Studying the role of the E3-Ubiquitin ligase Huwe1 in the regulation of pancreatic pro-endocrine factor Hgn3

In Type 1 Diabetes (T1D), pancreatic β-cells are destroyed due to an autoimmune reaction, resulting in impaired insulin production and dysregulated glucose metabolism. Traditional T1D treatments are associated with severe complications, therefore transplantation of pancreatic islets from healthy donors has been attempted, aiming to compensate for lost β-cells in diabetic patients. Although this strategy has helped to restore normoglycemia and insulin independence in some T1D patients, the effects are usually short-term. Moreover, a shortage of islets available for transplantation and the fact that the transplant must be accompanied by immunosuppression currently limits the clinical benefits of this therapy.

Although traditionally it was believed that terminally differentiated cells in the pancreas can only give rise to cells of the same type, plasticity between the different cell types has been documented in different mouse models. Furthermore, it has been shown that highly purified human adult pancreatic duct epithelial cells can transdifferentiate to functional, insulin-producing β-cells in vitro. Due to the relative abundance of ductal cells compared to β-cells in both the diabetic and the healthy pancreas, elucidating the molecular mechanisms that induce ductal-cell-to-β-cell plasticity could open new avenues for research into potential therapies for T1D.

Preliminary work in the lab suggests that an E3 ubiquitin ligase, Huwe1, may help ubiquitinate Ngn3 and target it for degradation. Huwe1 role in β-cell survival and apoptosis seems to be dichotomous, depending on physiological conditions. This project aims to investigate the role of Huwe1 in Ngn3 degradation and assess whether this Huwe1-dependent Ngn3 degradation pathway includes potential targets for inhibition or silencing that boosting β-cell neogenesis, either through trans-differentiation from ductal cells, or through differentiation from iPS cells.

In order to investigate whether Ngn3 interacts with Huwe1 directly, and whether this interaction is dependent on Ngn3 phosphorylation, we carried out immunoprecipitation experiments in HEK293A cells transfected with wildtype Ngn3 or Ngn3 phosphomutants. Our results show that Huwe1 coimmunoprecipitates with Ngn3, pointing to a direct interaction between the two proteins. While both Ngn3 types seem to interact with Huwe1, the affinity of the interaction is significantly decreased in the Ngn3 6SA mutant (protein mutated at 6 of its phosphorylation site). The decrease is not significant in the case of the Ngn3 AA mutant (protein mutated at Gsk3β phosphorylation site).

To analyse the effect of Huwe1 inhibition on Ngn3 stability, endocrine differentiation and insulin production, we carried out differentiation experiments in iPSC-derived pancreatic progenitors. Huwe1 was inhibited for 24h with 20µM Heclin at the end of the endocrine differentiation stage (D8 until D9 post-transfer to suspension culture). Our results suggest that Ngn3 is significantly stabilised by Huwe1 inhibition, but that the treatment does not seem to significantly increase β-cell numbers at D16.

Further experiments will look to establish what particular phosphorylation sites are involved in the Ngn3-Huwe1 interaction, as well as to confirm that the interaction leads to Ngn3 ubiquitination.

Publications:

Bench to bedside: Current advances in regenerative medicine.  G. Clarke, P. Harley, E.L. Hubber, T. Manea, L. Manuelli, E. Read, F.M. Watt. Curr Opin Cell Biol. 2018;55:59-66.

Stem/progenitor cells in normal physiology and disease of the pancreas.  M.E. Alvarez Fallas, S. Pedraza-Arevalo, A.M. Cujba, T. Manea, C. Lambert, R. Morrugares, R. Sancho. Mol Cell Endocrinol. 2021;538:111459.