Towards a systems-level understanding of the novel redox-regulated mitochondrial protein import and disulphide bond formation pathway

Mitochondria are vitally important organelles - often described as the powerhouses within eukaryotic cells. They generate the primary energy for biological activities that sustain human life, and regulate cell growth and cell death (apoptosis). Mitochondria are implicated in >40 human diseases, including diabetes, deafness, ageing and cancer. For example, a single Cys mutation in the mitochondrial deafness-dystonia protein 1 (DDP1) causes Mohr-Tranebjaerg/deafness-dystonia syndrome, an X-linked neurodegenerative disorder. Protein import is essential for the biogenesis of mitochondria, since 99% of mitochondrial proteins are synthesized in the cytosol and have to be imported into mitochondria for their biological function. Each mitochondrion is enclosed by a double membrane that divides the organelle into four sub-compartments: outer membrane, intermembrane space (IMS), inner membrane, and matrix. The mitochondrial IMS harbors many Cys-containing proteins that are essential for the biogenesis of mitochondria and viability of the cells. A recent important biological finding by us and others is the presence of novel redox regulated mitochondrial import and assembly (MIA) machinery in the IMS. The MIA machinery includes two essential component proteins, Mia40 and Erv1, which form a disulphide bond relay system responsible for the import and correct disulphide bond formation in the newly imported IMS proteins. However, the molecular basis for the function of this machinery is not clear. Many important questions, such as how are electrons and/or disulphide bonds transferred within the Mia40-Erv1 system; what regulates the disulphide bond transfer between the system and its substrate proteins; how are the protein-protein interactions and recognition regulated; is the harmful hydrogen peroxide (H2O2) the real product of the oxidation reaction, remain to be answered. Thus, this proposal will use a wide range of biophysical, biochemical, and biological methods to provide answers to these questions. This currently heated research topic has been a focus of many biologists. Biological studies on cellular systems often take the form of top-down approaches to identify new candidates and potential correlations in the systems. They are then formulated in terms of empirical relations, but rarely lead to the formulation of molecular mechanisms. Thus, a bottom-up mechanism-based study is timely and essential, which relies on knowledge of thermodynamics, kinetics, and measurable parameters of protein interactions. In this proposal, we will determine those functionally important thermodynamic, kinetic and structural properties of the proteins in order to define the molecular mechanism of the Mia40-Erv1 system. This study will provide insights in not only protein disulphide bond formation and mitochondrial biogenesis per se, but also redox regulation and cause of oxidative stress. All these processes are inextricably linked to mitochondrial physiology, ageing, dysfunction and therapeutic intervention.

Mitochondria are crucial in energy production, cell growth and apoptosis. Protein import is essential for the biogenesis of mitochondria, and disulphide bond formation is crucial for the import and folding of the mitochondrial intermembrane space (IMS) proteins. A novel mitochondrial sulfhydryl oxidoreductase system was identified recently, in which the proteins Mia40 and Erv1 play an essential role in import and oxidative folding of the IMS proteins. There is clear evidence that this mitochondrial oxidoreductase system exhibits major differences in the structures and mechanisms from other well-known sulfhydryl oxidoreductases. Many important questions about the functional mechanism of the Mia40-Erv1 system remain unanswered. In this proposal, we will use a wide range of biophysical and biological methods to define the molecular mechanism of Mia40-Erv1 system. The electron transfer mechanism, regulation of Mia40-Erv1 interactions, and the downstream substrate specificity of Erv1 will be investigated. The functionally important thermodynamic and kinetic parameters will be determined. Moreover, our in vitro finding will be tested using mitochondrial import assays and yeast genetic approaches in vivo. This study aims to provide essential knowledge for a systems-level understanding of this novel redox-regulated mitochondrial protein import and disulphide bond formation pathway.

The ultimate aim of this research project is to provide a detailed and systems-level analysis to understand the mechanisms of oxidative protein folding and redox regulation in the mitochondrial intermembrane space. This study will enhance our understanding of the molecular mechanisms of the biogenesis of mitochondrial proteins and the organelle. Thus, it will provide insight into the molecular basis of mitochondrial related diseases. This means that not only researchers in biology, biochemistry, and biomedicine will benefit from this study; the potential medical impact of this research will also benefit the public by enhancing the quality of our lives in long term. The mitochondrion is a vitally important organelle. It is essential for generating the primary energy to sustain our lives and plays important role in regulate the cell growth and death. Mitochondria are implicated in >40 human diseases, including diabetes, deafness, Alzheimer's disease, ageing, and cancer. It is a major source for oxidative stress caused by production of reactive oxygen species. This study will enhance our understanding in the molecular mechanisms of oxidative protein folding and biogenesis of mitochondria, and thus it will provide insight into the molecular basis of mitochondrial related diseases. Thus, the potential medical impact of this research will benefit the public and enhance quality of our lives in long term. The results of this study will also be benefit to biotechnology industry through improved knowledge-based strategies for design new catalysts and small molecule inhibitors of enzyme systems. Should new findings of the protein oxidation mechanism emerge from our work, it will have profound effects on how we prevent from oxidative stress, ageing, and target enzyme systems therapeutically. In addition, oxidative protein folding or protein disulphide bond formation is very important for the function of many proteins. The regulation of disulphide bond formation/breaking is crucial for the function of many redox-active proteins. Understanding the mechanism of oxidative protein folding and the functional roles of redox-active proteins has been slow because of the challenge in analysing the highly reactive thiol groups. In this study, we will define mechanisms of mitochondrial thiol-disulphide redox-regulation, providing important insights into oxidative protein folding. The results and the methods developed from this study will be of wide interest to cell biologists, biochemists, and protein chemists. Results of this study will primarily be communicated through publications in the scientific journals, presented in scientific conferences through oral and poster presentations by the applicants and PDRA. We will communicate our research with other academics working in similar areas and establish new collaborations positively. In addition, we will also take advantage of the 'Discover days' hosted by the Faculty of Life Sciences at Manchester and summer placements for 6th form students to introduce school children to the science underpinning mitochondria, proteins, ageing, and biological catalysis. We will explore other opportunities, such as the Royal Society annual science exhibition, writing short articles for magazines that targeted to school children and/or the public, to communicate our research to the public.