Elucidating the contributions of ubiquitin ligase complexes to endoplasmic reticulum associated degradation (ERAD)

Proteins are the molecular machines of cells, performing a wide range of essential functions. A broad range of inherited human diseases can be traced back to single proteins that do not fold correctly. A change in the genetic code results in a protein that is unable to reach the correct 3-dimensional structure necessary to function. Other disorders are associated with conditions within cells that are unsuitable for proteins in general to fold correctly. For those proteins that are secreted or expressed on the surface of cells, protein folding occurs in the intracellular compartment known as the endoplasmic reticulum (ER). Within the ER, each protein is evaluated by quality control mechanisms that decide whether it's folded correctly. When proteins misfold, a complex remediation scheme is engaged which removes those forms from the ER and destroys them. Collectively, this mechanism is known as ER-associated degradation (ERAD). As cell surface and secreted proteins represent approximately 30% of all the human proteins made, distinguishing and degrading such a wide range of potentially misfolded proteins presents a significant challenge. ERAD uses a network of interconnected factors able to identify, target, deliver and degrade non-native proteins with various configurations. A central component in this scheme is the family of ubiquitin ligases embedded in the ER membrane. One example is Hrd1, which forms nexuses of ERAD activity by scaffolding different accessory factors on both sides of the ER membrane. This MRC project aims to characterise both the composition and organisation of Hrd1-containing complexes and to ascertain the contributions that the entire family of ER-resident ubiquitin ligases make to ERAD.
The benefits of the proposed research will be to advance our understanding of how misfolded proteins are degraded in cells, and how this fundamental mechanism alleviates cellular stress that is the hallmark of many diseases. Our current view of ERAD has been based on a limited analysis of individual components and substrates. The proposed research aims to advance this picture by 1) characterising in detail, the functional Hrd1 complexes the carry out ERAD, 2) using a proven isolation strategy and mass spectrometry to characterise other ER-resident ubiquitin ligase complexes that participate in ERAD and 3) determining how cells use expression of the different ERAD components to respond to stress. As the number of diseases linked to protein misfolding grows, it is anticipated that the comprehensive analysis proposed here will advance our understanding of how these conditions may arise. Moreover, this study may help direct efforts to devise novel and alternative therapeutic strategies for the treatment of disorders of associated with protein misfolding and trafficking, such as cystic fibrosis and alpha1-antitrypsin deficiency.

ERAD represents the principal remediation mechanism for non-native, maturation-incompetent proteins whose unabated accumulation can disrupt optimal ER function. ERAD employs a network of accessory factors localised to both sides the ER membrane that coordinate with the ubiquitin-proteasome system (UPS) to degrade misfolded integral membrane and secreted proteins. Membrane embedded ubiquitin ligases (E3s) such as Hrd1 form nexuses for ERAD activity by assembling macromolecular complexes in the ER along the transmembrane axis. This proposal aims to identify the individual components and domains essential for formation of Hrd1-containing complexes using velocity sedimentation to monitor size, rationally designed truncations/deletions to map the interaction domains, RNAi (and possibly TALEN-based genomic editing) to determine essential accessory factors and radiolabeling-densitometry to ascertain stoichiometry. This proposal also draws on a proven affinity purification-LC-MS/MS strategy to establish the interaction networks (and potentially substrates) of the 26 ER-resident E3s putatively involved in ERAD. Quantitative analysis using the Nanostring hybridization platform will be used to track changes gene expression of components in the ERAD network (with a focus on ER-resident E3s and their interactors) that result in response to ER stress conditions. This study will provide the first comprehensive analysis of ER-resident E3 complexes, their organisation and their expression with the goal of ultimately determining the contributions they individually make to the ERAD capacity and its ability to restore ER functionality during periods of stress-induced global misfolding.

It is anticipated that this research will provide an immediate impact for the community of research scientists and health professionals by providing critical insights into the general mechanisms by which ERAD attains the capacity to recognise, deliver, target and degrade non-native proteins. Our work focuses on characterising the molecular components and regulation of ER-resident ubiquitin ligase complexes with the long-term view to determine how they collectively function to exercise constitutive ERAD activity as well as respond to provide the enhanced capacity needed during times when cells experience ER stress. This research will be of significant interest to both academic and industrial biologists both in the UK and abroad whose interests lie in protein quality control, protein folding diseases and conditions characterised by ER stress. Those researchers and clinicians specialising in protein folding disorders will find the data generated in the study of particular interest, as it will help to further define the molecular mechanisms governing the fate of their protein of interest. Moreover, the strategy proposed herein will lay the groundwork for future studies that will define the molecular rules governing how and why misfolded proteins require particular complexes for degradation. While the research plan presented in this proposal is unlikely to result in any directly patentable or otherwise commercially exploitable material in the near future, it may indirectly lead to the development of novel therapeutic strategies to treat protein misfolding disorders in the years following the conclusion of the project.

The impact of our work on society and economics here in the UK will be in the potential of these studies to identify novel molecular targets for therapies against protein folding disease such as cystic fibrosis, alpha1-antitrypsin disease and lysosomal storage diseases. In the future, our work may prompt pharmaceutical and biotech companies to target components within protein quality control mechanisms and the ERAD pathway in particular, to treat these and other diseases. Moreover, the role of ERAD as an alleviator of ER stress may have potential in therapeutics, as ER stress is increasingly being appreciated as a phenomenon associated with cancer, obesity, diabetes and conditions such as irritable bowel syndrome. If realised, the above examples would provide economic benefits locally and contribute to the improvement in quality of life for individuals in the UK, as well as abroad.