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Structure-function studies of the human FASTK family of mitochondrial proteins with putative novel RNA binding domains – the helical FAST motifs and the small RAP domain

Periodic Reporting for period 1 - FAST-RAP (Structure-function studies of the human FASTK family of mitochondrial proteins with putative novel RNA binding domains – the helical FAST motifs and the small RAP domain)

Reporting period: 2015-10-01 to 2017-09-30

Proteins play a fundamental role in all life processes and the human genome encodes tens of thousands of different proteins, but we still know very little about the function or structure of a large portion of them. A three-dimensional structural model of a protein can help explain its mechanism of action and enable the design of specific inhibitors that snugly fit the pockets in its shape. If the protein is engaged in a pathological process, such a small-molecule inhibitor of its function may become a useful therapeutic drug. Structural biologists are working towards obtaining structural models of all proteins, either through experimental methods such as protein crystallography or based on a computational analysis of known structures of similar proteins.
One important class of proteins are RNA-binding factors - enzymes and regulators of cellular metabolism – and recent studies of the human proteome have identified that there are still protein families left which contain RNA-binding domains of yet unknown structure. This project studies one of such protein families: Fas-activated Serine-Threonine Kinase (FASTK) and its relatives. Six such proteins can be found in the human mitochondria where they contribute to the regulation of the cellular respiration. The exact mechanism of action of FASTK proteins is not known, but misregulation of their expression may result in a mitochondrial disease or certain types of cancer. FASTK also has a pro-inflammatory effect due to its role in control of Fas, an important protein also called the death receptor. Mice devoid of Fastk protein were previously found resistant to immune-induced arthritis or pulmonary inflammation, which suggested that drugs developed to inhibit FASTK may be used to treat auto-immune or inflammatory disorders such as asthma, rheumatoid arthritis, lupus or type I diabetes. Proteins from the FASTK family play therefore a role both in the healthy and pathological states of the human body, and we were interested in describing their exact function and structure. Moreover, FASTK family members are expected to bind RNA through a new type of a structural domain, so that obtaining its 3D model could broaden our understanding of protein structures in general, and possibly give us new tips for engineering proteins, for example for use as tools in gene therapy.
None of the FASTK proteins have been successfully produced before in a test tube, and therefore their functions have not been confirmed in such a controlled environment, nor their 3D structure could be described or reliably predicted. This project focused on production of FASTK and related proteins, and using them to verify their function and to obtain experimental structural models. If the 3D structure of FASTK was known, this should help us understand what these proteins are doing in a human organism, how to treat mitochondrial, oncogenic or inflammatory disorders associated with their malfunction, and to see whether we can find new RNA-binding structures for protein engineering. The structural model could also be used in the future for designing anti-inflammatory drugs, for example to treat asthma or rheumatoid arthritis.
In addition, the project included the aspects of young researcher training and the transfer of the structural biology knowledge to establish the new laboratory for protein crystallography. This high technology is in demand for modern protein research and is still relatively underdeveloped in Poland. The availability of this method locally to support structural biology projects is valuable for medically-relevant research areas, such as drug design or the study of pathological mutations found in patients, or simply the understanding of function of medically important proteins.
As the outcome of this project, most of the proteins of the FASTK family were produced using a bacterial expression system and purified to high homogeneity. This achievement enabled for the first time structural and functional studies of these proteins in a test tube. We obtained a structural model of FASTK through small angle X-ray scattering and continue working towards a crystal structure. We also used bioinformatics analyses to identify and classify over a thousand relatives of FASTK proteins across all domains of life, including identification of bacterial homologs. This enabled us to understand better the evolutionary origin of these proteins and their function. In addition, the Fellow has established a Structural Biology Group with expertise in protein crystallography, which should strengthen this discipline in Poland.
We screened a large number of constructs to identify fragments of human FASTK proteins or homologs amenable to expression in bacteria and purification in large quantities. We successfully purified most of human FASTK proteins and used them for structural and functional studies. We studied oligomerization of the purified proteins and identified new complexes, performed secondary structure analysis and obtained a low resolution structural model of FASTK. In addition, we used bioinformatics to understand the distribution and function of FASTK homologs, and in the process developed a new method of detecting and classifying repeat motifs in protein sequences.
Several publications disseminating the results are under preparation or expected to be published in the future. The results of the project were presented at conferences such as the EMBO Workshop Molecular biology of mitochondrial gene expression or the EMBO Sectoral Meeting in Structural Biology and in Immunology. The Fellow co-organised conferences and workshops, for example the 7th European Charge Density Meeting, Bridging Science and Business and Standing up for Science EU workshops.
The Fellow improved both scientific (biophysical methods, bioinformatics) and soft skills (project and finance management, team supervision, writing, presentation and organisation skills). The Fellow supervised students, recruited additional team members and funding, and established Structural Biology Group with expertise in protein crystallography and biophysical characterisation of proteins. This project allowed the Fellow to advance her career towards a position of professional maturity and established valuable external collaborations.
For the first time, soluble constructs of FASTK family members were produced, which enabled the characterization of their structure and interactions. New collaborations regarding the function, structure and the bioinformatics analyses of FASTK proteins were established with neighbouring institutions. Through a bilateral transfer of knowledge, the project formed the basis for the formation of a new research group and introduction of a protein production and crystallization pipeline at the host department. The expertise in structural biology strengthened by this project will support medically-relevant and high-technology-based research at the host institution. The project was used for training of the Fellow and other research staff, in teaching students and in science popularisation. The Fellow participated actively in promoting interest in scientific careers through media appearances and outreach events, for example as Chair of the Polish Chapter of Marie Curie Alumni Association. All of the above should positively impact both on the excellence of science and its societal engagement, on a local and broader level within the European Research Area.