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Molecular and functional analysis of protein recognition and degradation in plants

 

Proteins belong to the fundamental equipment of each organism’s cell and play crucial roles in numerous biochemical and cell biological contexts. They are only able to function properly if their abundance and shape are correct. Protein folding is a major determinant of their stability and function, thus it is vital to know degradation mechanisms of proteins that need to be destructed and how intracellular protein abundance is controlled. Protein quality control (PQC) is necessary to respond to endogenous physiological cues and to environmentally harsh conditions. Erroneous PQC may lead to improper responses to biotic and abiotic stress in plants. Biotic and abiotic stimuli like drought, salinity, extreme temperatures, heavy metals, pathogen infection and chemicals lead to osmotic and oxidative stresses. These may irreversibly damage proteins by misfolding during formation and thus compromise the entire cell. Abiotic stress is considered to be the primary cause for adverse protein folding in plants and leads to a reduction of the average yields for major crop plants by more than 50% worldwide. Therefore, it represents a serious threat to agriculture and also the environment.

Special emphasis of our research lies on the so-called N-end rule pathway of targeted protein degradation (NERD). In plants, only little is known about its biological function, although mutations adversely influence cell proliferation, plant development and ageing, organ growth, and seed germination. In our lab, we set out to identify and characterize enzymatic NERD components both on biochemical and physiological level and assign their physiological substrates.

 

Plant PQC was shown to be important in breakdown of storage reserves in seeds, enabling germination, growth and seedling establishment. Noteworthy, the presence of functional plant proteins becomes increasingly relevant from a bioeconomical point of view as one of the premier storage units for energy, hallmarks of plant environmental stress tolerance, and plant-based biotechnological protein production. We have developed a transgenic in vivo protein stability reporter system as a molecular tool that allows screening for mutants defective in PQC and combine genetics, cell biology and state-of-the-art biochemistry. As experimental model system, we are using Arabidopsis thaliana, currently the best-understood plant organism, by combining genetics, cell biology and state-of-the-art biochemistry.

 Crystal structures of different kinasesCrystal structures of different kinases

A long-term goal is to understand the biological significance of plant PQC in the context of development and biotechnology by elucidating protein quality checkpoints and proteostatic control in general, but also focused on plant yield and stress signalling.

 

 

NERD – the N-end rule pathway of targeted protein degradation

NERD has the capacity of targeted recognition and removal of proteins that are flagged by specific destruction signals, so-called degrons. In several studied model systems, NERD functions include the control of chromosome segregation, DNA repair, apoptosis, meiosis, and diverse developmental processes.

Despite its clear involvement in cardinal cellular functions, in plants, NERD is poorly understood and only few biological roles were uncovered. However, it is known that this pathway comprises a hierarchical cascade of protein modifiers with multiple possible levels of regulation. Only recently, it has been identified as a major determinant for flooding tolerance in plants, amongst them highly important crops such as barley and rice. In Arabidopsis, NERD is required for seed ripening, lipid breakdown and germination. Moreover, plant NERD regulates leaf and shoot development, flower induction, cell division, and possibly plant-pathogen interaction. Most of these factors are – besides the general control of protein stability and abundance – highly important traits in agriculture. To date, the underlying molecular wiring remained obscure.

 

Our laboratory work is mainly focused on genetical and protein biochemical studies of enzymatic NERD components (E3 Ubiquitin ligases, arginyl-transferases, and amidases), their substrate proteins as well as biotechnological applications of NERD in plants. Moreover, we have successfully implemented a temperature-dependent protein accumulation technique in plants which relies on protein stability modulated by NERD. By using this technique, we can accumulate or deplete a specific artificial target protein fusion in living plants. This approach aims at the manufacture of plant-based molecules of interest, e.g. of proteins or chemical compounds that can be used in industrial or pharmaceutical biotechnology.

 

Our lab's main objectives

My lab is interested in proteostasis and posttranslational protein modification via the N-end rule pathway of targeted protein degradation as part of the Ubiquitin proteasome system. Our research includes: (1) enzymatic mechanisms and their regulation; (2) substrate identification and biological integration ('phenotypes’) of the N-end rule system; (3) roles under stress conditions versus the normal environment in plant development and fitness using Arabidopsis as the model; and (4) harnessing the N-end rule pathway for genetic and biotechnological applications in various multicellular organisms. We developed assays and artificial substrates highly specific for this pathway to identify and characterize novel enzymatic modifiers and approach potential substrates via differential proteomics and knowledge-based pipelines. We strive for biotechnological application and translation of our work which has allowed to generate "phenotypes on demand" by conditionally accumulating target proteins and by this switch both their function and activity.

 

Picture taken from a current review article (Dissmeyer & Schnittger (2011), The age of protein kinases, Methods Mol Biol. 2011;779: 7-52) which is publically available at Springer.