protein serve as """"""""kinetic guides"""""""" during folding - but be dispensible for the fuction of the protein once folded? Classical folding-pathway mutations have been identified in the trimeric tail-spike protein of phage P22. The proposed studies will test whether this paradigm generalizes to a monomeric folding reaction of central pharmaceutical and physiological importance. Of overarching interest would be the crystal structure of an """"""""unfoldable"""""""" protein. Our results promise to have implications for the evolution of insulin-like sequences and the possible misfolding of human proinsulin in type II diabetes mellitus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK069764-05
Application #
7474694
Study Section
Molecular and Cellular Endocrinology Study Section (MCE)
Program Officer
Sechi, Salvatore
Project Start
2004-09-30
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2010-07-31
Support Year
5
Fiscal Year
2008
Total Cost
$298,454
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Qi, Ling; Tsai, Billy; Arvan, Peter (2017) New Insights into the Physiological Role of Endoplasmic Reticulum-Associated Degradation. Trends Cell Biol 27:430-440
Cui, Jingqiu; Chen, Wei; Sun, Jinhong et al. (2015) Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos. J Biol Chem 290:28131-40
Avital-Shmilovici, Michal; Whittaker, Jonathan; Weiss, Michael A et al. (2014) Deciphering a molecular mechanism of neonatal diabetes mellitus by the chemical synthesis of a protein diastereomer, [D-AlaB8]human proinsulin. J Biol Chem 289:23683-92
Avital-Shmilovici, Michal; Mandal, Kalyaneswar; Gates, Zachary P et al. (2013) Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. J Am Chem Soc 135:3173-85
Wright, Jordan; Birk, Julia; Haataja, Leena et al. (2013) Endoplasmic reticulum oxidoreductin-1? (Ero1?) improves folding and secretion of mutant proinsulin and limits mutant proinsulin-induced endoplasmic reticulum stress. J Biol Chem 288:31010-8
Weiss, Michael A (2013) Diabetes mellitus due to the toxic misfolding of proinsulin variants. FEBS Lett 587:1942-50
Kiselar, Janna G; Datt, Manish; Chance, Mark R et al. (2011) Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling. J Biol Chem 286:43710-6
Liu, Ming; Haataja, Leena; Wright, Jordan et al. (2010) Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport. PLoS One 5:e13333
Luisier, Samuel; Avital-Shmilovici, Michal; Weiss, Michael A et al. (2010) Total chemical synthesis of human proinsulin. Chem Commun (Camb) 46:8177-9
Liu, Ming; Hua, Qing-xin; Hu, Shi-Quan et al. (2010) Deciphering the hidden informational content of protein sequences: foldability of proinsulin hinges on a flexible arm that is dispensable in the mature hormone. J Biol Chem 285:30989-1001

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