Wellcome Centre for Cell Biology

Georg Kustatscher

Proteome dynamics: The role of synthesis and degradation in regulating protein levels

Photo of Georg Kustatscher

Georg Kustatscher is an MRC Career Development Fellow at the Wellcome Centre for Cell Biology. He studied Molecular Biology at the University of Salzburg, Austria, and obtained a PhD from the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, working on epigenetics in the lab of Andreas Ladurner. From 2008 to 2020 he was a Postdoc in Juri Rappsilber’s group at the WCB.

In September 2020, with the help of an MRC Career Development Award, he joined the Institute for Quantitative Biology, Biochemistry and Biotechnology (IQB3) in Edinburgh as an independent group leader, before returning to the WCB in 2022. His lab combines proteomics and computational approaches to understand how cells regulate mRNA and protein levels from a systems perspective.

Lab members

Emmanuel Fiagbedzi, Van Kelly and Victoria Munro

Georg Kustatscher profile

Proteome dynamics: The role of synthesis and degradation in regulating protein levels

There is a major discordance between mRNA and protein expression levels in human cells. Why is this so and what mechanisms are behind it? Our aim is to understand the principles, mechanisms and regulators that shape the proteome at the level of translation and protein degradation. Despite their importance for cancer and other diseases, these regulatory processes remain poorly understood, leaving an enormous potential for therapeutic intervention unfulfilled. We aim to address three key questions:

1. What is the role of translation and degradation rates in regulating protein levels, for example when buffering the impact of chromosome abnormalities in glioblastoma stem cells.

2. Which proteins regulate translation and degradation rates, e.g. can we reveal regulatory networks between E3 ubiquitin ligases and their targets.

3. Which unconventional translation products exist in healthy and in cancer cells and what are their biological functions.

From a technological perspective we plan to address these questions using a combination of proteomics and computational approaches and, where necessary, RNA sequencing. We are currently at the beginning of these projects and focus on the development of the necessary proteomics techniques that will allow us to carry out these investigations. The Centre has recently obtained a Sciex tripleTOF mass spectrometer, which is suitable for high-throughput (HT) proteomics, a rapidly emerging mass spectrometry approach for the robust quantitation of proteomes in a matter of minutes. HT proteomics differs from conventional proteomics on every level of the experimental workflow: the chromatography, the mass spectrometer and the data processing. To harvest the power of HT proteomics for the analysis of proteome dynamics we are developing DIA-pulse-SILAC, a method that will allow the rapid quantitation of protein synthesis and degradation rates by mass spectrometry. 

An important aspect of HT proteomics, and indeed all proteomics experiments, is the statistical analysis and interpretation of the data. This is a second area of focus for our group. For example, we recently collaborated with the Earnshaw group to create an interactive proteomic map of chromatin transactions during mitotic entry. We are also working together with the Ralser lab (Charité, Berlin) to predict the potential function of uncharacterised yeast proteins based on the proteomic characterisation of thousands of yeast knock-out strains.

Interactive proteomic map of chromatin transactions during mitotic entry

Georg Kustatscher research illustration

Figure 1. Development of DIA-pulse-SILAC for the rapid and precise measurement of protein synthesis and degradation.

(A) SILAC titration series created by mixing defined ratios of light and heavy extracts from RPE1 cells. In a direct comparison our new DIA-SILAC workflow (DIA; data-independent acquisition) quantifies considerably more proteins than the traditional data-dependent acquisition (DDA) of SILAC samples. The DIA workflow also quantifies the same set of proteins more consistently across replicates

(B) and quantifies them more precisely (C).

(D) Schematic of a pulse-SILAC experiment.

(E) DIA-pulse-SILAC was used to quantify synthesis and degradation rates in RPE1 cells. Shown are two representative example proteins with slow and fast turnover rates, respectively.

Selected publications

Samejima, I., Spanos, C., Samejima, K., Rappsilber, J., Kustatscher, G.*, and Earnshaw, W.C.* (2022). Mapping the invisible chromatin transactions of prophase chromosome remodeling. Mol. Cell 82, 696–708.e4. * co-corresponding authors

Kelly, V., Al-Rawi, A., Lewis, D., Kustatscher, G., and Ly, T. (2022). Low Cell Number Proteomic Analysis Using In-Cell Protease Digests Reveals a Robust Signature for Cell Cycle State Classification. Mol. Cell. Proteomics 21, 100169.

Nakamura, K.*, Kustatscher, G.*, Alabert, C., Hödl, M., Forne, I., Völker-Albert, M., Satpathy, S., Beyer, T.E., Mailand, N., Choudhary, C., et al. (2021). Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination. Mol. Cell 81, 1084–1099.e6. *joint first authors