| 内容提要: |
Proteins containing Fe-S clusters are ubiquitously found in all kingdoms of life, they are involved in electron transfer, non-redox catalysis, protein synthesis, DNA replication and repair and many other essential biological processes. The biogenesis of Fe/S clusters requires complex machinery including cysteine desulfurase, scaffold proteins for assembling intermediates, chaperones, and electron donors, etc.. Mitochondria have all of these essential components and can synthesize Fe-S clusters on their own but cytosol cannot. Atm1 is a mitochondrial inner membrane transporter, presumably, it can transfer an unknown sulfur-containing compound from mitochondria to cytosol for further process to produce Fe-S clusters for cytosolic and nuclear proteins. Proteins involved in making cytosolic Fe-S clusters are so called CIA (Cytosolic Iron-sulfur cluster Assembly) components that have been identified since 2003. The defect of CIA pathway caused low activity of cytosolic and nuclear Fe-S cluster containing proteins, but does not affect mitochondrial Fe-S cluster containing proteins. Among the CIA components, Dre2 was first cloned in 2008. Dre2 itself contains a [2Fe-2S] and a [4Fe-4S] clusters. Another CIA component Tah18 was identified later. Tah18 is a diflavin reductase physically interacts with Dre2 and can use electrons from NADPH to reduce the [2Fe-2S] cluster in Dre2.
The bona fide electron recipient of Tah18/Dre2 complex has not been identified. And the physiological function of the [4Fe-4S] cluster in Dre2 is also unknown. It has been proposed that Tah18/Dre2 acts as a ferric reductase providing electrons for CIA and dinuclear iron containing enzyme ribonuclease reductase. The iron source of Fe-S cluster biosynthesis is mysterious. It has been known since 1980s that there is a labile iron pool existing in the eukaryotic cytosol. However, the nature of this iron pool and cellular iron trafficking is not well characterized. Recently, the Fe-S cluster located at the interface of monothiol glutaredoxin was proposed to be the long sought cytosolic labile iron pool, because Grx3/4 double mutants exihibit global iron utilization defects with all iron requiring enzymes and proteins including Fe-S, heme and non-heme mononuclear and dinuclear iron proteins. Grx3 and Grx4 belong to the large thioredoxin (Trx) fold family, and are composed of an N-terminal Trx domain and a C-terminal monothiol glutaredoxin (Grx) domain, they are capable of binding a bridging [2Fe-2S] cluster at the interface of the dimer with two cysteines, each from one monomer and glutathione (GSH) as ligands. Based on the study of the biosynthesis of differic-tyrosyl radical cofactor in class Ia RNR (ribonucleotide reductase), the Stubbe group proposed that Tah18-Dre2 complex reduces the Fe-S cluster on Grx3/4 and releases the iron for RNR. Class Ia RNR contains two homodimeric subunits α2 and β2, α2 contains the binding sites for substrates and allosteric effectors. β2 houses a diferric tyrosyl radical cofactor that is essential for initiation of nucleotide reduction in β2.
Reconstitution of this biological process in vivo is technically challenging and requires obtaining each of these recombinant proteins with stoichiometric cofactors. Towards this end, my master thesis aims to clone, express and purify Tah18 and Dre2 from E coli, reconstitute their cofactors. We will attempt to solve the crystal structures of both proteins separately or in a complex. We will also obtain Grx3/4 protein with [2Fe-2S] cluster bound and test if Tah18 and Dre2 can reduce the cluster in Grx3/4. It is known that the [2Fe-2S] cluster on Grx3/4 is reductively labile. This assumptive pathway has been supported by some others in a way. Human homolog of Dre2 is called Anamorsin (AM) which is a cell-death-defying factor. Knock out Dre2 in yeast can be complemented by Anamorsin from human. PICOT (PKCh interacting cousin of thioredoxin) is the human homolog of Grx3/4. Yuri Saito used yeast-two-hybrid assay and GST-Pull down assay showed that PICOT can preferentially bind to AM. This physical interaction further implied there maybe electron transfer between Dre2 and Grx3/4. But how they can bind to each other and why they can bind to each other? We need to do some experiments step by step to investigate it. We will use ferrozine assay to detect the release of iron upon the reduction of the [2Fe-2S] cluster. |