Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3.

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.

Unraveling the mechanism of [4Fe-4S] cluster assembly on the N-terminal cluster binding site of NUBP1.

[4Fe-4S] cluster assembly in human cytosol requires both a [2Fe-2S] cluster chaperone being able to donate two [2Fe-2S] clusters and an electron donor providing two electrons to reductively couple the two [2Fe-2S] clusters into a [4Fe-4S] cluster. The mechanism through which the cytosolic [4Fe-4S] cluster assembly works is still not defined. Here, we show that a hetero-tetrameric complex formed by two molecules of cluster-reduced [2Fe-2S] -anamorsin and one molecule of dimeric cluster-oxidized [2Fe-2S] -GLRX3 orchestrates the assembly of a [4Fe-4S] cluster on the N-terminal cluster binding site of the cytosolic protein NUBP1.

Copper Binding and Redox Activity of α-Synuclein in Membrane-Like Environment.

α-Synuclein (αSyn) constitutes the main protein component of Lewy bodies, which are the pathologic hallmark in Parkinson's disease. αSyn is unstructured in solution but the interaction of αSyn with lipid membrane modulates its conformation by inducing an α-helical structure of the -terminal region. In addition, the interaction with metal ions can trigger αSyn conformation upon binding and/or through the metal-promoted generation of reactive oxygen species which lead to a cascade of structural alterations.

The Intriguing mitoNEET: Functional and Spectroscopic Properties of a Unique [2Fe-2S] Cluster Coordination Geometry.

Despite the number of cellular and pathological mitoNEET-related processes, very few details are known about the mechanism of action of the protein. The recently discovered existence of a link between NEET proteins and cancer pave the way to consider mitoNEET and its Fe-S clusters as suitable targets to inhibit cancer cell proliferation. Here, we will review the variety of spectroscopic techniques that have been applied to study mitoNEET in an attempt to explain the drastic difference in clusters stability and reactivity observed for the two redox states, and to elucidate the cellular function of the protein.

Multiple Mitochondrial Dysfunction Syndrome Type 3: A Likely Pathogenic Homozygous Variant Affecting a Patient of Cuban Descent and Literature Review.

Multiple mitochondrial dysfunction syndrome type 3 (MMDS3) is a rare mitochondrial leukoencephalopathy caused by biallelic pathogenic variants in . Here, we describe a homozygous variant in , (NM_001010867.2): c.

Metal trafficking in the cell: Combining atomic resolution with cellular dimension.

Metals are widely present in biological systems as simple ions or complex cofactors, and are involved in a variety of processes essential for life. Their transport inside cells and insertion into the binding sites of the proteins that need metals to function occur through complex and selective pathways involving dedicated multiprotein machineries specifically and transiently interacting with each other, often sharing the coordination of metal ions and/or cofactors. The understanding of these machineries requires integrated approaches, ranging from bioinformatics to experimental investigations, possibly in the cellular context.

Molecular Basis of Rare Diseases Associated to the Maturation of Mitochondrial [4Fe-4S]-Containing Proteins.

The importance of mitochondria in mammalian cells is widely known. Several biochemical reactions and pathways take place within mitochondria: among them, there are those involving the biogenesis of the iron-sulfur (Fe-S) clusters. The latter are evolutionarily conserved, ubiquitous inorganic cofactors, performing a variety of functions, such as electron transport, enzymatic catalysis, DNA maintenance, and gene expression regulation.

The long-standing relationship between paramagnetic NMR and iron-sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?

Paramagnetic NMR spectroscopy and iron-sulfur (Fe-S) proteins have maintained a synergic relationship for decades. Indeed, the hyperfine shifts with their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues have been extensively used as a fingerprint of the type and of the oxidation state of the Fe-S cluster within the protein frame. The identification of NMR signals from residues surrounding the metal cofactor is crucial for understanding the structure-function relationship in Fe-S proteins, but it is generally impaired in standard NMR experiments by paramagnetic relaxation enhancement due to the presence of the paramagnetic cluster(s).

In Cellulo Mössbauer and EPR Studies Bring New Evidence to the Long-Standing Debate on Iron-Sulfur Cluster Binding in Human Anamorsin.

Human anamorsin is an iron-sulfur (Fe-S)-cluster-binding protein acting as an electron donor in the early steps of cytosolic iron-sulfur protein biogenesis. Human anamorsin belongs to the eukaryotic CIAPIN1 protein family and contains two highly conserved cysteine-rich motifs, each binding an Fe-S cluster. In vitro works by various groups have provided rather controversial results for the type of Fe-S clusters bound to the CIAPIN1 proteins.

GLRX3 Acts as a [2Fe-2S] Cluster Chaperone in the Cytosolic Iron-Sulfur Assembly Machinery Transferring [2Fe-2S] Clusters to NUBP1.

Human cytosolic monothiol glutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe-4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe-2S] clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron-sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe-2S] clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe-4S] clusters on both N-terminal CXCXCXC and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant.

Paramagnetic H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes.

Iron-sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Here we showed that H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate molecules with each of the two [4Fe-4S] clusters of human lipoyl synthase.

Correction to: The NMR contribution to protein-protein networking in Fe-S protein maturation.

The article "The NMR contribution to protein-protein networking in Fe-S protein maturation", written by Lucia Banci, Francesca Camponeschi, Simone Ciofi‑Baffoni, Mario Piccioli was originally published electronically on the publisher's internet portal (currently SpringerLink) on 22 March, 2018 without open access.

The NMR contribution to protein-protein networking in Fe-S protein maturation.

Iron-sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe-S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe-2S], [3Fe-4S] and [4Fe-4S] clusters bound to proteins.

Fragment-based approach to identify IDO1 inhibitor building blocks.

Indoleamine 2,3-dioxygenase 1 (IDO1) is attracting a great deal of interest as drug target in immune-oncology being highly expressed in cancer cells and participating to the tumor immune-editing process. Although several classes of IDO1 inhibitors have been reported in literature and patent applications, only few compounds have proved optimal pharmacological profile in preclinical studies to be advanced in clinical trials. Accordingly, the quest for novel structural classes of IDO1 inhibitors is still open.

Anamorsin/Ndor1 Complex Reduces [2Fe-2S]-MitoNEET via a Transient Protein-Protein Interaction.

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.

Copper(I) Forms a Redox-Stable 1:2 Complex with α-Synuclein N-Terminal Peptide in a Membrane-Like Environment.

α-Synuclein (αS) is the main protein component of Lewy bodies, characterizing the pathogenesis of Parkinson's disease. αS is unstructured in solution but adopts a helical structure in its extended N-terminal segment upon association with membranes. In vitro the protein binds avidly Cu(II), but in vivo the protein is N-acetylated, and Cu(II) binding is lost.

Elucidating the Molecular Function of Human BOLA2 in GRX3-Dependent Anamorsin Maturation Pathway.

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.

New Formulations of Polysaccharide-Based Hydrogels for Drug Release and Tissue Engineering.

Polysaccharide-based hydrogels are very promising materials for a wide range of medical applications, ranging from tissue engineering to controlled drug delivery for local therapy. The most interesting property of this class of materials is the ability to be injected without any alteration of their chemical, mechanical and biological properties, by taking advantage of their thixotropic behavior. It is possible to modulate the rheological and chemical-physical properties of polysaccharide hydrogels by varying the cross-linking agents and exploiting their thixotropic behavior.

The extracellular loop of IRT1 ZIP protein--the chosen one for zinc?

Zinc complexes with the extracellular loop of IRT1 (iron-regulated transporter 1), a ZIP (ZRT/IRT - Related Protein) family protein from Arabidopsis thaliana, have been studied. This unstructured fragment is responsible for metal selectivity and is located between the II and III transmembrane domains of IRT1. Zinc complexes with the Ac-(95)MHVLPDSFEMLSSICLEENPWHK(117)-NH2 peptide (IRT1), revealed surprisingly high thermodynamic stability.

Copper(I)-α-synuclein interaction: structural description of two independent and competing metal binding sites.

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.

Triple resonance cross-polarization for more sensitive 13C MAS NMR spectroscopy of deuterated proteins.

Save the last WALTZ for me: the use of simultaneous proton and deuterium cross-polarization for (13)C CPMAS NMR spectroscopy in highly deuterated proteins is discussed. The aim of the new method introduced herein, triple-resonance cross-polarization, is to increase the sensitivity of the carbon-detected methods in such systems.

The role of His-50 of α-synuclein in binding Cu(II): pH dependence, speciation, thermodynamics and structure.

Copper interaction with alpha synuclein (αS) has been shown to accelerate aggregation and oligomerization of the protein. Three different αS copper binding domains have been proposed: (i) the N-terminal residues (1-9) that represent the minimal copper binding domain; (ii) the His-50 imidazole and (iii) the Asp and Glu residues within the acidic C-terminal domain. The copper coordination at the N-terminus has been extensively characterized and it is generally accepted that it provides the highest affinity site.

Heteronuclear and homonuclear Cu2+ and Zn2+ complexes with multihistidine peptides based on zebrafish prion-like protein.

The homeostasis of metal ions, especially copper and zinc, is a major factor that may influence the prion diseases and the biological function of prion protein (PrP). The His-rich regions are basic sites for metal binding and antioxidant activity of the PrP structures. Animal prion-like proteins contain also His-rich domains, and their coordination chemistry may provide better insight into the chemistry and biology of PrP structures and related diseases.