New players in the beta cell ER stress response: UFM1 and UFBP1

Background and aims: To fulfil their demanding metabolic task – the regulated synthesis, storage and secretion of insulin – pancreatic beta cells possess a highly developed endoplasmic reticulum (ER). This may explain why beta cells are vulnerable for ER overload as was illustrated by heterozygous eIF2alphaser51ala mutant mice fed a high fat diet. Therefore, optimal functioning of the ER is essential for proper insulin folding and beta cell survival. Chronic imbalance between protein synthetic demands and ER folding capacity leads to ER stress and activation of the ER stress response to restore ER homeostasis. The aim of the present study was to identify new players in the ER stress response, starting from genome wide mRNA expression analysis and genes with unknown function which are highly expressed in islets. Ubiquitin-fold modifier 1 (UFM1), the most recently identified ubiquitin-like protein with unknown function, is proposed as being a new player in the beta cell ER stress response.Materials and methods: mRNA expression analysis was performed by Affymetrix microarrays and confirmed by RT-PCR. Using STrEP-tag affinity purification and mass spectrometry we identified a new target of UFM1. The cellular localisation of UFM1 and its target was identified via cellular fractionation and immunocytochemistry. The effect of free fatty acids and of cyclopiazonic acid on ER stress and on apoptosis was analysed in INS1 cells treated with Ufm1 specific siRNA. The influence of UFM1 and its target on ER associated protein degradation was analysed by overexpression of CD3delta, a known target of ERAD, in siRNA treated INS1 cells.Results: Among a panel of 20 investigated organs and tissues, UFM1 is highly expressed in pancreatic islets of Langerhans and in other protein-secreting exocrine cell types (pancreatic acini, salivary glands, and seminal vesicles), both at the mRNA and protein level. We identified a highly conserved, unknown protein as a target of UFM1 and named it UFBP1 for UFM1-binding protein 1 containing a PCI domain. Both UFM1 and UFBP1 co-localised in the ER. Pharmacological induction of ER stress by cyclopiazonic acid (25 µM) enhanced expression of both Ufm1 and Ufbp1 (respectively 3.7-fold and 5.3-fold) in INS1 cells. This pharmacolocial effect was partially mimicked by addition of the free fatty acids oleate and palmitate which enhanced Ufbp1 expression (respectively 1.7-fold and 2.4-fold). siRNA-mediated reduction of Ufm1 or Ufbp1 expression enhanced apoptosis upon ER stress stimulation and diminished ERAD as evidenced by less degradation of CDdelta. Conclusion: Our data show for the first time that UFM1 via its target protein UFBP1 is a new molecular player in ERAD and ER stress-induced beta cell apoptosis.

Publication year:2010