Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates1, including more recently the genomes of archaic hominins2,3. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for ~1% of autism cases4,5 and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. We reconstructed the evolutionary history of the locus and identified BOLA2 (bolA family member 2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95 kbp segment containing BOLA2 duplicated across the critical region ~282 thousand years ago (kya), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carry one or more copies of the duplication, which nearly fixed early in the human lineage—a pattern unlikely to have arisen so rapidly in the absence of selection (p < 0.0097). We show that the duplication of BOLA2 led to a novel, human-specific in-frame fusion transcript and that BOLA2 copy number correlates with both RNA expression (r = 0.36) and protein level (r = 0.65), with the greatest expression difference between human and chimpanzee in experimentally derived stem cells. Analyses of 152 patients carrying a chromosome 16p11.2 rearrangement showed that >96% of breakpoints occur within the Homo sapiens-specific duplication. In summary, the duplicative transposition of BOLA2 at the root of the Homo sapiens lineage ~282 kya simultaneously increased copy number of a gene associated with iron homeostasis and predisposed our species to recurrent rearrangements associated with disease.

In eukaryotes, the interaction between members of the monothiol glutaredoxin family and members of the BolA-like protein family has been involved in iron metabolism. To investigate the still unknown functional role of the interaction between human glutaredoxin-3 (GRX3) and its protein partner BOLA2, we characterized at the atomic level the interaction of apo BOLA2 with the apo and holo states of GRX3 and studied the role of BOLA2 in the GRX3-dependent anamorsin maturation pathway. From these studies, it emerged that apo GRX3 and apo BOLA2 form a heterotrimeric complex, composed by two BOLA2 molecules and one GRX3 molecule. This complex is able to bind two [2Fe-2S](2+) clusters, each being bridged between a BOLA2 molecule and a monothiol glutaredoxin domain of GRX3, and to transfer both [2Fe-2S](2+) clusters to apo anamorsin producing its mature holo state. Collectively, the data suggest that the heterotrimeric complex can work as a [2Fe-2S](2+) cluster transfer component in cytosolic Fe/S protein maturation pathways.

The aggregation of α-synuclein (αS) is a critical step in the etiology of Parkinson's disease. Metal ions such as copper and iron have been shown to bind αS, enhancing its fibrillation rate in vitro. αS is also susceptible to copper-catalyzed oxidation that involves the reduction of Cu(II) to Cu(I) and the conversion of O(2) into reactive oxygen species. The mechanism of the reaction is highly selective and site-specific and involves interactions of the protein with both oxidation states of the copper ion. The reaction can induce oxidative modification of the protein, which generally leads to extensive protein oligomerization and precipitation. Cu(II) binding to αS has been extensively characterized, indicating the N terminus and His-50 as binding donor residues. In this study, we have investigated αS-Cu(I) interaction by means of NMR and circular dichroism analysis on the full-length protein (αS(1-140)) and on two, designed ad hoc, model peptides: αS(1-15) and αS(113-130). In order to identify and characterize the metal binding environment in full-length αS, in addition to Cu(I), we have also used Ag(I) as a probe for Cu(I) binding. Two distinct Cu(I)/Ag(I) binding domains with comparable affinities have been identified. The structural rearrangements induced by the metal ions and the metal coordination spheres of both sites have been extensively characterized.

Human mitoNEET is a homodimeric protein anchored to the outer mitochondrial membrane and has a C-terminal [2Fe-2S] binding domain located in the cytosol. Recently, human mitoNEET has been shown to be implicated in Fe/S cluster repair of cytosolic iron regulatory protein 1 (IRP1), a key regulator of cellular iron homeostasis in mammalian cells. The Fe/S cluster repair function of mitoNEET is based on an Fe/S redox switch mechanism: under normal cellular conditions, reduced [2Fe-2S]-mitoNEET is present and is inactive as an Fe/S cluster transfer protein; under conditions of oxidative cellular stress, the clusters of mitoNEET become oxidized, and the formed [2Fe-2S]-mitoNEET species reacts promptly to initiate Fe/S cluster transfer to IRP1, recycling the cytosolic apo-IRP1 into holo-aconitase. Until now, no clear data have been available on which is the system that reduces the mitoNEET clusters back once oxidative stress is not present anymore. In the present work, we used UV-vis and NMR spectroscopies to investigate the electron transfer process between mitoNEET and the cytosolic electron-donor Ndor1/anamorsin complex, a component of the cytosolic iron-sulfur protein assembly (CIA) machinery. The [2Fe-2S] clusters of mitoNEET are reduced via the formation of a transient complex that brings the [2Fe-2S] clusters of mitoNEET close to the redox-active [2Fe-2S] cluster of anamorsin. Our data provide in vitro evidence of a possible direct link between the CIA machinery and the mitoNEET cluster transfer repair pathway. This link might contribute to recovery of CIA machinery efficiency to mature cytosolic and nuclear Fe/S proteins.