Choudhary Group – University of Copenhagen

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CPR > Research > Proteomics > Choudhary Group

Choudhary Group - Proteomics and Cell Signaling

Group Leader: Chunaram Choudhary, PhD


Biological systems respond to a wide range of extracellular and intracellular cues (signals), resulting in a myriad of diverse outcomes. Cells respond to these signals by modulating expression levels of protein as well as by regulating their functions via posttranslational modifications (PTMs). PTMs have critical roles in the dynamic regulation of protein activity, stability, spatial localization, and communication with other macromolecules within cells. Deregulation of PTM modifying enzymes, such as protein kinases, ubiquitin ligases, and lysine deacetylases, is often observed in human diseases further highlighting the importance of their regulatory functions. Thus, unraveling the scope of PTMs, their link to regulatory enzymes, and their dynamics in response to cellular perturbations is critically important for obtaining a better understanding of the principles of cellular governance.

A major focus of the Group is to obtain a `systems-wide` understanding of signaling networks. For this, our laboratory applies cutting-edge mass spectrometry-based proteomics technologies for unbiased, global, and quantitative analysis of key regulatory PTMs. We work closely with other groups within the Centre as well as with our national and international collaborators.


Recent advances in quantitative proteomics have opened new ways for unbiased analysis of proteomes and PTMs on a global scale (Choudhary and Mann, 2010, Nat Rev Mol Cell Biol). We use state-of-the-art, SILAC-based proteomics technology to quantify changes in expression of thousands of proteins and tens of thousands PTMs in a single experiment (Figure 1). This enables us to perform a ‘systems-wide' analysis of signaling pathways relevant to human health and diseases, such as cancer. Additionally, we use a range of biochemical and molecular biology approaches to investigate the functional relevance of dynamically regulated proteins and PTMs.

Figure 1. Work-flow of a typical quantitative proteomic experiment for large-scale PTM analysis.


Lysine acetylation

Lysine acetylation is a well-known epigenetic modification targeting lysines on histones. Using the proteomics technology we showed that acetylation is a widespread modification targeting proteins present in diverse subcellular compartments (Choudhary et al., 2009, Science). Furthermore, we found that many acetylation sites are evolutionary conserved from budding yeast to humans (Weinert et al., 2011, Sci. Signaling; Henriksen et al., 2012, Mol Cell Proteomics). A parallel quantification of acetylation, phosphorylation and proteome revealed their dynamic changes in response to DNA damage signaling and identified an important link between genotoxic stress and mRNA metabolism (Beli et al., 2012, Mol Cell).

Acetylation is frequently observed at mitochondrial proteins and metabolic intermediates are important regulators of this modification (Figure 2). Ongoing research is focused at understanding the link between cellular metabolism and lysine acetylation. We are also interested in investigating the dynamics of acetylation in various signaling pathways and its cross-talk with other regulatory PTMs.            

Figure 2. The role of acetyl-CoA and NAD+ in metabolism, and acetylation signaling.


Ubiquitin is a small, highly conserved protein that can be covalently conjugated to lysine side chains in a process termed as ubiquitylation or ubiquitination. It is catalyzed by concerted actions of E1, E2 and E3 ubiquitin conjugating enzymes and removed by deubiquitylases or deubiquitinases (DUBs). Proteomic analysis of endogenous ubiquitylation sites has been challenging in the past. Recently, our group has optimized proteomics workflow for global ubiquitylation analysis using the di-Gly approach (Wagner et al., 2011, Mol Cell Proteomics). This method allows identification and quantification of thousands of endogenous ubiquitylation sites in cells and tissues. Our results paint a broader picture of ubiquitylation landscape and reveals extensive overlap between ubiquitylation and acetylation sites (Figure 3).

Figure 3. An overview of ubiquitylation sites identified in human cells and their overlap with lysine acetylation sites.

We applied this method to investigate the dynamics of ubiquitylation in response to UV irradiation and identified many UV-regulated ubiquitylation sites (Figure 4) (Povlsen et al., 2012, Nat Cell Biol). We aim to further extend research in this area to investigating ubiquitylation dynamics in response to different cellular perturbations, and to identify potential targets of selected ubiquitin ligases and deubiquitylases.                                 

Figure 4. Analysis of functional associations among proteins with UV-upregulated ubiquitylation sites.


1. Weinert BT, Iesmantavicius V, Wagner SA, Gummesson B, Beli P, Nyström T, and Choudhary C. Acetyl-Phosphate is a Critical Determinant of Lysine Acetylation in E. coli. Mol Cell, 2013 Jul 25;51(2):265-72.

2. Weinert BT, Schölz C, Wagner SA, Iesmantavicius V, Su D, Daniel JA, Choudhary C. Lysine succinylation is a frequently occurring posttranslational modification in prokaryotes and eukaryotes and extensively overlap with acetylation. Cell Rep. 2013 Aug 29;4(4):842-51.

3. Povlsen LK, Beli P, Wagner SA, Sylvestersen KB, Poulsen SL, Poulsen JW, Nielsen ML, Bekker-Jensen S, Mailand N, Choudhary C. Systems-wide analysis of ubiquitylation dynamics reveals a key role of PAF15 ubiquitylation in DNA damage bypass. Nat Cell Biol. 2012 Sep 23;14(10):1089-98.

4. Beli P, Lukashchuk N, Wagner SA, Weinert BT, Olsen JV, Baskcomb L, Mann M, Jackson SP, Choudhary C.  Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response.  Mol. Cell   2012 Apr 27;46(2):212-25.

5. Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, Choudhary C. A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles. Mol Cell Proteomics. 2011 Oct;10(10):M111.013284.

6. Weinert BT, Wagner SA, Horn H, Henriksen P, Liu WR, Olsen JV, Jensen, LJ and  Choudhary C. Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation. Sci. Signaling. 4, ra48 (2011).

7. Wild P, Farhan H, McEwan DG, Wagner S, Rogov VV, Brady N, Richter B, Korac J, Waidmann O, Choudhary C, Dötsch V, Bumann D and Dikic I. Phosphorylation of the Autophagy Receptor Optineurin restricts Salmonella Growth. Science. 2011 Jul 8;333(6039):228-33.

8. Kaidi A, Weinert BT, Choudhary C, Jackson SP. Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science. 2010 Sep 10;329(5997):1348-53.

9. Choudhary C and Mann M. Decoding signaling networks by mass spectrometry-based proteomics. Nat Rev Mol Cell Biol. 2010 Jun;11(6):427-39.

10. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther T, Olsen JV, Mann M. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science; 2009 Aug 14;325(5942):834-40.

Research funding

Novo Nordisk Foundation Center for Protein Research is funded by a generous donation by the Novo Nordisk Foundation. In addition to this, research in the Choudhary group is funded by the following public and private funding sources:

  • the Danish Research Council
  • the Swedish Research Council
  • European Research Framework grants: SyBoSS and PRIME-XS

Link to Choudhary website outside University of Copenhagen (please note that this is a privately owned page exclusively under the responsibility of Professor Choudhary).