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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences


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.

Technical Summary

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.

Planned Impact

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.
Description Our findings provided a better understanding on the functional mechanism of the mitochondrial sulphydryl oxidase Erv1 at molecular and atomic levels. We also established a model for the molecular basis of a disease-associated R182H mutant, the mutation impairs the cofactor binding during the enzyme catalytic reaction. The model could potentially be extended to human ALR R194H and provides insights into the molecular basis of autosomal recessive myopathy. We also showed recently that Trp95 and Trp183 are important for the folding and oxidase function of Erv1.
Exploitation Route Most of our findings have be published with open access. However, it maybe better taken forward if we could attend more international or national meetings and give more oral/poster presentations.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

Description It has been used as case study in undergraduate teaching.
Sector Education
Description Collaboration with Yeast biologist Dr Lodi 
Organisation University of Parma
Department Department of Life Sciences
Country Italy 
Sector Academic/University 
PI Contribution Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.
Collaborator Contribution Our collaborator made the yeast mutant strain for us to test the function of wild-type and mutant Erv1.
Impact One paper was published @ It is a multi-disciplinary collaboration between biochemists and a yeast biologist.
Start Year 2013
Description Dr Pang - Computational analysis of Erv1 
Organisation University of Greenwich
Country United Kingdom 
Sector Academic/University 
PI Contribution Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp(95), no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. My research team performed various experiments to investigate the functions of Trp residues of Erv1. My collaborator performed computational analysis to enhance our understanding of the molecular mechanism in more detail.
Collaborator Contribution Computational analysis confirmed experimental observation and further indicates that Erv1 Trp95 plays a key role in stabilizing the cofactor binding to Cys133.
Impact One paper was published: Biosci Rep. 2015 Jul 28;35(4). pii: e00244. doi: 10.1042/BSR20150144. The collaboration is multi-disciplinary. While I am a experimental biochemist, the collaborator is a computational chemist.
Start Year 2011
Description Prof N Scrutton 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Perform various biological, biochemical and biophysical experiments to understand the functional mechanism of mitochondrial Mia40-Erv1 pathway
Collaborator Contribution Expertise on enzymology and kinetic studies
Impact One BBSR grant (BB/H017208/1) and two papers so far: Biochem J. 2014 Jun 1;460(2):199-210. doi: 10.1042/BJ20131540. Biochem J. 2014 Dec 15;464(3):449-59. doi: 10.1042/BJ20140679.
Start Year 2010