Flavin associated Sulfhydryl oxidase and Ero1β in Insulin activity in Type 2 Diabetes Mellitus

Introduction

Diabetes Mellitus is proving to be a global public health burden as this number is expected to rise to another 200 million by 2040. The hall mark of Diabetes Mellitus is Insulin deficiency which can be quantitative as seen in Type 1 Diabetes Mellitus (T1DM) or qualitative as seen in Type 2 Diabetes Mellitus (T2DM). in type 2 Diabetes Mellitus there is improper action of Insulin which is seen as Insulin resistance. Type 2 Diabetes Mellitus is the most common type of Diabetes accounting for about 90% of all Diabetes cases¹.

Pathophysiology of Insulin Resistance in Type 2 DM

With the elucidation of the amino-acid sequence of insulin by Sanger in the mid 1950’s it became known that insulin was a two-chain heterodimer consisting of a 21-residue A-chain linked to a 30-residue B chain by two disulfide bonds derived from cystine residues (A7-B7 and A20-B19). An intrachain disulfide bond also exists within the A-chain (A6-A11)². The three native disulfide bonds have been conserved in the insulin structure for more than half a billion years and are of major importance for the stability of the molecule. The structure of Insulin has been well characterized by X-ray crystallography and NMR spectroscopy. Structural and biological studies revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness³. A study showed that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP (porcine insulin precursor) from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin⁴. Another study revealed that the removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin⁵. It was documented by Vinther et al⁶ that several active four disulfide bonded insulin analogues markedly improved stability and gained insights into the instability of analogues with seven cysteine residues, importance of dimerization for stability, insulin fibril formation process, and the conformation of insulin binding to its receptor.

Hence, the importance of the disulphide linkages was well established with respect to stability and receptor binding activity of insulin resulting in its proper biological function.

Mechanistic link between flavin-dependent Endoplasmic Reticulum oxidoreductase 1 beta family of sulfhydryl oxidase and Insulin Resistance

Sulfhydryl oxidase 1 (QSOX1 or quiescin Q6) oxidizes sulfhydryl groups in peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide, permitting disulfide bond formation. QSOX1 is predominantly found in the Golgi and Endoplasmic reticulum⁷˒⁸. Sulfhydryl oxidases are flavin-dependent enzymes⁹.

Members of the Quiescin-sulfhydryl oxidase (QSOX) family catalyse the direct introduction of disulfide bonds into unfolded reduced proteins with the reduction of molecular oxygen to generate hydrogen peroxide¹⁰. The oxidase is shown to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase¹¹. Klyosova et al showed that polymorphism of the GFER gene encoding FAD-dependent sulfhydryl oxidase was associated with a low risk of Diabetes Mellitus in non-obese patients¹².

Endoplasmic reticulum oxidoreductase 1 beta (Ero1β) family of flavin-dependent sulfhydryl oxidase enzymes are believed to play key roles in disulfide generation in yeast and higher eukaryotes. Ero1β is particularly abundant in the pancreas where it is an important disulfide oxidase in insulin-producing β cells¹³. Mice homozygous for the Ero1β mutation developed a diabetic phenotype, as would be expected if this oxidase were a significant contributor to disulfide bond generation in proinsulin¹⁴. The defect in insulin is triggered by Ero1β deficiency which thus put emphasis on the special role of Ero1β in disulfide bond formation and protein folding homeostasis in the lumen of the ER of insulin-producing cells. Retarded oxidative folding of proinsulin in islets lacking ERO1-β are consistent¹⁵.

Riboflavin deficiency is highly prevalent in developing countries. In a study from India, it was shown that amongst all vitamin deficiencies, Vitamin B2(Riboflavin) accounted for about 50% of the cases¹⁶. Sulfhydryl oxidase is a Flavin dependent enzyme. Dietary Riboflavin deficiency leads to improper action of the enzyme resulting in abnormal disulphide linkage associated protein folding. This attributes to ineffective action of the protein molecule¹⁷˒¹⁸. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes¹⁹. Alam et al suggested supplementation with Riboflavin may help in reduction of Diabetic complications²⁰.

Conclusion

Hence, the mechanistic link between Sulfhydryl oxidase, Ero1 β and Riboflavin needs to be established in relation to ineffective Insulin action and resistance. The reduced Insulin activity may result due ineffective disulphide linkages integral to Insulin activity thus affecting its structural integrity. This may get aggravated in Riboflavin deficiency affecting the activity of Flavin dependent Sulfhydryl oxidase and Ero1β. Riboflavin supplementation in this scenario may prove to be beneficial in control of type 2 Diabetes Mellitus.

Conflict of Interest:

None

Funding Statement:

None.

Acknowledgements:

None.

References

1. Goyal R, Jialal I, Singhal M. Type 2 Diabetes. National Center for Biotechnology Information. Published June 23, 2023. https://www.ncbi.nlm.nih.gov/books/NBK513253/

2. Weiss M, Steiner DF, Philipson LH. Insulin Biosynthesis, Secretion, Structure, and Structure-Activity Relationships. Nih.gov. Published February 2014. https://www.ncbi.nlm.nih.gov/books/NBK279029/

3. Chang SG, Choi KD, Jang SH, Shin HC. Role of disulfide bonds in the structure and activity of human insulin. Mol Cells. 2003;16(3):323-330.

4. Guo ZY, Feng YM. Effects of Cysteine to Serine Substitutions in the Two Inter-Chain Disulfide Bonds of Insulin. Biological Chemistry. 2001;382(3). doi:https://doi.org/10.1515/bc.2001.054

5. Guo ZY, Jia XY, Feng YM. Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin. Biological chemistry. 2004;385 (12):1171-1175. doi:https://doi.org/10.1515/bc.2004.151

6. Vinther TN, Kjeldsen TB, Jensen KJ, Hubálek F. The road to the first, fully active and more stable human insulin variant with an additional disulfide bond. Journal of Peptide Science. 2015;21(11):797-806. doi:https://doi.org/10.1002/psc.2822

7. Heckler EJ, Alon A, Fass D, Thorpe C. Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis†. Biochemistry. 2008;47 (17):4955-4963. doi:https://doi.org/10.1021/bi702522q

8. Hoober KL, Glynn NM, Burnside J, Coppock DL, Thorpe C. Homology between egg white sulfhydryl oxidase and quiescin Q6 defines a new class of flavin-linked sulfhydryl oxidases. J Biol Chem. 1999;274(45):31759-31762. doi:10.1074/jbc.274.45.31759

9. Chakravarthi S, Jessop Catherine E, Willer M, Stirling Colin J, Bulleid Neil J. Intracellular catalysis of disulfide bond formation by the human sulfhydryl oxidase, QSOX1. Biochemical Journal. 2007;404(3):403-411. doi:https://doi.org/10.1042/bj20061510

10. Kodali VK, Thorpe C. Oxidative Protein Folding and the Quiescin–Sulfhydryl Oxidase Family of Flavoproteins. Antioxidants & Redox Signaling. 2010;13(8):1217-1230. doi:https://doi.org/10.1089/ars.2010.3098

11. Hoober KL, Sheasley SL, Gilbert HF, Thorpe C. Sulfhydryl Oxidase from Egg White. Journal of Biological Chemistry. 1999;274(32):22147-22150. doi:https://doi.org/10.1074/jbc.274.32.22147

12. E. Yu. Klyosova, Shkurat EA, Azarova YE, Polonikov AV. Polymorphism rs1046495 of the GFER Gene as a New Genetic Marker of Preposition to Type 2 Diabetes Mellitus. Bulletin of experimental biology and medicine. 2022;172(5):587-591. doi:https://doi.org/10.1007/s10517-022-05441-2

13. Zito E, Chin KT, Blais J, Harding HP, Ron D. ERO1-beta, a pancreas-specific disulfide oxidase, promotes insulin biogenesis and glucose homeostasis [published correction appears in J Cell Biol. 2010 May 17;189(4):769]. J Cell Biol. 2010;188(6):821-832. doi:10.1083/jcb.200911086

14. Motoharu Awazawa, Takashi Futami, Michinori Sakada, et al. Deregulation of Pancreas-Specific Oxidoreductin ERO1β in the Pathogenesis of Diabetes Mellitus. Molecular and cellular biology. 2014;34(7):1290-1299. doi:https://doi.org/10.1128/mcb.01647-13

15. Shergalis AG, Hu S, Bankhead A 3rd, Neamati N. Role of the ERO1-PDI interaction in oxidative protein folding and disease. Pharmacol Ther. 2020;210:107525. doi:10.1016/j.pharmthera.2020.107525

16. Sivaprasad M, Shalini T, Reddy PY, et al. Prevalence of vitamin deficiencies in an apparently healthy urban adult population: Assessed by subclinical status and dietary intakes. Nutrition. 2019;63-64:106-113. doi:10.1016/j.nut.2019.01.017

17. Hartl FU, Hayer-Hartl M. Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol. 2009;16(6):574-581. doi:10.1038/nsmb.1591

18. Manthey KC, Rodriguez-Melendez R, Hoi JT, Zempleni J. Riboflavin deficiency causes protein and DNA damage in HepG2 cells, triggering arrest in G1 phase of the cell cycle. J Nutr Biochem. 2006;17(4):250-256. doi:10.1016/j.jnutbio.2005.05.004

19. Liu M, Weiss MA, Arunagiri A, et al. Biosynthesis, structure, and folding of the insulin precursor protein. Diabetes Obes Metab. 2018;20 Suppl 2(Suppl 2):28-50. doi:10.1111/dom.13378

20. Alam MM, Iqbal S, Naseem I. Ameliorative effect of riboflavin on hyperglycemia, oxidative stress and DNA damage in type-2 diabetic mice: Mechanistic and therapeutic strategies. Arch Biochem Biophys. 2015;584:10-19. doi:10.1016/j.abb.2015.08.013