181 ROMANIAN JOURNAL OF RHEUMATOLOGY – VOLUME XXV, NO. 4, 2016 ORIGINAL ARTICLES EFFECT OF EDEM1 OVEREXPRESSION ON THE GENERATION AND ASSEMBLY OF MAJOR HISTOCOMPATIBILITY COMPLEXES Alexandra Circiumaru 1,2 , Gabriela Chiritoiu 1 , Livia Sima 1 , Mihai Bojinca 2 , Stefana Petrescu 1 1 Department of Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Bucharest, Romania 2 Department of Internal Medicine and Rheumatology, Dr. I. Cantacuzino Clinical Hospital, Bucharest, Romania Abstract Background. A better understanding of the role of endoplasmic reticulum degradation-enhancing alpha-manno- sidase – like protein 1 (EDEM1) in endoplasmic reticulum associated degradation (ERAD) may open new thera- peutic approaches in autoimmune diseases. Aim. To study ERAD and EDEM1 in the generation and assembly of MHC I and the potential role in the patho- physiology of autoimmune diseases. Materials and methods. HEK293T cell line (human embrionic kidney cells), A375 cell line (amelanotic melanoma cells) and THP-1 cell line (leukemic monocytes used both as undifferentiated and differentiated) underwent tran- sient transfection with EDEM1 and mock transfection with pTriEx. Western blot experiments assessed the total cellular MHC I levels in cell lysates, while expression on the cellular surface was quantified by flow cytometry of fixed cells. Results were analysed using the FACS Calibur and CellQuest Pro dedicated software. Experiments were done twice with duplicate probes for the Western blot assay and triplicate probes were used for flow cytom- etry. GraphPad Prism was used for data analysis. Results. MHC I plasma membrane routing and expression was similar in HEK293T and A375 both in mock trans- fected and non-transfected cells. Western blot assay for EDEM1 transfected cells showed bands corresponding to the total MHC I that migrated at 42kDa mass in non-transfected and mock transfected Hek293T, A375 and undifferentiated THP-1 cells. Mock transfected differentiated THP-1 cells showed a reduction of total MHC I. EDEM1 transfected Hek293T, A375 and undifferentiated THP-1 cells displayed higher levels of total MHC I, while differentiated THP-1 cells showed a marked reduction. Flow cytometry assay showed significantly reduced cell surface MHC I levels in Hek293T cell line. We observed a modest reduction of MHC I complexes on the cellular surface in undifferentiated THP-1 EDEM1 transfected cells, while there was no significant change in the A375 EDEM1 transfected cell line, as well as the differentiated THP-1 EDEM1 transfected cells. Conclusion. The impact of ERAD’s EDEM1 in MHC I reduction may have an important role in autoimmune dis- ease, making ERAD an interesting therapeutic target. Keywords: endoplasmic reticulum associated degradation, EDEM1, MHC I Correspondence address: Alexandra Circiumaru, Institute of Biochemistry of Romanian Academy, 296 Splaiul Independentei, Bucharest, Romania E-mail: alexandra.circiumaru@gmail.com INTRODUCTION The endoplasmic reticulum (ER) is a multifolded membraneous structure and the site of lipid and pro- tein biosintesys (1). It is estimated that aproximately one-third of the secreted and membrane proteins are synthesized and assembled in their native structure inside the ER lumen (2). Therefore, it is essential that a quality control system exists in order to maintain crucial cell homeostasis environment. Accumulation of unfolded intermediaries or misfolded proteins in the ER lumen leads to ER stress and triggers several stress sensors that act as the effectors of unfolded protein response (UPR). UPR is characterized by several cytoprotective pathways that ultimately lead to ER stress mitigation. This result is achieved by either increasing the protein folding capacity in the ER or decreasing the protein folding load through the activation of ER associated degradation (ERAD) or, when everything else fails, trigger apoptosis (3). The end result is to reestablish cell homeostasis. Three main effectors are described for UPR: acti- vating transcription factor 6 (ATF6), inositol-requir- ing enzyme 1 (IRE1) and PKR-like ER-localized eIF2α kinase (PERK), all residents of the ER and