A considerable fraction of all newly synthesized secretory polypeptides fail to attain their native conformation due to mutations, transcriptional and translational errors, folding defects or endoplasmic reticulum (ER) stress conditions.
A considerable fraction of all newly synthesized secretory polypeptides fail to attain their native conformation due to mutations, transcriptional and translational errors, folding defects or endoplasmic reticulum (ER) stress conditions. Besides the lack of function, the accumulation of aggregated proteins jeopardize the ER homeostasis and the cell functioning. Terminally misfolded polypeptides are retained and eventually removed by the ER associated degradation pathway (ERAD).
We have recently found that besides the mannosidase-like domain, EDEM1 protein, one of the important players of the ERAD has an intrinsically disordered domain with high prediction for favoring protein-protein interactions. Indeed, we showed that this domain is responsible for the interaction with a misfolded tyrosinase mutant. In sum, we suggest that the ER associated degradation of glycoproteins depends less on the glycan recognition signals while rather relying on the direct recognition of the misfolded region of the polypeptide.
The aim of this project is to validate this working hypothesis using ERAD substrates involved in tumor antigen degradation/peptide presentation and viral disassembly. Finally, we will use transgenic C.elegans as models for Huntington, Alzheimer and Parkinson diseases, to decipher the pathology and toxicity of protein aggregation.
Overall, our approach will reveal new insights into the mechanistic details of the ERAD pathway and help identify new therapeutic targets.
The role of ER mannose binding proteins in protein degradation is well documented. Our aim is not to address their role in ERAD, but to understand how the misfolded polypeptides are recognized by these lectins. It is therefore important to know whether the other ERAD recognition molecules may have domains similar to the IDR found in EDEM1 and whether they are involved in the interaction with the misfolded polypeptides.If the signal for degradation resides in the misfolded region of the polypeptide, what is the role of the de-mannosylated glycans? Misfolded polypeptides associate selectively with these lectins before being delivered to SEL1.
It is therefore important to know when and how the ERAD substrates select specific degradation pathways and how they function, with the following specific aims:
In sum, we suggest that the ER associated degradation of glycoproteins depends less on the glycan recognition signals while rather relying on the direct recognition of the misfolded region of the polypeptide. However, the role of the de-mannosylation process should not be disregarded because it may potentiate the ERAD pathway or, as our preliminary data suggest it may regulate the half-lives of the short lived ERAD components. Viral envelope glycoproteins and glycosylated melanoma antigens will be used as model proteins in this project that also aims at identifying therapeutical targets. To further understand the role of ERAD in a model organism, we will use Caenorhabditis elegans transgens with pathological accumulation of aggregates.
Accomplishments of major goals:
Our incursion into the ERAD pathway came from the investigation of glycoprotein folding. We are interested in understanding the folding and processing of tyrosinase, a tumor antigen of melanoma cells. While studying the half-life of this protein we found that its degradation is dependent on kifunensine, an inhibitor of ER mannosidase, but that its association with EDEM1 occurred even in the presence of the inhibitor. Single mutants of tyrosinase lacking individual N-glycans were immunoprecipitated with EDEM and a non-glycosylated tyrosinase mutant associated with the lectin. EDEM1 has been described as an ER resident protein whose expression is under the control of the UPR process and extracts the misfolded polypeptide from the calnexin cycle as a first step of the ERAD pathway. Its structural homology with the ER mannosidase lead to the hypothesis that the mannosidase-like domain catalyses its lectin or mannosidase function.
We hypothesize that besides the mannosidase domain, this protein has an intrinsically disordered domain (IDR) with high prediction for favoring protein-protein interactions. Indeed, we found that the IDR is responsible for the interaction with misfolded tyrosinase. Conversely we questioned if this might be relevant for other ERAD substrates. In this regard we extended our experiments to other ERAD substrates. Our results showed that EDEM1 overexpression enhances the degradation of the proteins used in our studies. Also, similar to tyrosinase EDEM1 is able to interact with other ERAD substrates employed here. We conclude that EDEM1 has the same behaviour with other substrates as observed for tyrosinase. The results have been communicated in two international meetings (See Conference Presentation: oral presentations 1and 2 and Poster presentations: posters 4, 6 and 7).
Our preliminary data indicate that EDEM1 has a distinct region for substrate recognition that is different from the one required for substrate extraction from calnexin cycle and delivery to SEL 1. Altogether these findings suggest that the EDEM1 IDR facilitates the direct retro-translocation of misfolded proteins towards the proteasomal degradation. To further identify ER sensors of protein misfolding we have investigated in detail the mannosidase domain, by generating a model of EDEM1 and a model of EDEM3 during the 2014 stage of this project (manuscript in preparation)
During this stage we have also focused on developping accurate methods to identify EDEM interactors by LC-MS using the Velos Orbitrap equipment recently acquired by IBAR. There is a complete list of interactors for the EDEM proteins with some interactors identified as ERAD components and a number of potential ERAD substrates that associate with the EDEM proteins during their processing pathway ( See the article published in Rom.J.Biochem, in 2015, Conference Presentation/Oral presentation-Conf.4, Poster presentations: posters 1, 6, 8, 9 and 10).
To determine the role of ERAD in the processing and assembly of the hepatitis B virus, we successfully established cell lines characterised by low- and over-expression of ERAD components, but able to undergo infection with the HBV. Using the generated cell lines we found that some ERAD components play special roles in the early stages of virus generation within the hepatic cell facilitating the assembly of the virus and the establishement of the chronic infection (manuscript under evaluation).
The other objective of this project is to investigate the aggregation diseases in the model organism, Caenorhabditis elegans. The first step was to investigate the pathology and toxicity of protein aggregation in the absence of ERAD components. Our preliminary data have shown that inactivation of three genes of the ERAD pathway resulted inan increased aggregation of poliQ protein and alpha-synuclein (See Invited presentation/Pres.1, 2 and Poster presentation/Poster 5). We report a role for ERAD in the degradation of the aggregates of alpha 1 antitrypsin and Q40::YFP (model of Huntington disease) but not for A..aggregates.found in Alzheimer disease (manuscript in preparation). This is an important finding with potential applications in the therapeutical approach for Huntington and Alzheimer diseases.
The Report frequency of the project is annual, with 2 reports submitted to the UEFISCDI agency and uploaded on the electronic platform in December 2013 (Raport stiintific_Petrescu_2013.pdf), December 2014 (RST2014_ID-4-0350.pdf) and December 2015(RST2015_ID-4-0350.pdf). December 2015 (RST2015_ID-4-0350.pdf) and December 2016 ( RST 2013-2016_ID-4-0350).
Opportunities for training and professional development
The project involved the active participation of 5 PhD students and 1 master student. The master thesis of Andrei Juncu entitled Purification of EDEM1 isolated from mammalina cells has been completed and presented at the University of Bucharest in June 2014. In September 2014 Marioara Marin(Chiritoiu) defended her thesis entitled The role of EDEM1 in glycoprotein degradation associated to the ER. In October 2016 Gabriela Chiritoiu defended publicly the thesis The Role of N-glycosylation and ERAD in tyrosinase immunogenicity modulatiom. All these theses were supervised by Dr. Stefana Petrescu. PhD students attained weekly seminars in the Department of Molecular Cell Biology of the Institute of Biochemistry and frequently presented their experimental data during these seminars. Each PhD student has presented in public sessions an annual report of his activity, according to the SCOSAAR rules and one annual presentation of a peer reviewed paper to the Journal Club Programme of the Institute, in sum 28 public oral communications.
New biological models, methods and reagents developped
These reagents obtained during the project are now available to the scientific community.
"Novel function of the endoplasmic reticulum degradation-enhancing .-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.", Lazar C, Uta M., Petrescu SM, Branza-Nichita N. Cell Microbiol., in press (2016)
"Comparison of protein extraction conditions for EDEM 3 interactors in melanoma cells", Cristian M. Butnaru, Cristian V.A. Munteanu, Gabriela Chiritoiu, Simona Ghenea, Andrei J. Petrescu, Stefana M. Petrescu, Rom.J.Biochem., 52, 1, 31-38 (2015)