Oligomerization of human and bacterial frataxin : Structural and functional studies

Abstract

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by defeciency in frataxin, a highly conserved protein central to iron homeostasis in mirochondria. The function of frataxin is related to its capability to bind, store and deliver iron to different biochemical process such as iron-sulfur cluster assembly and heme biosynthesis. Most FRDA pateints have extended GAA codon repeats in the first intron of the frataxin gene, which results in decreased protein expression. Low levels of frataxin lead to dysregulation of mitochondrial function, iron accumulation, increased levels of ROS production and subsequent mitochondrial protein and DNA damage.
Two isoforms of human frataxin, FXN42-210 and FXN56-210, as well as yeast frataxin Yfh1 have been shown to build up oligomeric species stabilized by a long non-conserved N-terminal extension. On the other hand, the short form of human frataxin FXN81-210, which is also the most abundant isoform of the protein in cells, appeared to function only as a monomer. Within this thesis we study the oligomerization of the short form of human frataxin FXN81-210 , and compare it to the oligomerization of bacterial frataxin CyaY. The study is based on several experimental methods, including dynamic light scattering (DLS), small angle X-ray scattering (SAXS), electron microscopy (EM), crosslinking mass spectrometry (CXMS), X-ray absorption spectroscopy (XAS), and nano-differential scanning fluorimetry (nano-DSF).
The results show that both FXN81-210 and CyaY are able to form oligomers in the presence of iron at aerobic conditions. However, while iron-induced CyaY oligomers had high stability over time, FXN81-210 oligomers dissociated into monomeric species after about 24 hours. Surprisingly, ferric iron chelators as well as hydrogen peroxide had the ability to enhance the formation of FXN81-210 oligomers. On the other hand, the presence of the same chelators resulted in the dissociation of higher order CyaY oligomeric into lower order oligomers and monomers. XAS studies indicated that large FXN81-210 oligomers are stabilized by a ferrihydrite mineral, which is presumably formed during oxidation of bound iron.
CXMS, SAXS and EM were used in the analysis of the oligomeric structures. The data suggested that for both FXN81-210 and CyaY the iron induced larger oligomers are built up by dimers arranged in a head-to-tail mode. SAXS data were also used to study the effect of iron concentration relative to protein concentration on iron-induced CyaY oligomers, showing increased build up of oligomers with increasing iron concentration.
Nano-DSF measurements on iron-induced CyaY oligomers were used for additional assessment of CyaY oligomer stabilty. The data showed two melting points a lower temperature point corresponding to oligomer dissociation and a high temperature point corresponding to monomer denaturation. This suggests that CyaY may build transient oligomers in cells in response to conditions of elevated iron content.

Abstract (Swedish)

Iron is a one of the critical elements in our life, being the most common element on earth, forming most of the inner and outer earth’s core. In our bodies iron is required in small amounts for biological processes to be performed. It is recommended to eat food containing iron to avoid common health problems such as anemia. A slight increase or decrease in iron levels above or below normal in living cells leads to various health problems. Iron can also be highly toxic if it is accumulated in cells in a free form. As such it may easily interact with different cellular components, including proteins and DNA.
Iron accumulation may occur in our bodies if iron is not properly treated physiologically. It may lead to many disorders and diseases such as thalassemia, Parkinson’s disease, and Friedreich’s ataxia (FRDA).
FRDA is a hereditary neurodegenerative disease that causes progressive damage to the nervous system. Many symptoms and problems were observed for FRDA patients, such as heart problems, skeletal deformation, speech problems, diabetes and sensory loss. No cure is known for FRDA till now.
The cause of FRDA is linked to deficiency in FRDA patients of a protein called frataxin. This protein is found inside the mitochondria (the factories of energy our body uses). When frataxin is present in our cells at lower levels than normal, increase of iron levels takes place. This increase leads to the formation of highly toxic molecules called radicals. These radicals may destroy mitochondrial DNA and proteins.
The disturbance in the balance of iron within the cells when frataxin is present at low levels is related to frataxin function. Frataxin is responsible for iron delivery to other proteins within the mitochondria. Frataxin has been suggested to be involved in iron storage by forming large complex within which iron is stored. These complexes of different sizes are called oligomers, which are built up by varied number of frataxin monomers. Therefore, low levels of frataxin in FRDA patients lead to imbalance of iron within cells. Two things happen when frataxin levels are low; first, the binding and transporting of iron to its target proteins will decrease. Second, the cell will translate this as an iron deficiency and will import more iron to the mitochondria. This will result in iron accumulation. This iron accumulation inside the mitochondria will increase the free iron content, leading to the formation of toxic radicals. If the mitochondria cannot deal with these radicals, the cell will die.
Towards finding a cure for FRDA, we need to have a clear picture of the exact role of frataxin. A main problem is that the full structures of frataxin oligomers are still unknown. Since protein function is always related to its structure, without clear knowledge of the structure of different oligomers it would be difficult to understand the exact role of frataxin.
Since frataxin from different organisms has similar structures, in this work we compare oligomerization of frataxin from human and Escherichia coli. Different experiments have been performed to characterize these two proteins in absence and presence of iron, to try to understand how those oligomers are formed and to describe the structures of the oligomers.
Iron chelators have been suggested for use as drugs for decreasing the symptoms of FRDA. These molecules have the ability to bind iron with strong affinity. They are introduced into the body to bind excess iron and to reduce the severe symptoms associated with iron accumulation. The effect of these chelators on frataxin oligomers was also studied during this work trying to understand how they work in vivo.
We have found that the increase of iron concentration increases the formation of human and bacterial frataxin oligomers. Some oligomers were formed of two, three or four frataxin monomers. Higher oligomers could be observed but their order and percentage could not be determined. The structure of dimeric bacterial frataxin was determined.
The iron chelators showed an opposite effect on both human and bacterial frataxin. For human frataxin they help in the building up of larger oligomers, while in bacterial frataxin they dissociate large oligomers into smaller ones. The stability of these oligomers was found to be low in general suggesting that they are formed in the cells for temporary functions then dissociate to single units to perform their main function in delivering iron to other proteins.
The effect of these iron chelators, which are used in chelation therapy, needs further studies. Even other chelators need to be studied in the future. We believe that having a clear picture of how these chelators work will help to identify more suitable drugs for patients suffering from FRDA.

• author: Abdalkhalik, Mostafa
• supervisor: Salam Al-Karadaghi; Derek Logan
• opponent: Professor Papageorgiou, Tassos, Åbo Akademi, University of Turku, Finland
• organization: Biochemistry and Structural Biology
• publishing date: 2017-02
• type: Thesis
• publication status: published
• subject: Natural Sciences
• keywords: Frataxin, Friedreich’s ataxia, iron, reactive oxiden species, protein oligomerization, SAXS, EM, cross-linking, XAFS, nano- DSF
• pages: 142 pages
• defense location: Lecture hall A, Center for chemistry and chemical engineering, Naturvetarvägen 14, Lund
• defense date: 2017-03-30 09:15:00
• ISBN: 978-91-7753-231-6
• ISBN: 978-91-7422-509-9
• language: English
• LU publication?: yes
• id: 31798a0c-10b5-404b-991b-bb9fba8b7a8a
• date added to LUP: 2017-03-03 15:40:56
• date last changed: 2018-11-21 21:30:25