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Peroxisomes: key to cell performance and health

Final Report Summary - PERFUME (Peroxisomes: key to cell performance and health)

PERFUME - PERoxisome formation, FUnction, MEtabolism
https://www.rug.nl/research/molecular-cell-biology/ITN-Perfume/index

Contact: info@itn-perfume.eu

Prof. Dr. Ida J. van der Klei
Molecular Cell Biology
University of Groningen
i.j.van.der.klei@rug.nl
P.O. Box 11103, 9700CC, Groningen, The Netherlands
Introduction
The PERFUME (PERoxisome formation, FUnction, MEtabolism) program is an interdisciplinary and intersectoral ITN providing state-of-the-art research training at the interface of medicine, plant and fungal biology. The PERFUME S&T specifically aims at increasing our knowledge on peroxisomes.
Peroxisomes are subcellular organelles that occur in almost all eukaryotic cells. They are involved in a large variety of metabolic functions such as methanol metabolism in yeast, photorespiration in plant and the biosynthesis of special lipids in mammals. Their importance is illustrated by the fact that peroxisome malfunction causes severe diseases in man and often is lethal. Consequently, research on peroxisomes is very relevant for human health and disease because the outcome can contribute to the development of new therapies and improved diagnosis. Peroxisomes also play crucial functions in plant, making peroxisome research also important for agriculture. Finally, in fungi peroxisomes are involved in the production of various important compounds, including the antibiotics. Therefore PERFUME research can contribute to improving fungal cell factories (in biotechnology) as well.

Training and research
The PERFUME consortium consists of eight scientific institutes, including two academic hospitals, together with two companies, located in four European countries. Fourteen Early Stage Researchers (ESRs) and three Experienced Researchers (ERs) have been trained by the PERFUME program. All fellows received optimal training by a comprehensive training program, which among others included training-through-research as well as participation in PERFUME workshops and courses in scientific and complementary skills. The fellows also attended two international PERFUME conferences and conducted multiple secondments to academic and industrial partners. The PERFUME research program aimed to fill the major gaps in our knowledge of peroxisome biology and consisted of four Work packages. In each of the Work packages important results were obtained.
WP1. Identification of novel peroxisome functions
Peroxisomal proteins contain information in their amino acid sequences (the so called Peroxisomal Targeting Signal, PTS) that is crucial to direct them to the correct organelle in the eukaryotic cell. In a bioinformatics approach, PTS prediction tools were applied to search in plant and yeast genomes for genes encoding proteins possessing a peroxisomal targeting signal. In a complementary approach peroxisomes were isolated from yeast or plant cells that were exposed to various stress conditions. Their protein content was subsequently analyzed by mass spectrometry (proteomics). Both approaches resulted in the identification of several novel plant and yeast peroxisomal proteins, including proteins involved in stress adaptation.
WP2. Role of compartmentalization
For many peroxisomal metabolic pathways it is speculative why they are contained in the peroxisomes. Advanced systems biology approaches were applied to understand this important question in peroxisome biology. The research in this WP aimed at constructing dynamic computer models of peroxisomal metabolism. An existing metabolic model of human cells (Recon2) was extended with enzymatic reactions involved in the oxidation of fatty acids, including reactions that occur in peroxisomes. Using this model cell lines of patients suffering from a severe peroxisomal disease were analyzed, resulting in a better understanding of this disease. The outcome of this research aids to the identification of new biomarkers that can be used in diagnosis of peroxisomal disorders.
WP3. Human peroxisomal disorders
The laboratory diagnosis of patients with known peroxisomal disorders is usually readily achieved by biochemical and genetic analysis. However, several patients have been identified which could not be diagnosed by these established methods. In order to improve diagnosis for these patients, detailed genetic, biochemical and cell biological studies were performed. The outcome of these studies contributes to a better understanding and hence improved tools for diagnosis, of three different peroxisome-related diseases. By comparing the total phospholipids profiles of healthy control individuals with those of patients with a severe or a relatively mild peroxisomal phenotype, we showed that specific phospholipid levels can be indicative for certain peroxisomal diseases. Importantly, patients with very mild phenotypes, that are difficult to pick up with standard diagnostic testing, could also be diagnosed with this method.
In the PERFUME program a new signaling pathway for peroxisome proliferation was identified. This pathway may serve as target to design novel drugs to treat peroxisome-related diseases that are accompanied by reduced peroxisome abundance.
WP4. Unravelling the molecular principles of peroxisome proliferation
WP4 aimed to understand the mechanisms of peroxisome multiplication at the molecular level. This research included the identification of proteins involved in fission of pre-existing organelles as well as proteins that play a role in the formation of peroxisomes from other cellular membranes (the so called de novo pathway).
Using biochemical approaches and mass spectrometry, novel binding partners of known proteins were identified. Various genetic approaches also resulted in the identification of novel genes. Their analysis contributed to the novel concept that peroxisomal membrane contact sites are important for peroxisome formation. Also, we observed that yeast mutants previously assumed to fully lack peroxisomes still contained peroxisomal remnant structures. The protein composition of these vesicles was established by detailed microscopy and biochemical approaches.
In-depth structure/function analysis was performed of known key proteins of peroxisome fission and de novo synthesis. This included bioinformatics analysis as well as biophysical approaches. For the latter, large quantities of the proteins were produced in bacteria or insect cells. After purification the proteins were crystallized in order to obtained detailed structural information by X-ray diffraction. This approach was successful to obtain low resolution structures for various human proteins that play a role in peroxisome formation.


Poster and photo of participants of the final PERFUME conference held in Hamburg June 20-22, 2016.