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EXPANDING THE KNOWLEDGE ON MAMMALIAN ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION (ERAD)

Final Report Summary - MAMMALIAN ERAD (Expanding the knowledge on mammalian endoplasmic reticulum-associated degradation (ERAD))

The MAMMALIAN ERAD project aims to facilitate reintegration of Dr Ballar, a researcher working on the degradation of misfolded proteins that fail to pass 'protein quality control' in mammalian cells, from United States to Turkey. The Endoplasmic reticulum (ER) comprises one third of the newly translated proteins in eukaryotic cells. After co-translational emerging into the ER, newly synthesised proteins undergo folding and posttranslational modifications. However, the folding process is not error-free and a significant fraction of proteins fails to reach their native folded state. The quality control mechanism of the ER distinguishes between properly and improperly folded proteins, and ensures that improperly folded or unassembled proteins are retained in the ER and subsequently degraded through the ubiquitin-proteasome pathway by the process called ER-associated degradation (ERAD). ERAD is a multi-step process involving selection of misfolded proteins in the ER, retrotranslocation from ER to the cytosol, polyubiquitination, deglycosylation, and degradation in the cytosol through the 26S proteasome.

The objectives of MAMMALIAN ERAD intend to identify substrate specific differences and regulations present in mammalian ERAD by utilisation of four substrates with different structural features.

These substrates are:

1. CD3d (unassembled membrane protein);
2. Z-variant a1-antitrypsin (ATZ, causes alpha-1-antitrypsin deficiency, ER luminal protein);
3. tyrosinase (C89R) (causes albinism, transmembrane protein with luminal folding defect);
4. mutant cystic fibrosis transmembrane conductance regulator protein (CFTRdF508) (causes cystic fibrosis, transmembrane protein with cytosolic folding defect).

Dr Ballar and her group firstly focused on the role of two ERAD ubiquitin ligases (E3) (gp78 and Hrd1) in the degradation of CFTRdF508, a well-known ERAD substrate. The deletion of phenylalanine 508 of Cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation associated with cystic fibrosis. This mutation renders otherwise functional protein susceptible to ERAD and prevents CFTR from exiting the ER and trafficking to the plasma membrane where it functions as a chloride channel. Dr Ballar demonstrated that silencing of gp78 leads to accumulation of CFTRdF508 protein. While overexpression of gp78 decreased CFTRdF508 levels in a dose-dependent manner, inactive gp78 Ring finger-mutant caused accumulation of CFTRdF508. Moreover, the results of Dr Ballar's studies suggest that gp78 facilitates the degradation of CFTRdF508 by enhancing both its ubiquitination and interaction with p97/VCP. Interestingly, overexpression of Hrd1 caused significant accumulation of CFTRdF508 suggesting that Hrd1 is a negative regulator of CFTRdF508 degradation. By designing suitable experiments, Dr Ballar identified Hrd1 as a regulator in CFTRdF508 degradation by targeting gp78 for degradation. This study was the first one reporting gp78 as the E3 targeting CFTRdF508 for degradation and Hrd1 as the inhibitor of CFTRdF508 degradation by acting as an E3 for gp78. Furthermore, she identified a second mechanism regulating CFTRdF508 degradation where endogenous ERAD inhibitor SVIP takes place. This finding has a great potential for clinical studies since the main reason of cystic fibrosis is the lack of CFTRdF508 on plasma membrane of the cells.

After showing that Hrd1 regulates gp78 by promoting its degradation with CFTRdF508, Dr Ballar focused on degradation of classic type-I oculocutaneous albinism-related tyrosinase C89R degradation. She has identified the mutant tyrosinase (C89R) as an Hrd1 substrate. Consistent with the results obtained in CFTRdF508 studies, Hrd1 silencing caused decreased CD3d and ATZ levels by elevating gp78 levels. This suggested that Hrd1 is not a specific regulator for CFTRdF508 degradation but a general regulator of gp78-mediated ERAD. To address the bidirectionality of this functional interaction between Hrd1 and gp78 she examined the effect of gp78 on the Hrd1 substrate, tyrosinase (C89R). The data from this study suggest that functional interaction between Hrd1 and gp78 is unidirectional rather than a crosstalk as silencing Hrd1 increased gp78 levels accompanied by an enhancement in the polyubiquitination of gp78 substrates; but silencing gp78 affected neither the levels nor polyubiquitination of tyrosinase (C89R) nor the substrate for Hrd1. By having model substrates both for Hrd1 and gp78-mediated degradation Dr Ballar studied the composition of retrotranslocation complex functioning in these two different ERAD pathways. Her results suggest that the Hrd1-mediated ERAD requires the well-established retrotranslocation machinery, the p97/VCP–Ufd1–Npl4 complex, whereas the gp78 pathway needs only p97/VCP and Npl4. This conclusion was reported as the first report suggesting different retrotranslocation complexes functioning in mammalian ERAD.

The last stage of the project aimed to decipher more molecular details of ERAD by utilising disease-related p97/VCP mutants. p97/VCP is a hexameric AAA type functioning as the key protein of retrotranslocation of misfolded proteins from ER to the cytosol. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder, which has been attributed to mutations in p97/VCP. Several missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Dr Ballar and her team analysed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, only one of them namely, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localisation. Almost all the mutants (except P137L) showed enhanced binding ability to the interacting proteins of p97/VCP, namely Ufd1-Npl4, p47 and ubiquitin. The P137L mutation completely lost its interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding, another interactor of p97/VCP. Similarly, while recombinant R155C mutant protein consistently interacts with both Ufd1 and VCP-interacting motif (VIM) of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. This was a very important finding, since it has been previously demonstrated that binding of Ufd1, p47 and the VIM-containing proteins SVIP and gp78 to the ND1 domain of p97/VCP is mutually exclusive. However, the data of Ballar's group suggests that replacing Proline 137 with a Leucine abolishes Ufd1-Npl4 and p47 binding while conserving its gp78 and VIM-binding ability. These differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD.

Besides the above-mentioned 'mammalian ERAD'-related studies, Dr Ballar participated in several projects during the last four-year period. By collaboration with Dr Shengyun Fang (Uni. of Maryland, USA) she participated in projects named 'the role of SVIP in the localisation of p97/VCP and autophagy' and 'an unanticipated role of importinß in ERAD'. Both of these studies are published. With the collaborations established within the Ege Uni., Dr Ballar was co-authors of some peer-reviewed publications. Examples of such publications are: 'Electrochemical based detection of microRNA, mir21 in Breast Cancer Cells' with Dr Mehmet Ozsoz (Ege Uni., Analytical Chemistry Dep.), 'Secondary metabolites with antiproteasomal activity from Centaurea urvillei DC. subsp. urvillei' with Dr Erdal Bedir (Ege Uni., Bioengineering Dep.), 'Synthesis and screening of cyclooxygenase inhibitor activity of some 1,3-dioxoisoindoline derivatives' with Varol Pabuccuoglu (Ege Uni., Pharmaceutical Chemistry Dep.). In her IBMPFD-related publication, Dr Ballar collaborated with Dr Devrim Gozuacik (Sabanci Uni., Istanbul, Turkey) and Dr Eric Toth (Uni. of Maryland, United States). In addition to her publications there are some other aspects reflecting the well reintegration of Dr Ballar.

She is the principal investigator of two national projects, the co-PI of two national projects, the co-applicant of an infrastructure grant that granted by national funding agencies. In 2011 Dr Ballar was assigned as the 'Head of Molecular Biology Laboratory' of Ege Uni. Pharmaceutical Sciences Research Center.

Moreover, she worked in several workshops related to Marie Curie actions in Turkey, and also presented her own success story as part of 'Destination Turkey' project in United States, where Turkish scientists in United States were informed about the opportunities offered by European Union and TUBITAK in order to encourage their return back to Turkey. Furthermore, Dr Ballar received L'oreal Turkey For Woman In Science Fellowship in 2010 and Turkish Pharmacists' Association Young Scientist Award in 2012. In 2010 she was elected as the chair organiser of the seventh annual meeting of INPROTEOLYS network composed of European scientists working on intracellular protein degradation and ubiquitin systems. She applied to the Federation of European Biochemistry Societies (FEBS) for financial support, and her project was awarded as FEBS special meeting with EUR 50 000 financial support. 'The FEBS Special Meeting on Protein Quality Control and Ubiquitin Systems in health and disease' was taken place as the seventh Inproteolys meeting in 14-16 November 2012 in Turkey. Over 200 scientists from 19 different countries participated this meeting.

Dr Ballar received the Associate Professor degree in 2012, which is given by a committee of external experts by Turkish Law. Considering her success in research and reintegration during the last four-years, Dr Ballar was hired into a permanent position as Associate Professor in Biochemistry Department of Faculty of Pharmacy, Ege University in February 2013.