Combining Genomics and Phosphoproteomics to Identify Kinase-Substrate Networks that Promote Mitochondrial

Mitochondria are specialized compartments found within cells that convert fuel into energy. During processes such as development, where energy demands are high, insulin activates multiple biological switches that ultimately generates new mitochondria. Decreases in mitochondrial number and function, and/or the accumulation of damaged mitochondria accelerate the ageing process. Moreover dysfunctional mitochondria can drive the onset of a chronic inflammatory state that underpins to the pathogenesis of type 2 diabetes, cancer, and neurodegenerative disorders. Insulin-resistance is a hallmark of such age-associated diseases strongly suggesting that suppression of insulin-mediated mitochondrial production is a common route to ageing and disease. However, the biochemical reactions that link insulin stimulation to mitochondrial health are largely unmapped. Gaining a systems-level view into the signaling networks that link insulin stimulation to mitochondrial production is essential.

We aim to use a combination of genetic and phosphoproteomic technologies to map key biochemical reactions that are critical for insulin-mediated mitochondrial homeostasis. We are optimistic that a comprehensive understanding of these reactions will allow us to ultimately develop means to manipulate insulin signalling networks to improve well-being during the ageing process.

Using a novel genetic screen that we have recently developed, we first aim to comprehensively identify all genes required for mitochondrial health. This cost-effective screen that use computational algorithms to automatically measure mitochondrial shape in millions of single cells following inhibition of each gene in the genome one at a time. If inhibition of a particular gene leads to abnormally shaped mitochondria this strongly suggests a role for this gene in promoting mitochondrial homeostasis.

We have recently developed a cutting-edge mass-spectrometry based technology to monitor levels of protein phosphorylation on all proteins in a cell. Kinase proteins phosphorylate different proteins as a means to turn different proteins "on" on "off". By monitoring all phosphorylation events that occur in cells following stimulation of insulin, we can then determine which proteins are likely to be regulated by insulin.

By completing these two aims we will thus identify sets of proteins that are both involved in mitochondrial health (through genetic screens completed in Aim 1), and that are regulated by insulin (through completion of Aim 2).

In order to determine the kinases that are responsible for phosphorylating substrates identified in Aims 1 and 2, we will then combine genetics and mass spectrometry to monitor protein phosphorylation following systematic inhibition of all kinases in insulin-treated cells.

In almost all cells insulin is a key factor in promoting mitochondrial biogenesis and function. In this proposal we aim to use a combination of functional genomic and phosphoproteomic technologies in order to describe, on a systems-level the key kinase-substrate reactions that promote mitochondrial homeostasis. Specifically we aim to implement a recently developed quantitative image-based readout to perform a high-throughput RNAi screen for regulators of mitochondrial morphology in Drosophila S2R+ cells. This screen uses automated image processing algorithms to extract dozens of different features that describe mitochondrial shape in single-cells. We have already demonstrated that we can use this screen to identify genes, such as InR, Mef2, and p38, that contribute to regulating mitochondrial shape - and thus function. In parallel we will determine all proteins that are phosphorylated following insulin stimulation using phospho-proteomics. We have established a protocol for purifying phosphorylated peptides from trypsin digested samples of S2R+ cells using a combination of Immobilised Metal Affinity Chromatography (IMAC) and Titanium Dioxide (TiO2). Phosphorylated peptides are separated by hydrophilic interaction liquid chromatography (HILIC) and identified by mass spectrometry. The completion of these two aims will thus lead to the identification of proteins that are both required for mitochondrial morphogenesis and phosphorylated following insulin stimulation. These proteins are thus excellent candidates for proteins that link insulin to mitochondrial homeostasis. In order to determine the kinases responsible for phosphorylating these proteins following stimulation we will then perform a systematic screen using in vitro phosphorylation of substrate peptides mixed with cell-lysates prepared following RNAi of all Drosophila kinases (KAYAK-RNAi screen). We will use classical methods such as combinatorial gene inhibition and genetic rescue to validate our findings.

The research described in this proposal aims to provide fundamental insight into the role of the biochemical networks that couple insulin signalling to mitochondrial homeostasis.

A number of groups will potentially benefit from this research including:

- staff working on the project will develop which develop research and professional skills they will be able to apply in the future across a number of different employment sectors
- academic researchers and industrial organization who will benefit from both the databases and novel methodologies developed as part of this grant
- an increasingly ageing British and international population
- the British economy as a whole

(1) Academic Researchers.
The two postdoctoral fellows supervised by Dr Bakal and Dr Jorgenesn who will work on this project will gain valuable and highly useful experience in: robotics, high-throughput screening techniques, phosphoproteomics, image-analysis, computational analysis. Moreover, both postdoctoral fellows will gain experience in project and budget management, industrial collaboration, and public speaking.

A number of academic researchers in the greater scientific community will benefit immediately from this work including those studying; academic researchers studying insulin signalling, mitochondrial biogenesis and function, aging, and signal transduction.

Specifically, the databases that we aim to generate as part of this work will be highly amenable for future use in both biological and computational studies of insulin signalling and mitochondrial homeostasis.

(2) Commercial Private Sector Beneficiaries.
The project outlined could potentially lead to the development of both single and combinatorial therapeutic targets to insulin activity by academic or industrial organizations. The Institute of Cancer Research Technology Transfer Enterprise Unit is well equipped to protect any Intellectual Property generated by this project and further pursue it in terms of commercial exploitation.

(3) Public Sector.
Completion of these studies pay potentially lead to therapeutic treatments, and/or intuition as to how to make lifestyle choices (e.g. nutritional changes) to manipulate and thus improve the quality of life of an ageing British populace.

(4) Potential to impact on the nation's health, wealth or culture.
By providing novel insights into the regulation of mitochondrial homeostasis this research could potentially lead to therapies and treatments which could dramatically improve the well-being, health, and creative output of an increasingly ageing British populace and in turn, the British economy.

The potential benefits for the academic and commercial sector will be realized almost immediately after completion of this research (5-10 years). Potential benefits to the health of the public sector would be realized over a longer period (10-15 years).

Research findings will be disseminated information through peer reviewed public journals, and public presentations (both to academic and public audiences). All datasets will be made publicly available via an ICR-maintained website.