Molecular pathways for the maturation of mitochondrial and cytosolic human Fe-S proteins

Iron-sulfur (Fe-S) clusters are simple inorganic protein cofactors supposed to be involved in the development of the very first forms of life. Indeed, they are ancient, versatile and ubiquitous prosthetic groups, which require numerous protein components for their biosynthesis and incorporation into scaffold apoproteins. The proteins responsible for this process cooperate within specific biogenesis machineries, which are the mitochondrial Fe-S cluster (ISC) assembly machinery and the cytosolic Fe-S cluster assembly (CIA) machinery, highly conserved in eukaryotes. Nowadays, the interest of the research on Fe-S proteins biogenesis is focused on the functional characterization of the involved proteins and on defining the pathway deputy to the Fe-S cluster biosynthesis and to the maturation of Fe-S proteins. The Fe-S clusters synthesis starts in the mitochondria; mutations in genes encoding for late-acting proteins in the ISC machinery were found to cause autosomal recessive diseases defined as Multiple Mitochondrial Dysfunctions Syndromes (MMDS). The MMDSs phenotypes are characterized by lower energy metabolism, impairment in neurologic development and respiratory failure, which are related to the impairment in the maturation of [4Fe-4S] proteins. The functioning of the ISC machinery is also fundamental for the maturation of cytosolic Fe-S proteins. The aim of the thesis work was the investigation of the molecular mechanisms underlying the maturation of human mitochondrial and cytosolic [4Fe-4S] proteins, using different spectroscopic and biochemical techniques, namely NMR, EPR, UV-vis and CD spectroscopies, mass spectrometry and size exclusion chromatography. This study led to elucidating the molecular basis of MMDS2 and MMDS3 caused by not yet investigated mutations in BOLA3 and IBA57 genes, but also to clarify the mechanism responsible for the maturation of [4Fe-4S]-NUBP1 in the cytosol, involving the protein chaperone GLRX3 and the partner protein anamorsin. Preliminary data were also obtained on the role of BOLA2 in the same process.