Phosphoprocessors by EU Research

Protein phosphorylation plays an important role in many cellular processes, now researchers in the Phosphoprocessors project aim to learn more about the molecular basis of this process. This work could have important implications for the design of circuits for synthetic biology, as Professor Mart Loog explains

Analysing the basis of protein phosphorylation Protein phosphorylation by

protein kinases plays a central role in many cellular processes. The addition of a negatively charged phosphoryl group can change the function of a protein, which then may lead on to further changes. “If you add another phosphate group to the protein, you can activate the protein or de-activate it, or localise it to other parts of the cell,” explains Professor Mart Loog, the Principal Investigator of the Phosphoprocessors project. Researchers in the project now aim to learn more about multisite phosphorylation, looking in particular towards three main objectives. “One is related to the regulation of cell division, and regulation of temporal order of individual molecular events in cell cycle and cell division,” outlines Professor Loog. “We’re looking at how the master regulators of this process, cyclindependent kinases (CDK), are able to

temporarily resolve these triggers and switches and other molecular events.” A second key objective in the project is to study the phosphorylation of proteins in kinetochores, a type of large protein structure. The main topic of interest here is the regulation of protein function through phosphorylation; the enzymes which catalyse this are protein kinases. “We are focusing on CDKs, as master regulators of the cell cycle. So far, we have studied this process with respect to individual substrate proteins, but now we are going to study it in the context of larger protein structures. We expect that we will find different rules and dynamics, as this is a new area of research,” says Professor Loog. The third objective is related to synthetic biology, synthetic circuit design.“We aim to apply the knowledge that we have gained from studying multi-site phosphorylation circuits in the cell

cycle – we aim to apply the same rules in designing regulatory circuits, in synthetic cellular systems,” continues Professor Loog.

Multi-site phosphorylation This work builds on continued research into the phosphorylation process. Within the project, researchers are looking in particular at multisite phosphorylation, in which the phosphoryl groups are attached at different sites in the proteins. “These sites can be distributed within the peptide chain in different patterns and clusters, with different distances between them, and different amino acids around them. These are the parameters that define the phosphorylation process – the kinetics of it, the dynamics of it, and basically, how its inputs and outputs are controlled,” explains Professor Loog. The aim in the project is to crack the ‘multisite phosphorylation code’ of CDK, while

EU Research

Professor Loog and his colleagues are also looking to apply their knowledge to design circuits for synthetic biology. “We can apply certain concentrations of kinase input, and depending on the information we encode in the amino-acids sequence, in terms of distances and patterns,” he says. “Depending on the design, and the ability of the kinase to read this kind of barcode, the protein will react in different ways.” The key issue here is to ensure that the circuit is sufficiently specific and well adapted to the environment in which it is to be introduced. A synthetic circuit put into a cell has to fulfil two main criteria. “One is that your own circuit or signalling system doesn’t affect the normal physiology of the cell,” outlines Professor Loog. Then it’s also important to ensure that cellular kinases won’t shoot their signals into the artificial system, that it’s almost insulated and protected from other similar signals in the cellular environment. “It’s really difficult to gain this specificity, because there are crossspecificities and similar, closely related kinases and substrates,” continues Professor Loog. “They compete with each other, and it’s very difficult to get rid of the noise of the other signals, it’s one of the biggest challenges in synthetic circuit design. With multi-step and linear coding, we can encode unlimited combinations into these proteins, so we can amplify the specificity and selectivity of signals at every step.” Researchers are working primarily with disordered proteins, which is why it is possible to encode patterns, networks and phosphorylation sites in a linear way, without the complexity of the 3D structure of proteins. The biochemical

www.euresearcher.com

rules on how to encode the signal have been identified; Professor Loog and his colleagues are building on this knowledge to design circuits for synthetic biology and develop a toolbox of synthetic parts. “The phosphorylation barcodes act like signal processors. Right now we are in the process of creating logic gates and different computational elements, which would be entirely unique technology,” he explains. This work reflects a wider change in biology, away from it being a purely descriptive science towards also becoming an engineering discipline. “We used to focus primarily on basic science, studying the cell cycle. Now, in this project, we are applying this

understanding of these processes, and now we can apply this knowledge for designing synthetic circuits. We understand it in greater depth now, so we have very predictable models and predictable switches,” he says. Researchers are working with yeast, which is the simplest eukaryotic system. “We are designing this system using yeast, because it’s the best option for doing very clean and simple experiments, for establishing a proof-of-concept,” says Professor Loog. “Once we’ve got our systems working as we’ve predicted, we’ll then go into stem cells and try to re-wire the stem cell differentiation pathways to see if we can create stem cells which are entirely programmeable by our inputs.”

We’ve been inspired by the signalling networks that have been created through evolution. We have gained very specialised understanding of these processes, and now we can apply this knowledge for

designing synthetic circuits

knowledge to engineer switches that are not found in nature, that can then mediate signals in cells and perform other biological functions,” continues Professor Loog. This work holds important implications beyond the research sphere. Virtually every signalling pathway or disease state is related to a phosphorylation process of some kind, underlining the wider relevance of Professor Loog’s research. “We’ve been inspired by the signalling networks that have been created through evolution. We have gained very specialised

Synthetic cells The next step could be to apply the technology in 3D tissue engineering, while Professor Loog is keen to explore further potential applications of the research. One major area of interest is sustainable bioprocessing and cell factories, using yeast in bioreactors. “With these systems, we can control the regulation of metabolic enzymes and metabolic pathways. This is very practical research,” explains Professor Loog. A longer-term goal within the synthetic biology field is the development of synthetic organs; while this remains a

At a glance Full Project Title Biological signal processing via multisite phosphorylation networks (Phosphoprocessors) Project Objectives Multisite phosphorylation of proteins is a powerful signal processing mechanism which plays crucial roles in cell division and differentiation, as well as in disease. The goal of the Phosphoprocessors project is to elucidate the molecular basis of this important mechanism. Project Funding ERC-CoG-2014 - ERC Consolidator Grant Contact Details Project Coordinator, Mart Loog Professor of Molecular Systems Biology Institute of Technology University of Tartu Nooruse 1, Tartu 50411, Estonia T: +37 2 517 5698 E: Mart.Loog@ut.ee W: www.looglab.com Kõivomägi M, Ord M, Iofik A, Valk E, Venta R, Faustova I, Kivi R, Balog ER, Rubin SM, Loog M. (2013) Multisite phosphorylation networks as signal processors for Cdk1. Nature Struct Mol Biol. 20(12), 1415-24. Kõivomägi, M., Valk, E., Venta, R., Iofik, A., Lepiku, M., Balog, E.R.M., Rubin, S.M., Morgan, D.O., and Loog, M. (2011). Cascades of multisite phosphorylation control Sic1 destruction at the onset of S phase. Nature, Oct 12. doi: 10.1038.

Professor Mart Loog

long way off, Professor Loog says the project’s research could have a practical impact, for example in cell factories. “Cell factories are being developed to synthesise certain chemicals and drugs,” he says. “The biggest bottleneck there is in regulating the metabolic pathways, so that the carbon flux goes to the compound, which then results in higher yields. This is where we are going to apply our research, in yeast in the first instance.”

vision, looking towards cell factories and sustainable bio-processing,” he outlines. There are plans to develop the Centre further, helping to establish it as a centre of excellence in synthetic biology, which could help translate research into practical applications. “We plan to create a pilot plant, a bioreactor facility for cell factories, and to also build a new centre for mammalian cell factories, microbial cell factories,” says Professor Loog.

We used to focus primarily on basic science, studying the cell cycle. Now, in this project, we are applying this knowledge to engineer switches that are not found in nature, that can then mediate signals in cells and perform other biological functions There is a huge market for these kinds of tools and synthetic circuits, as researchers seek to develop more sustainable methods of producing specific chemicals and pharmaceutical products. Over the next three years, researchers will be publishing results, and patenting and finalising the toolbox, while Professor Loog is also keen to lay the foundations for continued synthetic biology research. “We have established the Estonian Centre for Synthetic Biology, where we collaborate with many different research groups. We are introducing this industrial

This is a very active area of research, with scientists seeking to develop cells, control their functions, and harness their properties for specific biological applications. There is still a great deal to learn, yet Professor Loog believes that humans will eventually learn how to use synthetic cells, and the Estonian Centre for Synthetic Biology will play a prominent role in these terms. “We are in a very good position to be leaders in this field and to create an effective toolbox, which could have a wide range of biological applications,” he says.

Mart Loog is professor of molecular systems biology and head of the Estonian Centre for Synthetic Biology (ECSB). Mart received a PhD in medicinal biochemistry from Uppsala University, Sweden in 2002, followed by postdoctoral training at University of California, San Francisco. In 2006 Mart established his laboratory at the newly established Institute of Tehnology. He has received several international fellowships and awards including The Wellcome Trust Senior International Fellowship and a startup research grant from European Molecular Biology Organization (EMBO) and Howard Hughes Medical Institute (HHMI). In 2012 he received the Estonian National Science Prize in chemistry and molecular biology. In 2015 he was awarded the European Research Council (ERC) Consolidator Grant and became a principal coordinator of a H2020 ERA Chair project SynBioTEC to establish the multidisciplinary Centre of Synthetic Biology.

EU Research