11-13 May 2012 Helsingør - Denmark www.networkbio.org

MAREIKE WEIMANN*, JONAThAN WOODSMITh*, ARNDT GROSSMANN, zIYA özKAN, PETRA BIRTh,
DAVID MEIERhOfER, SASChA SAUER, ULRICh STELzL
OTTO-WARBURG LABORATORY, MAx-PLANCK INSTITUTE fOR MOLECULAR GENETICS (MPIMG), BERLIN
A Y2H-seq approach to define the protein methyltransferase interactome
Protein methylation, in particular on arginine and lysine residues, is a physiologically important post translational
modification (PTM). Whilst a large number of human methyltransferases (PMTs), potential demethylases
(DeMs) and methyl-recognition domain containing genes are annotated in the human genome,
systematic characterisation of this PTM remains poorly developed. Canonically studied with regards to its
role epigenetic regulation, methylation components or substrates have been shown to have diverse subcellular
localisations and molecular functions, indicating a wider role in cellular physiology. However, the lack
of affinity reagents and appropriate tools to detect methyltransferase substrate pairs has largely hampered
progress in defining the global role of non-histone protein methylation.
Here we present a novel proteome wide Y2H protein interaction screening approach involving a 2nd generation
sequencing readout that has a significantly improved sensitivity in comparison to our state of the art
Y2H matrix screening protocol. Furthermore, the sequencing readout provides a quantitation that correlates
very well with the retest success rate, indicative of the quality of the PPI information. Importantly, the workflow
presented here allows for rapid scalability using advances in sequencing technology to enable Y2H-seq
to accelerate large scale interactome mapping efforts.
We applied the Y2H-seq method to comprehensively screen proteins involved in either methylation or
demethylation. We present a network of >500 interactions involving 22 PMTs or putative DeMs and 324
potential methylation substrates. The network is validated using co-IP experiments and will serve as a major
informational resource to define cellular roles of protein methylation Furthermore, 7 candidate proteins are
characterized with respect to novel R and K methylation sites using a mass spectrometry approach, highlighting
the utility of the network to identify enzyme-substrate pairs.
abStractS For PoSterS abStractS For PoSterS
MOhAMMAD MOBAShIR AND DR. TILO BEYER
INSTITUTE Of MOLECULAR AND CLINICAL IMMUNOLOGY OTTO-VON-GUERICKE UNIVERSITY LEIPzIGER-
STRASSE 44 39120 MAGDEBURG GERMANY CONTACT: MOhAMMAD.MOBAShIR@MED.OVGU.DE AND
TILO.BEYER@MED.OVGU.DE
Unraveling of Network Motifs in Signal Transduction Networks Using Evolutionary Approach
Living organisms control their behavior and cellular function by propagating signals across multiple levels.
In T-cell signaling, the receptor, after receiving the signal activates downstream molecules that transmit the
signal to the nucleus, in order to control cellular functions such as proliferation, differentiation, and apoptosis.
During this process the change in the protein-levels, post-translational modification of the proteins, and
interaction strengths affect the final response of the cell. To investigate possible design priciples we developed
an in-silico model using evolutionary algorithm and ordinary differential equations. From our current
simulations, we found that (i) kinetics of the final response of signaling pathway is predominantly controlled
by the expression levels of proteins, (ii) the variations in protein-levels within the cell plays critical roles in
controlling the kinetic behavior of the cell and consequently the cellular functions, and (iii) an increase in
the interaction strengths up to a certain level leads to strong activation patterns, i.e. in the cells become
sensitive to stimuli. Thus, it appears that the kinetic properties of the signaling network mainly determine
the response threshold of a cell while the quantitative behavior is controlled by the expression levels of the
proteins.
50 / INB 2012 • 11-13 May 2012 www.networkbio.org / 51